Listeria, Listeriosis, and Food Safety, Third Edition (Food Science and Technology)

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Cover photo: An immunofluorescent image of L. monocytogenes (red) showing cell-to-cell spread via polymerized actin tails (green). Photo courtesy of Dr. Pascale Cossart, Institut Pasteur, Paris.

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2007 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8247-5750-5 (Hardcover) International Standard Book Number-13: 978-0-8247-5750-2 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Listeria, listeriosis, and food safety / editors, Elliot T. Ryser and Elmer H. Marth. -- 3rd ed. p. ; cm. -- (Food science and technology ; 160) Includes bibliographical references and index. ISBN-13: 978-0-8247-5750-2 (hardcover : alk. paper) ISBN-10: 1-4200-1518-4 (hardcover : alk. paper) 1. Listeriosis. 2. Listeria monocytogenes 3. Foodborne diseases. 4. Food--Microbiology. I. Ryser, Elliot T., 1957- II. Marth, Elmer H. III. Title. IV. Series: Food science and technology (Taylor & Francis) ; 160. [DNLM: 1. Listeria Infections--etiology. 2. Food Contamination--prevention & control. 3. Food Microbiology. 4. Listeria monocytogenes--pathogenicity. W1 FO509P v.160 2007 / WC 242 L773 2007] QR201.L7R9 2007 615.9’52937--dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

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IN MEMORIAM

After conceiving the idea for a third edition of Listeria, Listeriosis and Food Safety in June 2002, Elmer Marth and I were in biweekly contact by phone to ensure continued progress toward its completion. This daunting project involved 33 contributors, 3 of whom in addition to myself (Robert E. Brackett, Jeffrey L. Kornacki, and Ahmed E. Yousef) earned their doctorates in the laboratory of Elmer Marth. As the book was rapidly nearing completion in Spring 2006, I was deeply saddened to learn that Elmer had become seriously ill. On June 19, 2006, Emeritus Professor Elmer H. Marth died in Madison, Wisconsin at age 78 while I was editing the chapter proofs for the book. Consequently, it is only fitting that the third edition of Listeria, Listeriosis and Food Safety be dedicated in memory of Dr. Elmer H. Marth who grew up on a small dairy farm in Grafton, Wisconsin to become one of the most preeminent dairy microbiologists of our time.

Elliot T. Ryser

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Preface to the Third Edition Since the second edition of Listeria, Listeriosis and Food Safety was published in 1999, the United States has seen a 40% decrease in the incidence of listeriosis, with the current annual rate of illness rapidly approaching the 2010 target of 2.5 cases per million population. This reduction in the rate of listeriosis would not have been possible without the combined effort of researchers, academicians, commodity organizations, and various regulatory authorities. We would like to believe that the first and second editions of our book also played a role in this outcome by providing needed information to concerned persons. Despite considerable progress in understanding the sources, spread, control, and pathogenicity of Listeria monocytogenes, research on this foodborne pathogen has continued unabated, with more than 5,000 publications on Listeria and foodborne listeriosis appearing in the scientific literature since the second edition of this book was published. A portion of this work was fueled by a series of widely publicized outbreaks of listeriosis involving ready-to-eat meat products in the United States that began in the late 1990s. Over the last 5 years, increasing emphasis has been given to development of risk assessments that can be used to focus limited financial resources on certain high-risk foods, such as soft cheeses, ready-to-eat meats, and smoked fish, that support growth of the pathogen. Efforts ultimately have had an impact on public policies regarding allowable levels of L. monocytogenes in these and other foods. The third edition of Listeria, Listeriosis and Food Safety again summarizes much of the newly published literature and integrates this information with earlier knowledge to present readers with a complete and current overview of foodborne listeriosis. The 17 chapters in the second edition have been retained; all are updated and expanded as appropriate. A total of 33 authors have lent their expertise to preparing this book, with new authors contributing to Chapters 1, 2, 4, 6, 7, 8, 10, 13, and 17. Sometimes these authors have collaborated with the original authors to develop the revised chapter. Two new chapters, Chapter 18 and Chapter 19, have been added to the book. Chapter 18 deals with risk assessment, cost of foodborne listeriosis outbreaks, and regulatory control of the Listeria problem in various countries. In Chapter 19, four experts point out specific data gaps and where, in their view, research efforts should be directed. As was true for the earlier editions, this book will be useful to advanced undergraduate students in food science, microbiology, and public health; graduate students in these same disciplines; and practitioners in food or dairy microbiology, food or dairy science, bacteriology or microbiology, public health, epidemiology, risk assessment, meat science, animal science, and veterinary medicine. It will also be helpful to personnel in the food and dairy industries and the food service industry, and to researchers in industrial, governmental, and university laboratories. Elliot T. Ryser Elmer H. Marth

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Preface to the Second Edition Listeriosis and Listeria monocytogenes continue to be of worldwide interest to the food industry and regulatory agencies, scientists in various disciplines, and consumers of food. Such interest is prompted by the occasional appearance of L. monocytogenes in ready-to-eat foods, leading to the removal of these products from the marketplace. Furthermore, sporadic cases of listeriosis continue to occur and several food-associated outbreaks of the disease have occurred since the first edition of this book was published. Scientists in several disciplines are still studying different aspects of the listeriosis problem. Their efforts have resulted in the development of much new information that has appeared in hundreds, if not thousands, of papers published since the first edition of this book was completed in 1990. This explosion of information warranted publication of a second edition. The second edition differs markedly from the 1991 edition. Whereas we were the authors of the earlier edition, chapters in this edition have been prepared by various experts in the field. We now serve as editors, although one of us (ETR) has revised several chapters. The contributions of this edition’s authors have resulted in an improved book that contains timely topics. The chapters in the first edition have been retained; each has been revised and expanded with new information when appropriate. Two new chapters deal with typing methods and pathogenesis. Thus, this book contains 17 chapters addressing the following topics: description of L. monocytogenes occurrence and behavior of this pathogen in various natural environments animal and human listeriosis pathogenesis of L. monocytogenes characteristics of L. monocytogenes important to food processors conventional and rapid methods to isolate, detect, and identify L. monocytogenes strain-specific typing of L. monocytogenes foodborne listeriosis incidence of behavior of L. monocytogenes in unfermented and fermented dairy products, meat, poultry (including eggs), fish and seafood, and products of plant origin incidence and control of this pathogen within various types of food-processing facilities This book will be useful to advanced undergraduate students, graduate students, and practitioners in food or dairy microbiology, food or dairy science, bacteriology or microbiology, public health, dietetics, meat science, poultry science, and veterinary medicine. It also will be helpful to personnel in the food and dairy industries and regulatory agencies, as well as researchers in industrial, governmental, and university laboratories. Elliot T. Ryser Elmer H. Marth

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Preface to the First Edition Interest in the occurrence of Listeria in food, particularly Listeria monocytogenes, escalated rapidly during the 1980s and continues unabated as a result of several major outbreaks of foodborne listeriosis. The first of these occurred during 1981 and involved consumption of contaminated coleslaw. In 1983, the reputation of the American dairy industry for producing safe products suffered when epidemiological evidence showed that 14 of 49 people in Massachusetts died after consuming pasteurized milk that was supposedly contaminated with L. monocytogenes. Two years later, consumption of contaminated Mexican-style cheese manufactured in California was directly linked to more than 142 cases of listeriosis, including at least 40 deaths. Heightened public concern regarding the prevalence of L. monocytogenes in food prompted the United States Food and Drug Administration to initiate a series of Listeria surveillance programs. Subsequent discovery of this pathogen in many varieties of domestic and imported cheese, in ice cream, and in other dairy products prompted numerous product recalls, which in turn have led to staggering financial losses for the industry, including several lawsuits. These listeriosis outbreaks, together with a subsequent epidemic in Switzerland involving consumption of Vacherin Mont d’Or soft-ripened cheese and discovery of L. monocytogenes in raw and ready-to-eat meat, poultry, seafood, and vegetables, have underscored the need for additional information concerning foodborne listeriosis. In 1961, Professor H. P. R. Seeliger, now retired from the University of Würzburg, published his time-honored book, Listeriosis. His monograph has provided scientists, veterinarians, and the medical profession with much needed information regarding Listeria and human and animal listeriosis, as well as pathological, bacteriological, and serological methods to diagnose this disease. However, documented cases of foodborne listeriosis were virtually unknown 30 years ago. Although much information in his book is still valid today, some of the knowledge regarding media and methods used to isolate, detect, and identify L. monocytogenes in clinical and, particularly, nonclinical specimens is now largely out of date. The emergence of L. monocytogenes as a serious foodborne pathogen together with the virtual flood of Listeria-related papers that have appeared in scientific and trade journals as well as numerous conference proceedings, prompted us to review and summarize the current information so that food industry personnel, public health and regulatory officials, food microbiologists, veterinarians, and academicians have a ready source of information regarding this now fully emerged foodborne pathogen. This book consists of 15 chapters that address the following topics: L. monocytogenes as the causative agent of listeriosis occurrence and survival of this pathogen in various natural environments human and animal listeriosis characteristics of L. monocytogenes important to food processors conventional and rapid methods for isolating, detecting, and identifying L. monocytogenes in food recognition of cases and outbreaks of foodborne listeriosis incidence and behavior of L. monocytogenes in fermented and unfermented dairy products, meat, poultry (including eggs), seafood, and products of plant origin incidence and control of this pathogen within various types of food-processing facilities

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It is evident that major emphasis has been given to information directly applicable to food processors. Information concerning the bacterium and the disease has been admirably reviewed by Professor Seeliger and others, so our discussion of these topics should not be considered exhaustive. Thus, the first four chapters of this book supply only pertinent background information to complement our discussion of foodborne listeriosis. Although many in the scientific community must be commended for the extraordinary progress made since 1985 toward understanding foodborne listeriosis, the continuing “explosion” of information concerning Listeria and foodborne listeriosis has made the 3-year task of compiling an upto-date review of this subject quite difficult. Therefore, to produce as current a document as possible, we have included a bibliography of references that have appeared since the book was completed. We acknowledge with gratitude the many investigators whose findings made this book necessary and possible. Special thanks go to individuals who shared unpublished information with us so that we could make the book as up to date as possible. Our thanks also go to the scientists who provided photographs or drawings; each person is acknowledged when the appropriate figure appears in the book. We thank Barbara Kamp, Pat Gustafson, Beverly Scullion, and Judy Grudzina for typing various parts of the manuscript. Illustrations were prepared by Jennifer Blitz and Suzanne Smith; their help is acknowledged and appreciated. Special thanks to Dr. Ralston B. Read, Jr., former director of the Microbiology Division of the Food and Drug Administration and now deceased, who in 1984 encouraged development of a research program on foodborne Listeria at the University of Wisconsin–Madison, and to Dr. Joseph A, O’Donnell, formerly with Dairy Research, Inc. and now director of the California Dairy Foods Research Center, for his early interest in and support of research on behavior of L. monocytogenes in dairy foods. Research done in the Department of Food Science at the University of Wisconsin-Madison and described in this book was supported by the U.S. Food and Drug Administration; National Cheese Institute; the National Dairy Promotion and Research Board; the Wisconsin Milk Marketing Board; Kraft, Inc.; Carlin Foods; Chr. Hansen’s Laboratory, Inc.; the Aristotelian University of Thessaloniki, Greece; the Cultural and Educational Bureau of the Egyptian Embassy in the United States; the Malaysian Agricultural Research and Development Institute; the Korean Professors Fund; and the College of Agricultural and Life Sciences, the Center for Dairy Research, and the Food Research Institute—all of the University of Wisconsin. We thank these agencies for their interest in and support of research on L. monocytogenes. Our book is dedicated to all persons who have contributed to a better understanding of foodborne listeriosis so that control of this disease is facilitated. Elliot T. Ryser Elmer H. Marth

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Editors Elmer H. Marth, Ph.D., (1927–2006), a native of Jackson, Wisconsin, was emeritus professor of food science and bacteriology at the University of Wisconsin–Madison. He earned his B.S. (1950), M.S. (1952), and Ph.D. (1954) from the University of Wisconson–Madison in bacteriology with an emphasis on food and dairy bacteriology. After 3 years as instructor of bacteriology, he joined the R&D Division of Kraft Foods in Geneva, Illinois, in 1957. He rose through the ranks and in 1966 was named associate manager of the microbiology laboratory. Also in 1966, Dr. Marth returned to the University of Wisconsin–Madison as associate professor of food science with joint appointments in bacteriology and food microbiology and toxicology. He was promoted to professor in 1971 and, upon retirement in 1990, was named emeritus professor. In 1981, Dr. Marth was a visiting professor at the Swiss Federal Institute of Technology in Zürich. From 1967 to 1987, he was editor of the Journal of Food Protection. At the University of Wisconsin–Madison, Dr. Marth taught courses in food sanitation, food fermentations, farm bacteriology, and writing scientific reports; he lectured in seven other courses and in five short courses. His research program included studies on food spoilage, food fermentation, and foodborne disease organisms, including Listeria monocytogenes. During his career, he was the author, co-author, editor, or co-editor of more than 660 scientific publications, including research papers, review papers, books, chapters in books, patents, and abstracts of papers given at meetings of professional organizations. Dr. Marth served as major professor for 32 students who received M.S. degrees and 32 students who earned Ph.D. degrees; in addition, he supervised 17 postdoctoral researchers who worked in his laboratory. Dr. Marth was named a fellow of the Institute of Food Technologists (IFT) (1983), the International Association for Food Protection (IAFP) (1998), and the American Dairy Science Association (ADSA) (1998). He was also a member of the American Society for Microbiology and the Council of Science Editors. From the ADSA he received Pfizer (1975), Dairy Research Foundation (1980), Borden (1986), and Kraft Teaching (1988) awards. The IAFP honored him with Educator (1977), Citation (1984), Honorary Life Member (1987), and NFPA Food Safety (2000) awards. The IFT presented him with the Nicolas Appert (1987) and Babcock–Hart (1989) awards. In 2002, the Institute for Scientific Information designated Dr. Marth as a highly cited researcher, worldwide, in the agricultural sciences. The National Cheese Institute presented its highest honor, the Laureate Award, to Dr. Marth in 2004. Elliot T. Ryser, Ph.D., a native of Milwaukee, Wisconsin, is an associate professor in the Department of Food Science and Human Nutrition and the National Food Safety and Toxicology Center at Michigan State University. He earned his B.S. (1979) in biology from Carroll College, Waukesha, Wisconsin, and his B.S. (1980) in bacteriology, and M.S. (1982) and Ph.D. (1990) in food science, from the University of Wisconsin–Madison, with an emphasis on microbial safety of food and dairy products. Following a 1-year appointment as a research scientist at Institute National de la Recherche Agronomique, Station de la Recherches Laitieres, Jouy-en-Josas, France, Dr. Ryser joined Silliker Laboratories Group, Inc. in Chicago Heights, Illinois, where he worked for 2 years as a research project manager. In 1994 he left Silliker and began his academic career as a research associate in the Department of Animal and Food Sciences at the University of Vermont, working in the laboratory of Dr. Catherine Donnelly. Dr. Ryser joined Michigan State University as an assistant professor in 1998 and was promoted to associate professor in 2004. He teaches courses on foodborne diseases, food safety, and HACCP.

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Dr. Ryser’s research program is focused on the incidence, survival, transfer, and eradication of Listeria monocytogenes and other foodborne pathogens from various foods, including dairy products. He has authored, co-authored, or co-edited more than 140 scientific publications, including research papers, review papers, books, chapters in books, patents, and abstracts of work presented at professional meetings. He has served thus far as the major professor for four Ph.D. and six M.S. students and supervised the work of four postdoctoral researchers. Dr. Ryser is a member of the International Association for Food Protection, Institute of Food Technologists, American Society for Microbiology, and American Dairy Science Association. He received the National Milk Producers Federation Richard M. Hoyt Award in 1988. In addition, he served as a scientific editor for the Journal of Food Science from 2000 to 2005 and is currently a scientific editor for the Journal of Food Protection.

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Contributors Robert E. Brackett Centers for Food Safety and Applied Nutrition U.S. Food and Drug Administration College Park, Maryland Christopher R. Braden Enteric Diseases Epidemiology Branch Division of Foodborne, Bacterial and Mycotic Diseases National Center for Zoonotic, Vectorborne and Enteric Diseases Centers for Disease Control and Prevention Atlanta, Georgia Byron F. Brehm-Stecher Department of Food Science and Human Nutrition Iowa State University Ames, Iowa Carmen Buchrieser Laboratoire de Genomique des Microorganismes Pathogenes Institut Pasteur Paris, France Catherine W. Donnelly Department of Nutrition and Food Science University of Vermont Burlington, Vermont Mel W. Eklund U.S. National Marine Fisheries Service (retired) Mel Eklund and Associates, Inc. Seattle, Washington Jeffrey M. Farber Microbiology Research Division Bureau of Microbial Hazards Food Directorate Health Canada Banting Research Centre Ottawa, Ontario, Canada

Werner Goebel Department of Microbiology University of Würzburg Würzburg, Germany Lewis M. Graves Enteric Diseases Laboratory Response Branch Division of Foodborne, Bacterial and Mycotic Diseases National Center for Zoonotic, Vectorborne and Enteric Diseases Centers for Disease Control and Prevention Atlanta, Georgia Joshua Gurtler Department of Food Science University of Georgia Griffin, Georgia Susan B. Hunter Coordinating Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia Karen C. Jinneman Seafood Products Research Center, Pacific Regional Laboratory—Northwest Office of Regulatory Affairs U.S. Food and Drug Administration Bothell, Washington Eric A. Johnson Departments of Food Microbiology and Toxicology and Bacteriology University of Wisconsin–Madison Madison, Wisconsin Sophia Kathariou Department of Food Science North Carolina State University Raleigh, North Carolina Jeffrey L. Kornacki Kornacki Microbiology Solutions, LLC McFarland, Wisconsin

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Michael Kuhn Biozentrum University of Würzburg Würzburg, Germany

Brian D. Sauders Department of Food Science Cornell University Ithaca, New York

Beatrice H. Lado Nestle Research and Development Centre Shanghai Ltd., China

Chris Scherf National HIV and Retrovirology Laboratory Public Health Agency of Canada Ottawa, Ontario, Canada

Elmer H. Marth University of Wisconsin–Madison Madison, Wisconsin

Laurence Slutsker Foodborne and Diarrheal Diseases Branch Centers for Disease Control and Prevention Atlanta, Georgia

Dawn M. Norton Enteric Diseases Epidemiology Branch Division of Foodborne, Bacterial and Mycotic Diseases National Center for Zoonotic, Vectorborne and Enteric Diseases Centers for Disease Control and Prevention Atlanta, Georgia David G. Nyachuba Department of Food Science University of Massachusetts Amherst, Massachusetts Franco Pagotto Listeriosis Reference Service Bureau of Microbial Hazards Health Products and Food Branch Health Canada Ottawa, Ontario, Canada John Painter Foodborne and Diarrheal Diseases Branch Centers for Disease Control and Prevention Atlanta, Georgia Jocelyn Rocourt Institut Pasteur Paris, France Elliot T. Ryser Department of Food Science and Human Nutrition Michigan State University East Lansing, Michigan

Bala Swaminathan Foodborne and Diarrheal Diseases Branch Division of Bacterial and Mycotic Diseases National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia Ewen C.D. Todd National Food Safety and Toxicology Center Michigan State University East Lansing, Michigan R. Bruce Tompkin Food Safety Consultant LaGrange, Illinois Marleen M. Wekell Center for Veterinary Medicine U.S. Food and Drug Administration Laurel, Maryland Irene V. Wesley Enteric Diseases and Food Safety Research Unit National Animal Disease Center Agricultural Research Service, USDA Ames, Iowa Martin Wiedmann Department of Food Science Cornell University Ithaca, New York Ahmed E. Yousef Department of Food Science and Technology The Ohio State University Columbus, Ohio

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Contents Chapter 1 The Genus Listeria and Listeria monocytogenes: Phylogenetic Position, Taxonomy, and Identification.............................................................................................................1 Jocelyn Rocourt and Carmen Buchrieser Chapter 2 Ecology of Listeria Species and L. monocytogenes in the Natural Environment..........................21 Brian D. Sauders and Martin Wiedmann Chapter 3 Listeriosis in Animals ......................................................................................................................55 Irene V. Wesley Chapter 4 Listeriosis in Humans ......................................................................................................................85 John Painter and Laurence Slutsker Chapter 5 Molecular Virulence Determinants of Listeria monocytogenes ....................................................111 Michael Kuhn and Werner Goebel Chapter 6 Characteristics of Listeria monocytogenes Important to Food Processors...................................157 Beatrice H. Lado and Ahmed E. Yousef Chapter 7 Conventional Methods to Detect and Isolate Listeria monocytogenes .........................................215 Catherine W. Donnelly and David G. Nyachuba Chapter 8 Rapid Methods for Detection of Listeria .....................................................................................257 Byron F. Brehm-Stecher and Eric A. Johnson Chapter 9 Subtyping Listeria monocytogenes ................................................................................................283 Lewis M. Graves, Bala Swaminathan, and Susan B. Hunter Chapter 10 Foodborne Listeriosis.....................................................................................................................305 Dawn M. Norton and Christopher R. Braden

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Chapter 11 Incidence and Behavior of Listeria monocytogenes in Unfermented Dairy Products .....................357 Elliot T. Ryser Chapter 12 Incidence and Behavior of Listeria monocytogenes in Cheese and Other Fermented Dairy Products .............................................................................................................405 Elliot T. Ryser Chapter 13 Incidence and Behavior of Listeria monocytogenes in Meat Products ........................................503 Jeffrey M. Farber, Franco Pagotto, and Chris Scherf Chapter 14 Incidence and Behavior of Listeria monocytogenes in Poultry and Egg Products ......................571 Elliot T. Ryser Chapter 15 Incidence and Behavior of Listeria monocytogenes in Fish and Seafood....................................617 Karen C. Jinneman, Marleen M. Wekell, and Mel W. Eklund Chapter 16 Incidence and Behavior of Listeria monocytogenes in Products of Plant Origin ........................655 Robert E. Brackett Chapter 17 Incidence and Control of Listeria in Food Processing Facilities .................................................681 Jeffrey L. Kornacki and Joshua Gurtler Chapter 18 Listeria: Risk Assessment, Regulatory Control, and Economic Impact ......................................767 Ewen C.D. Todd Chapter 19 Perspectives on Research Needs....................................................................................................813 Elmer H. Marth, Robert E. Brackett, R. Bruce Tompkin, Sophia Kathariou, and Ewen C.D. Todd Index ..............................................................................................................................................843

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The Genus Listeria and Listeria 1 monocytogenes : Phylogenetic Position, Taxonomy, and Identification Jocelyn Rocourt and Carmen Buchrieser CONTENTS History ................................................................................................................................................1 Phylogenetic Position of the Genus Listeria .....................................................................................3 Numerical Taxonomy ...............................................................................................................3 Chemotaxonomy.......................................................................................................................3 rRNA Sequencing.....................................................................................................................4 Whole Genome Sequencing .....................................................................................................4 Conclusion ................................................................................................................................4 Taxonomy of the Genus Listeria .......................................................................................................5 L. monocytogenes, L. ivanovii, L. welshimeri, and L. seeligeri ..............................................6 L. grayi (and “L. murrayi”)......................................................................................................7 L. denitrificans/Jonesia denitrificans .......................................................................................8 Present State of the Taxonomy of the Genus Listeria ............................................................8 Identification of Bacteria of the Genus Listeria................................................................................9 Genus Characteristics ...............................................................................................................9 Morphology ..................................................................................................................9 Culture ..........................................................................................................................9 Nutritional Requirements ...........................................................................................10 Metabolism and Biochemical Characteristics............................................................10 Species Identification..............................................................................................................10 Conclusion........................................................................................................................................12 References ........................................................................................................................................12

HISTORY Murray, Webb, and Swann first published a description of Listeria monocytogenes in 1926 [112]. Several earlier reports may have described Listeria isolation [62,156]; the most plausible is certainly that by Hulphers [73]. However, the authors of these reports did not deposit their isolates in a permanent collection, so no subsequent investigations or comparisons with further strains were possible. Murray and colleagues observed six cases of sudden death of young rabbits in 1924 in the animal breeding establishment of the Department of Pathology at Cambridge and many more in 1

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2

Listeria, Listeriosis, and Food Safety

the succeeding 15 months. The interesting characteristics presented by the disease and the increasing mortality prompted investigation. The authors wrote at that time [112]: Both the natural and the experimental disease have interesting and characteristic features and their consideration has forced us to the conclusion that the causative organism either has not been described previously, or has been inadequately described and so cannot be traced in the literature. In either case, we feel justified in naming it. Its salient character is the production of a large mononuclear leucocytosis. This is far the most important and most striking character we have discovered and we name the microorganism we shall describe in this paper “Bacterium monocytogenes.” The question of the generic name is more difficult and we have not succeeded in associating our organism with many other genera proposed in Bergey’s Manual of Determinative Bacteriology (1925). We propose for the present to use the undefined Bacterium ([...], for, if the present chaos is to be resolved and if the classification adopted by the American Society of Bacteriologists is to be improved, it will be achieved only by co-operation and with this end in view we cannot use the term Bacillus.

In 1927, during investigations of unusual deaths observed in gerbils near Johannesburg, South Africa, Pirie [130] discovered a new microorganism, agent of what he called “the Tiger River disease.” He named this new agent Listerella hepatolytica 1: “The causative organism is a Grampositive bacillus for which, from its most striking pathogenic effect, I propose the specific name hepatolytica, and the generic name Listerella, dedicating it in honour of Lord Lister, one of the most distinguished of those concerned with bacteriology whose name has not been commemorated in bacteriological nomenclature.”2 Discoverers Murray and Pirie [130] sent their strains to the National Type Collection at the Lister Institute in London. Dr. Leningham, the director, was struck by the similarity of the two microorganisms and put Murray and Pirie into contact. Because the identity was clear, they decided to call this bacterium Listerella monocytogenes [111,130,159]. However, in 1939, the Judicial Commission of the International Committee on Systematic Bacteriology rejected the generic name Listerella because it had previously been used for a mycetozoan in 1906 in honor of Arthur Lister (younger brother of Lord Lister) and for a species of foraminifer in 1933 in honor of Joseph Jackson Lister (father of Lord Lister). As noted by Gibbons in 1972 [54], it is certainly unique that the same name was chosen for three quite different groups of microorganisms to honor contributions of a father and his two sons. In 1940, Pirie proposed the name Listeria [129]. Before and even after this date, numerous names were used to designate L. monocytogenes: Bacterium monocytogenes hominis and later Listerella hominis by Nyfeldt, who considered that it was the agent of infectious mononucleosis [116,117] Corynebacterium parvulum by Schultz et al. in 1934 [154] Listerella ovis by Gill in 1937 [55] Listerella bovina, L. gallinaria, L. cunniculi, and L. gerbilli by Nyfeldt [117,118] Erysipelothrix monocytogenes by Wilson and Miles in 1946 [187] Corynebacterium infantisepticum by Potel (1951) during his first observations of fetal and neonatal listeriosis in Germany [132] Unlike some pathogenic agents responsible for large outbreaks that have marked the history of humans for centuries, the history of L. monocytogenes and listeriosis is recent: It begins officially in 1924. The first confirmed diagnosis in a human was that of a soldier suffering from meningitis at the end of World War I (retrospective identification of the strain [28]). Before this case, there are no validated observations. Interestingly, however, a historian has suggested that L. monocytogenes could have been the cause of Queen Anne’s 17 unsuccessful pregnancies [150].

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The Genus Listeria and Listeria monocytogenes

3

PHYLOGENETIC POSITION OF THE GENUS LISTERIA The relationships of Listeria to other bacteria remained obscure until the 1970s. Absent from the three first editions of Bergey’s Manual of Determinative Bacteriology published in 1923, 1925, and 1930, the genus Listeria was included in the tribe Kurthia of the Corynebacteriaceae family in the next edition in 1934. In the sixth and seventh editions (published in 1948 and 1957, respectively), Listeria was still a member of the Corynebacteriaceae; however, in the next edition (1974), it was considered a genus of uncertain affiliation and placed with Erysipelothrix and Caryophanon after the family of Lactobacillaceae [3–6]. Finally, Listeria was classified with Lactobacillus, Erysipelothrix, Brochothrix, Renibacterium, Kurthia, and Caryophanon in the section of “regular, nonsporing, Gram-positive rods” in Bergey’s Manual of Systematic Bacteriology [7]. How can these repeated reclassifications be explained? On the basis of morphological resemblances (Gram-positive, non-spore-forming rod), Listeria has long been associated with the coryneform group of bacteria. However, with the successive introduction and development of numerical taxonomy, chemotaxonomy, DNA/DNA hybridization, and, more recently, rRNA (ribosomal RNA) and DNA sequencing, the phylogenetic position of Listeria has been more clearly determined.

NUMERICAL TAXONOMY Numerical taxonomy provided the first attempts to investigate in depth the phylogenetic position of Listeria among Gram-positive bacteria. In the first studies, Listeria was included among coryneform bacteria and actinomycetes and, consequently, was located with the corynebacteria [13,31] or in an indefinite position [18,71]. In contrast, from 1969, more natural relationships were described when Listeria was compared to various representatives of lactic acid bacteria [33,172,173]. In 1975, the close relatedness with these microorganisms was clearly demonstrated by the broader numerical taxonomic survey of Jones, who studied 173 characteristics of 233 strains of various genera, including coryneform and lactic acid bacteria [79]. The refined position of Listeria was later investigated by Wilkinson and Jones in 1977 and Feresu and Jones in 1988 [43,186]. From these works, it became clear that Listeria is distinct from other known genera, including Erysipelothrix and Brochothrix thermosphacta (formerly Microbacterium thermosphactum) and is closely related to Lactobacillus and Streptococcus. Consequently, Wilkinson and Jones [186] suggested that Listeria, Gemella, Brochothrix, Streptococcus, and Lactobacillus be classified in the family Lactobacillaceae. In spite of some imprecision over the exact position with regard to the higher taxonomic relationships—especially with Brochothrix, certain lactobacilli, and Carnobacterium [43,186]—conclusions based on numerical analysis of data for large numbers of phenotypic features were precursors of the current phylogenetic classification of the genus Listeria.

CHEMOTAXONOMY A number of chemotaxonomic markers have been especially useful for solving the phylogenetic position of the genus Listeria, reinforcing its distinctness from coryneform bacteria and its relatedness to the lactic acid bacteria. The G + C percent DNA content of L. monocytogenes isolates ranges from 36 to 42% [43,142,172], indicating that Listeria belongs to the low G + C percent DNA content (<55%) group of Gram-positive bacteria. Lipoteichoic acids (amphilic polymers of the cytoplasmic membranes) have been isolated from L. monocytogenes [68,148,178]. These acids consist of hydrophilic poly(glycerophosphate) chains covalently attached to glyco- or phosphatidylglycolipids (the hydrophilic moieties of the molecules) and exhibit structural analogies with lipoteichoic acids from other bacteria. Although lipoteichoic acids show a distinct structural diversity in their hydrophilic and lipophilic portions, a given lipoteichoic acid is known to be a fairly stable characteristic and may be used as a taxonomic marker.

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In the case of Listeria, the presence of particular lipoteichoic acids provides further evidence that the genus is a biochemically coherent taxon [148]. In addition, lipoteichoic acids are absent from coryneform bacteria but are found in Bacillus, Staphylococcus, Streptococcus, and Lactobacillus, indicating that Listeria should be grouped with these microorganisms [148]. With the exception of one report [106], free mycolic acids, which are specific for high G + C percent DNA content Gram-positive bacteria, have not been detected in Listeria [43,81]. The presence of respiratory menaquinones (seven isoprene units) of predominantly methyl-branched cellular fatty acids and meso-DAP as major peptidoglycan diamino acid support the close relatedness of Listeria and Brochothrix and the greater distance from lactobacilli [24,26,43,46,134]. Analysis of low-molecularweight RNA profiles also supports the independent identity of L. monocytogenes among other Gram-positive taxa [171].

rRNA SEQUENCING Analysis of the 16S and 23S rRNA of L. monocytogenes has further clarified the position of Listeria with regard to other genera of Gram-positive bacteria. In 1986, using the 16S rRNA cataloguing approach (partial sequencing), Ludwig et al. unambiguously demonstrated that Listeria forms with Brochothrix thermosphacta one of the several sublines within the Clostridium subdivision [100]. They did not detect any relationship with the coryneform organisms (except for highly conserved 16S RNA sequences universal to all eubacteria and those of Gram-positive bacteria, respectively). Reverse transcriptase sequencing of 16S rRNA data confirmed this phylogenetic position of Listeria and indicated that the Listeria–Brochothrix subline is approximately equidistant from the Bacillus and Enterococcus–Carnobacterium sublines [26]. The close relationship with Bacillus was later confirmed [38]. On the basis of rRNA sequencing data and chemotaxonomic properties, Collins et al. [26] considered that Listeria is phylogenetically remote from Lactobacillus and should not be included in the Lactobacillaceae family and that the Listeria–Brochothrix subline probably merits a separate family, the Listeriaceae. This great distance between Lactobacillus and Listeria has been recently confirmed by sequencing of the 23S rRNA Listeria exhibiting the highest similarity with Bacillus and Staphylococcus [149].

WHOLE GENOME SEQUENCING Recently, the complete genome sequences of L. monocytogenes and L. innocua have been determined [56]. Comparison of the gene content and organization of the two sequenced Listeria genomes, as well as comparison with that of other genomes, allows insight into phylogenetic relationships (Figure 1.1). The two Listeria genomes show a perfect conservation of the order and the relative orientation of orthologous genes, indicating high stability in genome organization and a close phylogenetic relationship. When the Listeria genomes were compared to those of B. subtilis and B. halodurans, 1,428 and 1,296 orthologous genes, respectively, were predicted (on the basis of Blastp comparisons) [104]. Furthermore, high synteny in genome organization was observed between Listeria and Bacillus genomes. The same results were obtained when the genome organization of L. monocytogenes and Staphylococcus aureus were compared. In contrast, comparison of the L. monocytogenes genome with Streptococcus agalactiae (another phylogenetically closely related member of the group of Gram-positive, low GC content bacteria) or Lactococcus lactis showed no synteny [17]. Thus, recent data from complete genome comparisons confirmed that Listeria exhibits the highest similarity with Bacillus and Staphylococcus.

CONCLUSION Data accumulated during the last three decades clearly demonstrate that Listeria is a welldefined taxon that possesses a number of features distinguishing it from neighboring taxa. It is not

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Origin

2 944 528 bp

Terminus FIGURE 1.1 Circular genome map of L. monocytogenes strain EGDe showing position and orientation of genes. From the outside: circle 1: L. monocytogenes genes on the + and – strands, respectively; color code: red = L. monocytogenes genes; black = rRNA operons; circle 2: genes present in L. monocytogenes but absent in L. innocua; circle 3: genes present in L. innocua but absent in L. monocytogenes; circle 4: G + C content of L. monocytogenes with <32.5% G + C in light yellow, between 32.5 and 43.5% in yellow, and >43.5% G + C in greenish yellow. The putative origin of replication and terminus are indicated on the outside.

a coryneform bacterium as evidenced by numerical phenetic studies, chemotaxonomic properties, and various rRNA and DNA sequencing analyses. However, the exact phylogenetic position of this genus still remains controversial. Although it is generally agreed that its nearest neighbor is Brochothrix, its relationships with other members of the low G + C percent DNA content Gram-positive bacteria, especially with Lactobacillus, need further clarification.

TAXONOMY OF THE GENUS LISTERIA For many years after its discovery, the genus Listeria was monospecific, containing only the L. monocytogenes species. Because of its ability to reduce nitrates, L. denitrificans was added in 1948 [172]; L. grayi was added in 1966 in honor of M. L. Gray, an American microbiologist [98]. L. murrayi was added in 1971 (in honor of E. G. D. Murray, a Canadian microbiologist) [185] and L. innocua (named thusly because of its harmlessness) in 1981 [155]. In 1985, L. ivanovii was added in honor of I. Ivanov, a Bulgarian microbiologist [161]; L. welshimeri was added in 1983 (in honor of H. J. Welshimer, an American microbiologist) and L. seeligeri (in honor of H. P. R. Seeliger, a German microbiologist) in 1983 [143].

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As for the phylogenetic analysis of Listeria, the introduction of molecular biology methods allowed a better appreciation of the diversity within the genus Listeria, which now contains six species: L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seeligeri, and L. grayi.

L.

MONOCYTOGENES,

L.

IVANOVII,

L.

WELSHIMERI, AND

L.

SEELIGERI

Serotyping was the first approach used to elucidate the infrageneric structure of the genus Listeria. The first antigenic scheme was devised by Paterson [123,124], who described the first four serovars. This scheme was later extended by Donker-Voet and Seeliger with the addition of new serovars [36,160]. Until 1960, Listeria was nearly exclusively isolated from pathological samples—thus the isolation of nearly only L. monocytogenes strains. In 1962, Ivanov observed atypical L. monocytogenes strains isolated from aborted sheep and proposed to allocate them to a new species, L. bulgarica, on the basis of their strong hemolytic activity and their new antigenic structure (serovar 5) [75,76]. Years later, with development of selective media, numerous strains were isolated from various environmental sources. Seeliger serotyped hundreds of strains collected between 1965 and 1980 and observed that they were nonhemolytic, characterized by particular antigenic factors (serovars 6a and 6b [formerly 4f and 4g] and undesignated serovars), and apparently nonpathogenic. He proposed to name them L. innocua in 1981 [155]. Thus, simple phenotypic methods, serotyping and hemolysis, led to the demonstration that the species L. monocytogenes as defined in the eighth edition of Bergey’s Manual of Determinative Bacteriology [6] was heterogenous, covering a number of different species. Early DNA/DNA hybridizations (filter study) by Stuart and Welshimer in 1974 showed L. monocytogenes to be heterogenous [175]. However, the number of DNA hybridization groups in their collection of strains could not be ascertained because only one DNA from L. monocytogenes was labeled; moreover, serovars were not indicated. Further DNA/DNA hybridization studies (S1 method) that aimed to resolve the genomic heterogeneity of the so-called L. monocytogenes and evaluate the validity of the new species L. bulgarica and L. innocua (not officially validated at that time) were undertaken in 1982 with many strains of various origins [142]. Five DNA relatedness groups were found among strains formerly identified as L. monocytogenes: Genomic group 1 contained the type strain of L. monocytogenes, thus corresponding to L. monocytogenes, sensu stricto; it included strains belonging to serovars 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7. Genomic group 2 was constituted by strongly hemolytic strains, all belonging to serovar 5 (for which Ivanov suggested the name L. bulgarica) and deserved species rank [75]; they were named L. ivanovii in 1985 [161]. Further investigations of strains of this species using multilocus enzyme electrophoresis, DNA/DNA hybridizations, and rRNA gene restriction patterns described two subspecies: L. ivanovii subsp. ivanovii (ribose positive) and L. ivanovii subsp. londoniensis (ribose negative) [10]. Genomic group 3 contained nonhemolytic and nonpathogenic (for mice) strains of serovars 4ab, 6a, 6b, and undesignated serovars, including the two strains that Seeliger previously proposed as reference strains for L. innocua [155]. Therefore, this group corresponded to L. innocua. Based on these data, this species was officially validated in 1983 [179]. Genomic group 4 contained nonhemolytic strains of serovars 6a and 6b; these strains produced acid from D-xylose and were nonpathogenic for mice [139,144]; they therefore corresponded to a group of strains previously described by Groves and Welshimer in 1977 [65]. The group was given species status and called L. welshimeri in 1983 [143]. Genomic group 5, an unexpected group, included hemolytic and nonpathogenic strains of various serovars (1/2b, 4c, 4d, 6b, and undesignated serovars) [139,144]. It was subsequently named L. seeligeri [143].

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Numerical taxonomic surveys confirmed that L. monocytogenes, as defined in the eighth edition of Bergey’s Manual of Determinative Bacteriology, was not a single taxon. However, this method is of limited sensitivity for bacteria that differ by a small number of characteristics and these studies were of little help in resolving the heterogeneity [43,82]. DNA/DNA homology experiments (optical method) in 1993 supported these results and confirmed that L. monocytogenes, L. innocua, L. ivanovii, L. welshimeri, and L. seeligeri each corresponded to a single cluster with no overlaps among them [67]. This classification was later sustained by data using a multilocus enzyme electrophoresis study, two-dimensional protein mapping, and sequencing of the 16S rRNA gene and 16S-23S rRNA operon intergeneric spacer region [11,57,60,181]. More recently, DNA/DNA hybridization of macroarrays containing Listeria-specific genes from the genome sequence of L. monocytogenes and L. innocua were used to predict absence and presence of genes studied in 110 Listeria strains [37]. A matrix of binary data (absence/ presence) was analyzed by hierarchical clustering and neighbor joining to illustrate possible phylogenetic relationships among the different listeriae. Important gene conservation within each species of the genus Listeria and within distinct groups of L. monocytogenes strains was identified. Furthermore, this analysis revealed that, without exception, the tested strains of L. monocytogenes, L. innocua, L. ivanovii, L. welshimeri, and L. seeligeri clustered according to their species definition. Each species was defined by a combination of genes specifically present or absent [37]. Furthermore, DNA/DNA hybridization showed that serogrouping of the different Listeria species as well as of the species L. monocytogenes is reflected in the genetic content and evolution of the different serotypes. Ninety-three L. monocytogenes strains tested by DNA/DNA hybridization of macroarrays were grouped into three evolutionary lineages correlated with serovars. Lineage I comprised serovars 1/2a, 1/2c, 3a, and 3c, lineage II contained strains of serovars 1/2b, 3b, 4b, 4d, 4e, and 7, and lineage III contained serovars 4a and 4c. In addition, this allowed two subdivisions within lineages I, II, and III to be distinguished clearly. Lineage I was subdivided into two groups. One consisted of the strains of serovars 1/2a and 3a; the other consisted of the strains of serovars 1/2c and 3c. Lineage II was subdivided into two groups. One group consisted of the strains of serovars 4b, 4d, and 4e; the other group was made up of strains of serovars 1/2b, 3b, and 7. Lineage III was subdivided into lineage IIa, containing serovar 4a strains, and lineage IIIb, which contained serovar 4c strains. Each lineage and subgroup is characterized by highly conserved genes representing specific markers for L. monocytogenes strains belonging to lineages I, II, or III [37]. Similar results are obtained by comparison of almost full-length iap (invasion-associated protein) gene sequences that grouped the L. monocytogenes strains into the same three distinct genotypes, correlating well with distinct serotypes. These results were corroborated by further comparative sequence analysis of genes encoding two phospholipases, pIcA and pIcB [151].

L.

GRAYI (AND

“L.

MURRAYI”)

The long controversy about the taxonomic position of L. grayi and L. murrayi started when DNA/DNA homology studies of Stuart and Welshimer in 1974 demonstrated low DNA relatedness between L. monocytogenes on the one hand and L. grayi and L. murrayi on the other and high genomic homology between L. grayi and L. murrayi. These data were supported by a numerical phenetic analysis [174,175]. The authors proposed to transfer L. grayi and L. murrayi to a new genus, L. murraya, with L. grayi as the type species, divided into two subspecies: L. grayi subsp. grayi and L. grayi subsp. murrayi. L. grayi, L. murrayi, and L. monocytogenes share a number of similarities: They cluster in all numerical taxonomic studies [79,173,175,186] and possess lipoteichoic acid [148], teichoic acid of the polyribitol phosphate type [47], peptidoglycan of the A1 gamma variation [46], nonhydrogenated

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menaquinones of the MK-7 type [24,43], and the same cytochromes (a1bdo) [43]. However, other features support the distinctness of the L. grayi and L. murrayi pair within the genus Listeria: slight difference in G +C percent DNA content (42 vs. 36 to 38) [43,175] small number of biochemical reactions [144] nature of substitution of the lipoteichoic acids [148] slight differences of protein electrophoregrams [97] cellular fatty acid composition [114] antigenic structure [160] low DNA homology values [174] Finally, the 16S rRNA oligonucleotide cataloguing of L. murrayi placed it close to L. monocytogenes. These data, together with the substantial phenotypic similarity with L. monocytogenes, provided no support for exclusion of L. murrayi (and the closely related species L. grayi) from the genus Listeria [145]. This was later confirmed by 16S rRNA gene sequencing [181]. Various investigations offered evidence of the close relationship between L. grayi and L. murrayi. They fall into a single distinct cluster in numerical taxonomic analysis [43,186], in multilocus enzyme electrophoresis analysis [11], and in DNA/DNA hybridization studies [174]; in addition, they share a number of chemotaxonomic properties that distinguish them from the other Listeria species: the same DNA base composition values [43,174], the same substitution of lipoteichoic acids [148], the same patterns of cellular fatty acids and fatty aldehydes [84], and a common antigenic pattern structure in spite of small differences [160,182]. They have been distinguished from each other only on the basis of nitrate reduction [185]. Reexamination of the genomic relatedness of L. grayi and L. murrayi using DNA/DNA hybridizations and multilocus enzyme electrophoresis indicated that they should be considered members of a single species: L. grayi [140]. These data are consistent with 16S and 23S rRNA sequencing and cellular protein electrophoretic pattern data [26,88,149] as well as with DNA/DNA macroarray studies in which L. grayi and the former L. murrayi exhibit a closely related hybridization profile distinct from the other Listeria species [37].

L.

DENITRIFICANS/JONESIA DENITRIFICANS

Although only a single isolate is currently known for this species, an amazing number of papers have dealt with its taxonomic position. As early as 1966, a numerical taxonomic study showed that L. denitrificans clustered with certain coryneform bacteria; this was later confirmed [18,79,173,174]. The results of chemotaxonomic studies and DNA/DNA hybridization further emphasized the phenotypic differences between L. denitrificans and other members of the genus Listeria [24,25,46,47,148,175]. In 1987, 16S rRNA cataloguing confirmed that this species is not a member of the genus Listeria and belongs to the coryneform group of bacteria [146]. It was later demonstrated that Jonesia differs from members of the family Cellulomonadaceae and that its closest relatives are Dermobacter hominis and Brachybacterium faicium [136].

PRESENT STATE

OF THE

TAXONOMY

OF THE

GENUS LISTERIA

Finally, the genus Listeria currently contains six species—L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seeligeri, and L. grayi—as evidenced by DNA homology values, 16S rRNA and DNA sequencing, chemotaxonomic properties, and multilocus enzyme analysis. Based on DNA/DNA hybridization, DNA/DNA macroarray hybridization, 16S rRNA cataloguing, reverse transcriptase sequencing of 16S and 23S rRNA, sequencing of 16S–23S rRNA operon intergenic spacer region, and protein mapping, the genus embraces two closely related but obviously distinct lines of descent. One contains L. grayi and the other L. monocytogenes, L. ivanovii, L. innocua,

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L. welshimeri, and L. seeligeri. Within this line, the species can be divided into two groups: L. monocytogenes and L. innocua, on the one hand, and L. ivanovii, L. seeligeri, and L. welshimeri on the other [26,37,59,142,145,149,181].

IDENTIFICATION OF BACTERIA OF THE GENUS LISTERIA GENUS CHARACTERISTICS Morphology Listeria is a small (0.5 µm in diameter and 1 to 2 µm in length), regular Gram-positive rod with rounded ends. Cells are found as single units or in short chains, or they may be arranged in V and Y forms or in palisades. Sometimes, cells are coccoid, averaging about 0.5 µm in diameter and may be confused with streptococci. In old cultures, some cells lose the ability to retain Gram stain and may be occasionally mistaken for Hemophilus. Long, thin, filamentous cells appear in old and rough culture and after osmotic shock [66,83,93]. Listeria does not produce spores and capsules are not formed [159]. Listeria is motile from its few peritrichous flagella when cultured at 20 to 25°C (not or very weakly motile at 37°C) [51]. Hanging-drop preparations of a fresh culture in tryptose phosphate broth incubated at 20°C show a characteristic tumbling motility: Cells start with twisting and wriggling movements that increase to fast, eccentric rotations before they suddenly move quickly in various directions. Stab cultures in semisolid motility medium produce a typical picture of an “umbrella” or inverted “pine tree” about 0.5 cm below the surface because of the microaerophilic nature of the organism. A recent report indicates that L. monocytogenes and L. innocua differ markedly in motility and flagellin production at 37°C: L. monocytogenes strains are virtually nonmotile and produce little or no detectable flagellin, whereas strains of L. innocua are frequently motile and produce substantial amounts of flagellin [89]. Culture On nutrient agar, colonies are 0.2 to 0.8 mm in diameter, smooth, punctiform, bluish gray, translucent, and slightly raised with a fine surface texture and entire margin after 24 h of incubation. After 5 to 10 days, well-separated colonies may be 5 mm or more in diameter. When cultures of Listeria grown for 18 to 24 h at 37°C on a clear medium are examined with a binocular microscope under obliquely transmitted light, the smooth colonies exhibit a typical blue-green iridescence [61,95,110]. Even when the population of contaminants is rather high and that of Listeria is low, Listeria can be recognized because of this characteristic [61,99]. Rough colonies may occasionally be observed [63]. Conversion of smooth colonies to rough colonies is not reversible [156]. Differences in virulence between rough and smooth colonies have been observed [70,93,96]. Petite colony formation by strains grown on esculin-containing agar has been described [167]. Listeria usually grows well on most commonly used bacteriological media. The growth rate is increased by the presence of fermentable sugar, particularly glucose. On plate culture, Listeria has a particularly penetrating acid odor that may result from formation of carboxylic acids, hydroxy acids, and alcohols [32]. In broth, the medium becomes turbid after 8 to 24 h of incubation at 37°C. Profuse growth is always observed slightly below the clear area near the surface of the medium, indicating the propensity for Listeria to grow better at oxygen tensions lower than that in air [156]. The temperature limits of growth are 1–2°C to 45°C [85,158]. The ability to grow at very low temperature was first used by Gray [64] for selective enrichment of a contaminated sample. In broth, Listeria grows between pH 4.5 and pH 9.2, optimally at pH 7 [53,119,125]. It can grow in 10% (w/v) NaCl and survive at higher concentrations [158,163]. Survival at low pH and high salt concentration depends strongly on temperature [23]. Listeria is one of the few foodborne pathogens that can grow at aw below 0.93 [40,125].

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Nutritional Requirements According to published data, growth factors include cystine, leucine, isoleucine, arginine, methionine, valine, cysteine, riboflavin, biotin, thiamine, and thioctic acid [133,164,184]. Growth is stimulated by Fe3+ and phenylalanine [133,164]. In some experiments, virulent strains grew faster in the presence of iron than did the avirulent strains [29]. Glucose and glutamine are required as primary sources of carbon and nitrogen [133,158]. Chemically defined media have been described for Listeria [133,135,165]. Metabolism and Biochemical Characteristics Listeria is aerobic, microaerophilic, facultatively anaerobic, catalase positive (rare catalase-negative strains have been observed), and oxidase negative. Although Feresu and Jones [43] reported cytochrome a1bdo, presence of cytochrome is controversial [122,177]. Listeria is homofermentative and oxidizes glycolytic intermediate compounds [28]. It possesses glucose oxidase and NADH oxidase activities [122]. All strains grow on glucose, forming lactate, acetate, and acetoin under aerobic conditions as main end products [127,147]. Acetoin is not produced under anaerobic conditions. Only hexoses and pentoses support growth anaerobically; maltose and lactose support growth of some strains aerobically, but sucrose does not [127]. Catabolism of glucose proceeds by the Embden-Meyerhof pathway aerobically and anaerobically [158]. L. monocytogenes imports glucose by a high-affinity phosphoenolpyruvate-dependent phosphotransferase system and a low-affinity proton motive forcemediated system [19,120]. All strains are methyl red and Voges-Proskauer test positive. Acid is also produced from amygdalin, cellobiose, fructose, mannose, salicin, maltose, dextrin, alpha-methyl-D-glucoside, and glycerol. Acid production from galactose, lactose, melezitose, sorbitol, starch, sucrose, and trehalose is variable. Acid is almost never produced from adonitol, arabinose, dulcitol, erythritol, glycogen, inositol, inulin, melibiose, raffinose, or sorbose. Phenylalaninedeaminase, ornitine-, lysine-, and arginine-decarboxylases are not produced; H2S is not produced. Urea is not hydrolyzed and indole is not produced. Additional information on biochemical tests is available [43,87,141,158,162,166,186].

SPECIES IDENTIFICATION All Listeria species are phenotypically very similar, but can be distinguished by combinations of the following tests: hemolysis, acid production from D-xylose, L-rhamnose, alpha methylD-mannoside, and mannitol [144]. Phenotypic similarities are consistent with high genomic homologies between the different species [26,144,149]. Hemolysis is a key characteristic for allocating an isolate to a species and is obviously the most difficult characteristic of the identification gallery. During a collaborative study on Listeria identification, Higgins and Robinson [69] noted that a large percentage of errors in the identification of L. seeligeri and L. ivanovii was caused by inaccurate reading of the CAMP test and hemolysis. Isolates of L. monocytogenes and L. seeligeri show narrow, slight clearing zones of beta-hemolysis. L. ivanovii shows wide, clearly delineated zones of beta-hemolysis. In contrast, L. innocua, L. welshimeri, and L. grayi are not hemolytic. L. innocua can produce a green zone of hemolysis according to the medium used [131,169]. L. monocytogenes is hemolytic for blood of various origins: sheep, horse, cow, guinea-pig, piglet, and human [152,156,168,180]. Various methods have been developed to determine hemolytic activity, especially for weakly hemolytic strains (L. seeligeri and some L. monocytogenes isolates): examination of hemolysis below the colonies, prolonged incubation (48 h), incubation for a few hours at 4°C, thin layer blood agar plates, several media [50], tube tests and microplate techniques with erythrocyte suspension [34,35,176], addition of an exosubstance from Rhodococcus equi, S. aureus, or L. ivanovii to the blood agar [107,168,180], and the CAMP test with Staphylococcus aureus or

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R. equi [15,48,65,74,75,108,109,113,144,170]. Positive CAMP tests are indicated by an enhanced zone of beta-hemolysis at the intersection of the test strains L. monocytogenes, L. ivanovii, and L. seeligeri with S. aureus and L. ivanovii with R. equi. Conflicting readings of the CAMP test with R. equi have been reported; some authors consider L. monocytogenes to be positive [44,153,183] and others consider it negative [144,158]. The typical positive CAMP test with R. equi, as observed with L. ivanovii, gives a shovel-like shape. In contrast, when this test is positive with L. monocytogenes, the shape is that of an onion. This could reflect different abilities of R. equi strains to interact with L. monocytogenes because of different amounts of listeriolysin O secreted by L. monocytogenes strains or to variation in the capability of R. equi strains to secrete cholesterol oxidase [45,183]. However, whether or not positive, this test is not essential because L. monocytogenes and L. ivanovii can be easily distinguished by acid production from D-xylose, L-rhamnose, and alpha-methyl-D-mannoside. The L. monocytogenes exosubstance involved in the CAMP reaction with S. aureus and R. equi is listeriolysin O [137]. The exosubstances of S. aureus and R. equi are sphingomyelinase C and cholesterol oxidase, respectively [45,107,137]. Hemolysin is a major virulence factor of L. monocytogenes (see the chapter on L. monocytogenes virulence factors). Three species—L. monocytogenes, L. ivanovii, and L. seeligeri—are hemolytic and possess the virulence gene cluster as recently demonstrated [58]; however, only two, L. monocytogenes and L. ivanovii, are naturally and experimentally pathogenic [103,139]. L. ivanovii is mainly responsible for abortion in animals. Therefore, pathogenicity should not be presumed on the observation of hemolysis alone. Few nonhemolytic L. monocytogenes isolates have been observed. The best known example is the type strain of this species [70,80,90]. Dissociation between hemolytic and nonhemolytic colonies is rarely observed [128]. Nonhemolytic and a number of weakly hemolytic strains are not or are weakly pathogenic [12,27,34,42,70,94,126,176]. In spite of these atypical strains, routine pathogenicity testing for L. monocytogenes is not recommended [99]. Additional tests, especially to distinguish L. monocytogenes from L. innocua, have been proposed, such as detection of phospolipase C activity [22,115], hydrolysis of D-alanine-p-nitroanilide [20,87], hydrolysis of DL-alanine-beta-naphthylamide [20], and hydrolysis of a naphthylamide substrate (API Listeria [8]). Several commercially miniaturized culture or enzyme multitest assays have been tested or used for Listeria identification because conventional culture procedures for identification are tedious and time consuming. They include API 5O CH [92,141], APl-ZYM [141], API 20 STREP [102], API Listeria [8,9,50,121], API Coryne [91], Micro-ID Listeria [2,8,69,138], Mast ID [92], RAPID CORYNE [52], RAPID ID 32 Strep [49], and a microtiter plate method [167]. Information provided by API ZYM, API 20 STREP, and RAPID ID 32 Strep distinguishes isolates at the genus level, whereas API Listeria, Mast-ID, API Coryne, and API 50 CH are more appropriate for genus and species identifications. Phenotypic markers are often used for routine identification of Listeria isolates in food and clinical laboratories. More sophisticated methods, often based on genotypic markers, have been proposed; according to the goal, some of them can be used for rapid identification of isolates— alone or in association with other methods—or can help allocate atypical isolates to known species. These methods include 16S rRNA sequencing [30] sequence analysis of the 16S-23S internal transcribed spacer loci [39,59] automated RNA probe [101] amplification of the amplified product of a small 16S rRNA gene fragment [105] ribotyping [77] pulsed-field gel electrophoresis (PFGE) [14] random amplification of polymorphic DNA [40] repetitive element sequence-based PCR [78]

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multiplex PCR based on a single reaction [16] denaturing gradient gel electrophoresis (DGGE) based on the PCR amplification of the iap gene [21] multilocus enzyme electrophoresis [11] cellular protein electrophoretic pattern analysis [88] enzymatic profiling using fluorogenic substrates [86] analysis of fermentation products by frequency-pulsed electon-capture gas-liquid chromatography [32] Fourier transform infrared spectroscopy analysis [72] thermogram determination [1] Genome-based typing using macro- or microarray techniques is not used for routine identification. However, it is a very promising approach and first results indicate its power and usefulness.

CONCLUSION Studies on the phylogenetic position of Listeria started when numerical phenetic studies were applied to Gram-positive bacteria. These first studies were of primary importance in demonstrating that Listeria was not a coryneform bacterium. The data were confirmed by 16S rRNA cataloguing and whole genome comparisons. The refined location of Listeria within the low G + C percent DNA content Gram-positive bacteria were later determined by reverse transcriptase 16S and 23S sequencing data; this was later confirmed. Numerical taxonomic studies revealed a certain heterogeneity within this genus and the exact species content was determined by DNA/DNA hybridization, rRNA sequencing, and, more recently, by genome sequencing. On the basis of this genomic dissection, the genus contains six species divided into two sublines of descent. The present state of Listeria taxonomy is the result of studies over more than 25 years by various laboratories in different countries, using as many methods as possible. The distinction between L. monocytogenes and nonpathogenic species was already defined when foodborne listeriosis became a public health problem with a major economic impact on the food industry. This allowed restricting efforts to food contaminated with L. monocytogenes because food contaminated by other Listeria species was of no concern.

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99. Lovett, J. 1988. Isolation and identification of Listeria monocytogenes in dairy products. J. Assoc. Off. Anal. Chem. 71:658–660. 100. Ludwig, W., K.-H. Schleifer, and E. Stackebrandt. 1984. 16S rRNA analysis of Listeria monocytogenes and Brochothrix thermosphacta. FEMS Microbiol. Lett. 25:199–204. 101. Mabilat, C., S. Bukwald, S. Machabert, S. Desvarennes, R. Kurfurst, and P. Cros. 1996. Automated RNA probe assay for the identification of Listeriamonocytogenes. Int. J. Food Microbiol. 28: 333–340. 102. MacGowan, A. P., R. J. Marshall, and D. S. Reeves. 1989. Evaluation of API 20-STREP system for identifying Listeria species. J. Clin. Pathol. 42:548–550. 103. Mainou-Fowler, T., A. P. MacGowan, and R. Postlethwaite. 1988. Virulence of Listeria spp.: Course of infection in resistant and susceptible mice. J. Med. Microbiol. 27:131–140. 104. Maitournam, A., C. Buchriese, F. Kunst, L. Frangeul, and P. Glaser. Analysis of orthologous positions among Bacillus subtilis, Bacillus halodurans and Listeria monocytogenes: A statistical approach. Submitted. 105. Manzano, M., L. Cocolin, C. Cantoni, and G. Comi. 2000. Temperature gradient gel electrophoresis of the amplified product of a small 16S rRNA gene fragment for the identification of Listeria species isolated from food. J. Food Prot. 63:659–661. 106. Mara, M., and C. Michalec. 1977. Chromatographic study of mycolic acid-like substances in lipids of Listeria monocytogenes. J. Chromat. 130:434–436. 107. McKellar, R. C. 1994. Identification of the Listeria monocytogenes virulence factors in the CAMP reaction. Lett. Appl. Microbiol. 18:79–81. 108. McKellar, R. C. 1993. Novel mechanism for the CAMP reaction between Listeria monocytogenes and Corynebacterium equi. Int. J. Food Microbiol. 18:77–82. 109. McKellar, R. C. 1994. Use of the CAMP test for identification of Listeria monocytogenes. Appl. Environ. Microbiol. 60:4219–4225. 110. Moura, S. M., M. T. Destro, B. D. G. M. Franco, and R. M. Brancaccio. 1991. Low-cost illumination system for Listeria spp. research. Rev. Microbiol. Sao Paulo. 22:75–77. 111. Murray, E. G. D. 1963. A retrospect of listeriosis. In Second symposium on listeric infection, ed. M. L. Gray. Bozeman, MT: Artcraft Printer, pp. 3–6. 112. Murray, E. G. D., R. A. Webb, and M. B. R. Swann. 1926. A disease of rabbit characterized by a large mononuclear leucocytosis, caused by a hitherto undescribed bacillus Bacterium monocytogenes (n. sp.). J. Pathol. Bacteriol. 29:407–439. 113. Nakazawa, M., and H. Nemoto.. 1980. Synergistic hemolysis phenomenon of Listeria monocytogenes and Corynebacterium equi. Jpn. J. Vet. Sci. 42:603–607. 114. Ninet, B., H. Traitler, J. M. Aeschliman, D. Hartmann, and J. Bille. 1992. Quantitative analysis of cellular fatty acids (CFAs). Composition of the seven species of Listeria. Syst. Appl. Microbiol. 15:76–81. 115. Notermans, S. H. W., J. Dufrenne, M. Leimeisterwachter, E. Domann, and T. Chakraborty. 1991. Phosphatidylinositol-specific phospholipase-C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species. Appl. Environ. Microbiol. 57:2666–2670. 116. Nyfeldt, A. 1929. Etiologie de la mononucléose infectieuse. C. R. Soc. Biol. 101:590–592. 117. Nyfeldt, A. 1932. Klinische und experimentelle Untersuchungen über die Mononukleosis infectiosa. Folia Haematol. 47:1–144. 118. Nyfeldt, A. 1937. Studier over den infectiose mononucleoses etiologi. Hygiena 99:432–456. 119. Parish, M. E., and D. P. Higgins. 1989. Survival of Listeria monocytogenes in low pH model broth systems. J. Food Prot. 52:144–147. 120. Parker, C., and R. W. Hutkins. 1997. Listeria monocytogenes Scott A transports glucose by highaffinity and low-affinity glucose transport systems. Appl. Environ. Microbiol. 63:543–546. 121. Paziak-Domanska, B., E. Boguslawska, M. Wieckowska-Szakiel, R. J. Kotlowski, B. Rozalska, M. Chmiela, J. Kur, W. Darowki, and W. Rudnicka. 1999. Evaluation of the API test, phosphatidylinositolspecific phospholipase C activity and PCR method in identification of Listeria monocytogenes in meat foods. FEMS Microbiol. Lett. 171:209–214. 122. Patchett, R. A., A. F. Kelly, and R. G. Kroll. 1991. Respiratory activity in Listeria monocytogenes. FEMS Microbiol. Lett. 78:95–98. 123. Paterson, J. St. 1939. Flagellar antigens of organisms of the genus Listerella. J. Pathol. Bacteriol. 48:25–32.

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150. Saxbe, W. B., Jr. 1972. Listeria monocytogenes and Queen Anne. Pediatrics 49:97–101. 151. Schmid, M., M. Walcher, A. Bubert, M. Wagner, M. Wagner, and K. H. Schleifer. 2003. Nucleic acidbased, cultivation-independent detection of Listeria spp. and genotypes of L. monocytogenes. FEMS Immunol. Microbiol. 1474:1–11. 152. Schuch, D. M. T., J. Moore, R. H. Madden, and W. E. Espie. 1992. Hemolytic reaction of Listeria monocytogenes on bilayer Columbia agar plates with defibrinated Guinea-pig blood. Lett. Appl. Microbiol. 15:78–79. 153. Schuchat, A., B. Swaminathan, and C. V. Broome. 1991. Listeria monocytogenes CAMP reaction. Clin. Microbiol. Rev. 4:396. 154. Schultz, E. W., M. C. Terry, A. T. Brice, Jr., and L. P. Gebjardt. 1934. Bacteriological observations on a case of meningo-encephalitis. Proc. Soc. Exp. Biol. Med. 31:1021–1023. 155. Seeliger, H. P. R. 1981. Apathogene Listerien: L. innocua sp. n. (Seeliger et Schoofs, 1977). Zbl. Bakteriol. Hyg. I. Abt. I Orig. A 249:487–493. 156. Seeliger, H. P. R. 1961. Listeriosis. Basel: Karger. 157. Seeliger, H. P. R. 1979. Listeriosis. Still of interest? In Problems of listeriosis, ed. I. Ivanovii. Sofia: National Agroindustrial Union, Center for Scientific Information. 158. Seeliger, H. P. R., and D. Jonesy. 1986. Genus Listeria pirie 1940. In Bergey’s manual of systematic bacteriology, vol. 2, eds. P. H. A. Sneathy, N. S. Mair, N. E. Sharpe, and J.G. Holt. 159. Seeliger, H. P. R., and J. Bockemühl. 1968. Kritische Untersuchungen zur Frage einer Kapselbildung bei Listeria monocytogenes. Zbl. Bakteriol. Parasit. Infekt. Hyg., I. Orig. 206:216–227. 160. Seeliger, H. P. R., and K. Höhne. l979. Serotyping of and related species. In Methods in microbiology, eds. T. Bergan and J. Norris. New York: Academic Press, pp. 33–48. 161. Seeliger, H. P. R., J. Rocourt, A. Schrettenbrunner, P. A. D. Grimont, and D. Jones. l984. Listeria ivanovii sp. nov. Int. J. Syst. Bacteriol. 34:336–337. 162. Seller, H., and M. Busse. 1989. Biochemische Differenzierung von Listerien aus Käse. Berl. Münch. Tierärztl. Wschr. 102:166–170. 163. Shahamat, M., A. Seaman, and M. Woodbine. 1980. Survival of Listeria monocytogenes in high salt concentrations. Zbl. Bakteriol. Hyg. I. Abt. Orig. A 246:506–511. 164. Siddiqi, R., and M. A. Khan. 1989. Amino acid requirement of six strains of Listeria monocytogenes. Zbl. Bakteriol. 271:146–152. 165. Siddiqi, R., and M. A. Khan. 1982. Vitamin and nitrogen base requirements for Listeria monocytogenes and haemolysin production. Zbl. Bakteriol. Hyg. I. Abt. Orig. A 253:225–235. 166. Siragusa, G. R., L. A. Elphingstone, P. L. Wiese, S. M. Haefner, and M. G. Johnson. 1990. Petite colony formation by Listeria monocytogenes and Listeria species grown on esculin-containing agar. Can. J. Microbiol. 36:697–703. 167. Siragusa, G. R., and J. W. Nielsen. 1991. A modified microtiter plate for biochemical characterization of Listeria spp. J. Food Prot. 54:121–125. 168. Skalka, B., and J. Smola. l982. Hemolytic properties of exosubstance of serovar 5 Listeria monocytogenes compared with beta toxin of Staphylococcus aureus. Zbl. Bakteriol. Hyg. I. Abt. Orig. A 252:17–25. 169. Skalka, B., J. Smola, and K. Elischeroval. 1983. Hemolytic phenomenon under the cultivation of Listeria innocua. Zbl. Bakteriol. Hyg. I. Abt. Orig. A 253:559–565. 170. Skalka, B., J. Smola, and K. Elischerova. 1982. Routine test for in vitro differentiation of pathogenic and apathogenic Listeria monocytogenes strains. J. Clin. Microbiol. 15:503–507. 171. Slade, P. J., and D. L. Collins-Thompson. 1991. Differentiation of the genus Listeria from other Grampositive species based on low molecular weight (LMW) RNA profiles. J. Appl. Bacteriol. 70:355–360. 172. Sohier, R., F. Benazet, and M. Piechaud. 1948. Sur un germe du genre Listeria apparemment non pathogene. Ann. Inst. Pasteur 74:54–57. 173. Stuart, M. R., and P. E. Pease. 1972. A numerical study on the relationships of Listeria and Erysipelothrix. J. Gen. Microbiol. 73:551–565. 174. Stuart, S. E., and H. J. Welshimer. 1974. Taxonomic reexamination of Listeria pirie and transfer of Listeria grayi and Listeria murrayi to a new genus Murraya. Int. J. Syst. Bacteriol. 24:177–185. 175. Stuart, S. E., and H. J. Welshimer. 1973. Intrageneric relatedness of Listeria pirie. Int. J. Syst. Bacteriol. 23:8–14.

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176. Tabouret, M., J. Derycke, A. Audurier, and B. Poutrel. 1991. Pathogenicity of Listeria monocytogenes isolates in immunocompromised mice in relation to listeriolysin production. J. Med. Microbiol. 34:13–18. 177. Trivett, T. L., and E. A. Meyer. 1967. Effect of erythritol on the in vitro growth and respiration of Listeria monocytogenes. J. Bacteriol. 93:1197–1198. 178. Uchikawa, K.-L., I. Sekikawa, and I. Azuma. 1986. Structural studies on lipoteichoic acids from four Listeria strains. J. Bacteriol. 168:115–122. 179. Validation of the publication of new names and new combinations previously effectively published outside the USB. List n° 10. 1983. Int. J. Syst. Bacteriol. 33:438–440. 180. Van der Kelen, D., and J. A. Lindsay. 1990. Differential hemolytic response of Listeria monocytogenes strains on various blood agars. J. Food Safety, 11:9–12. 181. Vaneechoutte, M., P. Boerlinl, H.-V. Tichy, E. Bannerman, B. Jager, and J. Bille. 1998. Comparison of PCR-based DNA fingerprinting techniques for the identification of Listeria species and their use for atypical isolates. Int. J. Syst. Bacteriol. 48:127–139. 182. Vazquez-Boland, J. A., L. Dominguez-Rodriguez, J. F. Fernandez-Garayzabal, J. L. Blanco Cancelo, E. Gomez-Lucia, V. Briones Dieste, and G. Suarez Fernandez. 1988. Serological studies on Listeria grayi and Listeria murrayi. J. Appl. Microbiol. 64:371–378. 183. Vazquez-Boland, J. A., L. Dominguez, J. F. Fernandez-Garayzabal, and G. Suarez. 1992. Listeria monocytogenes CAMP reaction. Clin. Microbiol. Rev. 5:343. 184. Welshimer, H. J. 1963. Vitamin requirements of Listeria monocytogenes. J. Bacteriol. 85:1156–1159. 185. Welshimer, H. J., and A. L. Meredith. 1971. Listeria murrayi: A nitrate-reducing mannitol-fermenting Listeria. Int. J. Syst. Bacteriol. 21:3–7. 186. Wilkinson, B. J., and D. Jones. 1975. Some serological studies on Listeria and possibly related bacteria. In Problems of listeriosis, ed. M. Woodbine. Leicester: University of Leicester, p. 261. 187. Wilson, G. S., and A. A. Miles. 1946. Topley and Wilson’s principles of bacteriology and immunity, 3rd ed., vol. 1. London: E. Arnold & Co.

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Ecology of Listeria Species 2 and L. monocytogenes in the Natural Environment Brian D. Sauders and Martin Wiedmann CONTENTS Introduction ......................................................................................................................................21 Methods to Study the Ecology of Listeria Species ........................................................................22 Traditional Enrichment and Isolation Methods .....................................................................22 Enumeration Methods ............................................................................................................23 Limitations and Pitfalls of Current Environmental Characterization Methods for Understanding the Ecology of Listeria.............................................................24 Molecular Detection and Subtyping Methods .......................................................................25 Ecology of Listeria Species in Different Environments .................................................................27 Survival and Multiplication of Listeria spp. under Environmentally Relevant Stress Conditions ..............................................................................................................27 Listeria spp. in Soil and Vegetation.......................................................................................28 Listeria spp. in Sewage ..........................................................................................................30 Listeria spp. in Water .............................................................................................................31 Listeria spp. in Animal Feeds ................................................................................................32 Regulation of Listeria Gene Expression during Environmental Survival and Multiplication....................................................................................................34 Transmission Dynamics ...................................................................................................................35 Natural Environment and Transmission of Human Listeriosis .............................................35 Transmission of Listeria between Natural and Food-Processing Environments ..............................................................................................................36 Ecology of Human Outbreak-Associated L. monocytogenes Strains........................37 Ecology of L. monocytogenes Strains Associated with Sporadic Human Listeriosis Cases ............................................................................................38 Natural Environment and Transmission of Listeria in Animals............................................40 Transmission of Listeria within Natural Environments ........................................................42 Defining Global Listeria Transmission Pathways and Reservoirs ........................................42 Acknowledgments ............................................................................................................................44 References ........................................................................................................................................44

INTRODUCTION Most members of the genus Listeria, including L. monocytogenes, appear to be widely distributed in many different environments. Due to the importance of L. monocytogenes as a human foodborne and animal pathogen most studies on Listeria in the environment have focused on food processing 21

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and farm environments. Because the presence and ecology of Listeria in food processing environments is covered extensively in other chapters of this book, this chapter will focus on the ecology of Listeria in environments outside food processing and preparation operations. A broad understanding of the distribution and ecology of Listeria spp. and L. monocytogenes in the natural environment will be critical to provide for better control of this and other foodborne and environmental pathogens.

METHODS TO STUDY THE ECOLOGY OF LISTERIA SPECIES Studies on the ecology of Listeria spp. critically depend on our ability to detect and characterize these organisms in different environments. Other chapters in this book will provide a detailed overview of detection and characterization methods for Listeria spp. and L. monocytogenes from food, food-processing environments, and clinical specimens. Here, we will critically review selected detection and characterization methods, highlighting their application for isolation and characterization of Listeria spp. from samples collected from natural environments.

TRADITIONAL ENRICHMENT

AND ISOLATION

METHODS

Despite wide distribution in nature, L. monocytogenes and other Listeria spp. occur in small numbers in most natural habitats. Detection methods for Listeria spp. thus generally include selective enrichment, followed by plating on selective and differential media. Commonly used selective and differential plating media (e.g., Oxford, lithium-chloride-phenylethanol-moxalactam [LPM]) allow for isolation of Listeria spp., but do not provide for specific identification of L. monocytogenes. Listeria-like colonies must be further characterized biochemically or genetically to determine the species isolated. Because Listeria can survive and multiply at refrigeration temperatures (1 to 8°C), cold enrichment at 5°C represented the selective enrichment methodology of choice for many years. This technique required incubation and subsequent periodic plating for several months to one year [85]. Newer media use selective agents to allow for enrichment of Listeria spp. at higher temperatures (30 and 35°C). Selective agents widely used in different enrichment media include acriflavin (to inhibit many Gram-positive bacteria) and nalidixic acid (to inhibit Gram-negative bacteria), among others. A wide variety of different one-step and two-step enrichment and isolation media and protocols for Listeria have been described (see Chapter 7); three commonly used isolation methods are briefly described here. All three methods covered here were originally developed for detection of Listeria spp. and L. monocytogenes from foods and food-processing environments; however, these methods (or adaptations of these methods) also have been used successfully for isolation of L. monocytogenes from environmental samples [12,206]. All three methods use selective enrichment but the concentrations of the selective agents vary to strike a balance between recovery of injured cells and suppression of other microorganisms. For example, the U.S. Food and Drug Administration (FDA) uses primary enrichment for 4 h at 30°C without selective agents to increase recovery of potentially injured L. monocytogenes. After 4 h of incubation, acriflavin, nalidixic acid, and cycloheximide are added, followed by incubation for an additional 44 h (48 h total). Enrichments are plated at 24 and 48 h onto Oxford (OX) agar, and LPM or polymyxin-acriflavin-lithium chloride-ceftazidime-esculinmannitol (PALCAM) agar [186]. The U.S. Department of Agriculture (USDA) uses primary enrichment in University of Vermont (UVM) broth for 24 h at 30°C, with subsequent plating onto modified Oxford agar (MOX) plates and transfer of the primary enrichment to Fraser broth (FB) for secondary enrichment. FB enrichments are plated onto MOX plates after 24 h at 30°C [36]. The Netherlands Government Food Inspection Service (NGFIS) method [187] uses PALCAM-egg yolk broth incubated at 30°C for 24 to 48 h with subsequent plating onto PALCAM agar at 24 and 48 h. The U.S. Centers for

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Disease Control and Prevention reported that utilizing the USDA and NGFIS methods provided for 90% sensitivity for isolating L. monocytogenes from food and fecal samples [23]. Although many media, including those mentioned in the previous paragraphs, have been widely evaluated and used for isolation of Listeria spp. and L. monocytogenes from foods, food-processing environments, and fecal materials [23,57,62,95,113], very few studies have evaluated different enrichment procedures for their ability to detect Listeria spp. and L. monocytogenes in the natural environment. Initial work in the 1970s by Weis [196] compared the use of an early selective enrichment medium (potassium thiocyanate medium with and without 10 µg/mL of acriflavin per milliliter) with cold enrichment in tryptose broth and in brain–heart infusion broth for isolation of Listeria spp. and L. monocytogenes from plant and soil materials. The study concluded that selective enrichment provided a suitable method for isolation of Listeria from environmental samples. A more recent study by van Renterghem [187] compared four different enrichment procedures for their ability to detect L. monocytogenes in manure samples and samples from natural environments and concluded that cold-enrichment procedures were unsatisfactory for isolation of L. monocytogenes from natural environments. This is consistent with an evaluation of cold enrichment and the USDA selective enrichment procedure on food samples, which showed that the selective enrichment procedure provided significantly better results for isolation of L. monocytogenes than cold enrichment [92]. Many publications [32,34,58] report the use of cold enrichment for detection of Listeria and L. monocytogenes from samples collected from natural environments (e.g., silage); however, selective enrichment likely provides a better method for isolation of Listeria and L. monocytogenes from these sample types. The most commonly used enrichment procedure for samples from natural environments includes selective enrichment in LEB (although with varying concentrations of selective agents), followed by plating on selective and differential media (most often Oxford or LPM) [102,206]. Definitive recommendations on the most effective protocols for detection of Listeria and L. monocytogenes in natural environments are not possible until comparative evaluations of different detection protocols for different sample types (silage, sewage, soil, etc.) are performed. Based on experiences with development and evaluation of enrichment protocols for food samples, it is unlikely that any single method will allow for sensitive detection of Listeria in different sample types. A combination of different enrichment and plating procedures will likely provide for the most sensitive detection in natural environments, but may not be feasible for many larger studies on Listeria in the natural environment. Although maceration and suspension of samples from natural environments (e.g., soil, silage, plant materials) in selective enrichment media represent a common and suitable approach for detection of Listeria, certain sample types may require additional bacterial cell concentration steps. For water samples, filtration is often used to concentrate bacterial cells before culture. For example, Watkins and Sleath [195] used filtration combined with cold enrichment and subculturing onto selective media for detection of Listeria in water samples. Colburn et al. [34] also used filtration and cold enrichment to isolate Listeria spp. from water samples. Immunomagnetic separation (IMS) using magnetic beads with antibodies specific to Listeria provides another approach to concentrating Listeria cells [174]. For example, IMS has been used to concentrate Listeria cells after incubation in enrichment broth and before plating [174]. IMS has also been used to concentrate Listeria cells after primary enrichment of human [86] and animal fecal specimens [16].

ENUMERATION METHODS Quantification of Listeria spp. and L. monocytogenes is sometimes critical to understanding the ecology and transmission of Listeria in natural environments. Although a most-probable-number (MPN) approach using selective enrichment methods must be used for quantification of Listeria populations <100 CFU/g, direct plating can be utilized if Listeria populations >100 CFU/g are present [36]. MPN methods have been used to quantify Listeria spp. and L. monocytogenes in food

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samples [211], although few have used this approach to quantify Listeria spp. and L. monocytogenes in samples from natural environments [20]. Enumeration of Listeria spp. in samples contaminated with >100 CFU/g can easily be achieved by direct plating on selective and differential media, which allow specific detection of Listeria spp. (e.g., Oxford). On the other hand, specific enumeration of L. monocytogenes by direct plating is not practically feasible unless plating media are available that allow for specific detection of L. monocytogenes. Vazquez-Boland et al. [188] demonstrated that a method using direct plating on Listeria selective agar followed by 37- to 48-h incubation at 37°C with a subsequent overlay with blood agar allowed for quantification of hemolytic Listeria in silage samples. This method can thus be used to enumerate L. monocytogenes if the presence of hemolytic L. seeligeri and L. ivanovii can be excluded. More recently, several selective and differential chromogenic agars were developed to allow specific detection of L. monocytogenes. L. monocytogenes plating medium (LMPM; Biosynth) contains the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate to allow detection of phosphatidylinositol phospholipase C (PI-PLC) activity, which, among the Listeria spp., is unique to L. monocytogenes and L. ivanovii [157]. In combination with rapid biochemical tests for differentiation of L. ivanovii and L. monocytogenes (e.g., L. monocytogenes confirmation media, LMCM, Biosynth), this selective and differential medium thus provides a tool for more rapid quantification of L. monocytogenes by direct plating or MPN procedures.

LIMITATIONS AND PITFALLS OF CURRENT ENVIRONMENTAL CHARACTERIZATION METHODS FOR UNDERSTANDING THE ECOLOGY OF LISTERIA Despite the continued development of improved selective and sensitive isolation procedures for Listeria spp. and L. monocytogenes, culture-based methods show certain limitations that may affect our ability to understand the ecology of Listeria. There is considerable evidence that use of a single enrichment method may limit recovery of injured Listeria cells (if the medium is too selective) or may not allow for effective recovery of Listeria cells in the presence of high levels of other microorganisms (if less selective compounds are used). Although two-step enrichment procedures may ameliorate these problems to a certain extent, recovery of injured Listeria cells in samples with high levels of competing microorganisms represents a particular challenge. Furthermore, a putative viable but not culturable (VBNC) state was recently proposed for L. monocytogenes [21]; cells in this state are unlikely to be recovered by any currently used enrichment procedures. There also is considerable evidence that many environments as well as samples collected from these environments can contain multiple Listeria species and/or multiple Listeria strains [151,161,162]. For example, Ryser et al. [162] performed enrichment of ground beef, pork sausage, ground turkey, and chicken, followed by ribotyping of up to 10 isolates per sample. Over half of all positive samples contained more than one L. monocytogenes ribotype and the choice of enrichment medium affected the subtypes recovered from a given sample. In some instances, detection of certain L. monocytogenes ribotypes was only possible when 10 isolates from a sample were typed. This clearly shows the challenging nature of characterizing the Listeria microbial diversity using current detection methods. Although it is increasingly recognized that collection and characterization of multiple isolates may be necessary to evaluate the Listeria diversity in a given sample, enrichment methods appear to affect recovery of different Listeria species and strains selectively. In addition to the fact that L. innocua may outcompete L. monocytogenes during enrichment [51,126], it also appears that different enrichment media lead to recovery of different bacterial subtypes from the same sample [161]. Future application of molecular methods will provide an opportunity to improve our ability to probe and understand the ecology of Listeria in natural environments. Similar to commonly used 16S rDNA-based approaches to characterize bacterial population structures, Listeria- and/or L. monocytogenes-specific PCR primers [30,166,194] could be used to detect and characterize

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Listeria isolates and their diversity directly, including through construction and sequencing of clone libraries. The full power of these molecular methods can only be realized as sensitive, inexpensive, and rapid methods for isolation from natural environments of DNA suitable for PCR amplification are developed and applied [39,42,107,155,169].

MOLECULAR DETECTION

AND

SUBTYPING METHODS

Chapter 9 provides a comprehensive summary of subtyping methods for L. monocytogenes; application of molecular subtyping methods is critical to our ability to understand the ecology and transmission of L. monocytogenes in natural environments. Methods commonly used to subtype L. monocytogenes include ribotyping, pulsed-field gel electrophoresis (PFGE), and, more recently, multilocus sequence-based typing (MLST). Ribotyping [204,206] and PFGE [122,190] have been applied in studies of the diversity of L. monocytogenes in natural environments. These DNA-based methods define bacterial subtypes using restriction digestion of bacterial DNA to generate DNA fragment banding patterns. DNA fragment size-based subtyping methods have significant drawbacks. For example, despite the existence of software packages for data normalization and analyses (e.g., Bionumerics, Applied Maths, Sint-Martens-Latem, Belgium), these subtyping methods are often difficult to standardize. As a consequence, the ease of exchanging and comparing subtype data among laboratories can be severely limited. Although DNA fragment size-based subtyping methods have been used for cluster analyses, they generally do not provide information amenable to inference of primary genetic characteristics (i.e., nucleotide sequences) for evolutionary analyses. Long-term studies on the ecology and evolution of Listeria require subtyping data that can be used to infer and quantify the genetic relatedness of isolates, so DNA fragment size-based subtyping methods have limited utility for these applications. DNA sequencing-based methods are being developed and increasingly used for subtyping and characterizing L. monocytogenes [30,165]. With these methods, complete or partial nucleotide sequences are determined for one or more bacterial genes or chromosomal regions, thus providing unambiguous and discrete data. Sequencing can target a single gene (single locus approach) or multiple genes. Advantages of sequencing methods over DNA fragment-sized typing methods include their ability to generate unambiguous data that are portable through Web-based databases and that can be used for phylogenetic analyses [54]. Although a variety of DNA sequence-based subtyping strategies targeting virulence genes, housekeeping genes, or other chromosomal genes and regions is feasible, multilocus sequence typing (MLST), which is an extension of multilocus enzyme electrophoresis (MLEE), represents a widely used strategy [30,48,53,54]. Ultimately, MLST may lead to integration of PCR-based detection and subtyping in a single format, eliminating the need for culturing for certain applications [53]. Molecular subtyping methods have been widely used and developed for L. monocytogenes [78,202], but only limited information is available on molecular subtyping methods for Listeria spp. [154,176,193]. Further development, particularly of MLST-based subtyping methods, is necessary to allow comprehensive studies of the ecology of Listeria spp. in natural environments. Our knowledge of the transmission and ecology of Listeria spp. and L. monocytogenes has critically relied on application of classical and molecular subtyping methods for strain differentiation. For the purpose of studying the ecology of L. monocytogenes in the natural environment or otherwise, it is particularly important to bear in mind the limitations of all subtyping methods currently used for L. monocytogenes strain differentiation and also to consider the advantages and disadvantages of any specific subtyping method. As outlined later, subtyping data using a variety of different methods have, for example, shown that human disease-associated strains can also be found in farm and natural environments (see Figure 2.1 for examples); however, one must bear in mind the relative discriminatory power of different subtyping methods when interpreting these findings.

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A Location (city, state)

Ribotype

1985

Los Angeles, CA

DUP-1038B

Jul 1998

Cortland, NY

DUP-1038B

animal (bovine)

Jul 2001

Brooktondale, NY

DUP-1038B

animal (caprine)

Jan 2002

Burdette, NY

DUP-1038B

food (hummus)

Jun 1997

Albany, NY

DUP-1038B

Strain No.

Source

Date collected

FSL J1-119

human epidemic

FSL C1-057

human sporadic

FSL E1-128 FSL N3-080 FSL J1-179 FSL L4-170

processing plant drain

Oct 2002

Unknown, US

DUP-1038B

FSL N3-796

farm (water trough)

May 2002

Perry, NY

DUP-1038B

FSL S4-049

urban lake water

Jun 2001

Syracuse, NY

DUP-1038B

FSL S4-169

urban playground soil

Aug 2001

Rochester, NY

DUP-1038B

FSL S4-257

urban park A soil

Nov 2001

Albany, NY

DUP-1038B

FSL S4-628

urban park B soil

Jun 2002

Albany, NY

DUP-1038B

FSL S4-774

urban sidewalk

Aug 2002

Syracuse, NY

DUP-1038B

FSL S4-780

urban drain water

Aug 2002

Syracuse, NY

DUP-1038B

FSL S4-926

urban stream water

Oct 2002

Rochester, NY

DUP-1038B

FSL S6-096

urban park vegetation

Nov 2002

Albany, NY

DUP-1038B

FSL S6-116

urban sidewalk

Nov 2002

Albany, NY

DUP-1038B

FSL S6-136

urban runoff water

Nov 2002

Albany, NY

DUP-1038B

FSL S4-941

pristine vegetation

Oct 2002

Tompkins county, NY* DUP-1038B

Strain No.

Source

Date collected

Location (city, state)

Ribotype

FSL C1-103

human epidemic

Oct 1998

Troy, NY

DUP-1044A

FSL J1-193

human sporadic

Jun 1997

Manhasset, NY

DUP-1044A

FSL E1-054

animal (bovine)

Mar 1999

Unknown, NY

DUP-1044A

FSL N3-038

animal (caprine)

Oct 2001

Lake Ariel, PA

DUP-1044A

FSL F2-008

food (hispanic cheese)

Jul 1999

New York, NY

DUP-1044A

FSL H1-139

processing plant drain

Mar 2000

Unknown, US

DUP-1044A

FSL N3-243

farm (haylage)

Mar 2002

Cazenovia, NY

DUP-1044A

FSL S4-728

urban sidewalk

Jul 2002

Albany, NY

DUP-1044A

FSL S6-134

urban river water

Nov 2002

Albany, NY

DUP-1044A

Ribotype Pattern Will be run 4/15/03

B Ribotype Pattern

FIGURE 2.1 Ribotypes of Listeria monocytogenes isolated from epidemic and sporadic listeriosis, foods, foodprocessing environments, and the natural environment. Examples demonstrate that ribotypes that have caused human epidemic listeriosis are temporally and geographically widely distributed among animal cases of listeriosis, foods, food-processing environments, and the natural environment. Ribotype and associated isolate information were obtained from the PathogenTracker database (www.pathogentracker.net). (A) Ribotype DUP-1038B was implicated as cause of a human listeriosis outbreak in Los Angeles, California (1985), linked to consumption of Mexican-style cheese. (Linnan, M. J. et al., 1988, N. Engl. J. Med. 319:823–828.) (B) Ribotype DUP-1044A was implicated as the cause of a multistate human listeriosis outbreak in the United States (1998 to 1999) linked to consumption of contaminated hotdogs. (Anonymous, 1999, MMWR 47:1117–1118.) (*) = Sample collected from the Connecticut Hill Wildlife Management area; county location is provided because city was not applicable.

Although use of less discriminatory methods (e.g., MEE, single enzyme ribotyping) to characterize a given set of isolates may show that the same strains are found among isolates from human clinical cases and natural environments, application of more discriminatory subtyping methods (e.g., PFGE) may further differentiate strains. By no means does this make MEE or ribotyping data meaningless; rather, it indicates different levels of relatedness. We do not yet understand the specific levels of relatedness determined by each subtyping method. However, less discriminatory methods will generally indicate that two indistinguishable isolates may share a less recent common ancestor as compared to two indistinguishable isolates using a very discriminatory method. Subtype characterization by a less discriminatory method thus can still provide a very powerful tool to probe the distribution of different clonal groups (i.e., a group of genetically

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closely related isolates) and to differentiate broadly distributed clonal groups (e.g., clonal groups found in natural environments, among human and animal clinical cases and foods) from clonal groups that occupy a specific niche (e.g., cause disease in only a single specific host species). The power of less discriminatory subtyping methods has been demonstrated by the broad contributions of MEE studies to our understanding of bacterial population genetics [17,24,62,63, 113,142,160].

ECOLOGY OF LISTERIA SPECIES IN DIFFERENT ENVIRONMENTS Listeria spp. are generally considered ubiquitously distributed in the natural environment. Listeria spp. and L. monocytogenes have specifically been isolated from many different environments including soil [196], water [195], vegetation [196,199], sewage [3,4,34,67,195], animal feeds [32,58,60,121,162,175,188,190,203,205], farm environments [45,63,101,102,179,184,192], and food-processing environments [43,89,95,105,112,151,160]. Although many reports indicate that L. monocytogenes and L. innocua may represent the most common Listeria spp. in many natural environments, others have reported considerable prevalence of other Listeria spp. in specific natural environments. For example, a survey of an urban setting showed that L. ivanovii and L. seeligeri represented the most common Listeria spp. isolated from soil samples [127]. Cox et al. (1989) isolated L. monocytogenes and L. innocua from environmental samples collected from a sawmill; however, L. ivanovii represented the most prevalent Listeria spp. isolated [37]. Interestingly, although L. ivanovii, as well as L. welshimeri and L. seeligeri (both of which were not isolated in their study) ferment xylose, a common decomposition product of wood, L. monocytogenes and L. innocua do not ferment xylose. Further studies on the distribution of different Listeria spp. and subtypes in different natural environments will be needed to develop a true understanding of the ecology of Listeria and L. monocytogenes, including the reservoirs and niches of the different Listeria spp. and different clonal groups. This knowledge will also be necessary to understand transmission pathways of the pathogenic Listeria and the sources and spread of human and animal infections.

SURVIVAL AND MULTIPLICATION OF LISTERIA SPP. UNDER ENVIRONMENTALLY RELEVANT STRESS CONDITIONS L. monocytogenes is distinguished among foodborne pathogens in that it can tolerate high (up to 20%) salt concentrations, can multiply over a wide range of temperatures (1 to 45°C), and can adapt to and survive acid stress. In contrast to other non-spore-forming bacteria that cause foodborne illness, L. monocytogenes appears to be able to survive longer under adverse environmental conditions [195]. The ability of L. monocytogenes to colonize, multiply, and persist in the food-processing environment and on food-processing equipment likely also reflects its ability to survive in the natural environment for extended periods. Data available on stress resistance and survival characteristics of Listeria spp. are more limited than those available for L. monocytogenes. In general, it is hypothesized that other Listeria spp. show resistance to environmental stress (acid, salt, temperature, etc.) similar to that observed for L. monocytogenes because L. innocua and other Listeria spp. are commonly found in foods [9,31,149,209] and food-processing environments [152,181]. Experimental studies comparing stress resistance between L. monocytogenes and other Listeria spp., including survival and growth of temperature stress [147], salt stress [18], low water activity [140]; in the presence of various antimicrobial agents [183]; and under various combined stress conditions [114,156,163], also support that all Listeria spp. share similar stress resistance characteristics. Recent sequencing of the genome of one L. monocytogenes strain and one L. innocua strain [76] also showed that these two species share a large number of predicted genes that encode various

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components of potential stress response systems including regulatory proteins, transporters, and transcriptional regulators [29,76]. Further comparative genomic analyses on presence and transcription of various stress response genes in different Listeria spp. and subtypes will provide a unique opportunity to increase our understanding of the stress response pathways that facilitate Listeria survival in the environment and under stress conditions. Laboratory studies on survival under controlled environmental stress conditions can help to understand the biology of Listeria, but data on Listeria survival in natural environments also critically contribute to our understanding of Listeria transmission and ecology. The following sections attempt to summarize our current knowledge on distribution, prevalence, and survival of L. monocytogenes and Listeria spp. in different natural environments. In addition, Table 2.1 provides a summary of data from various studies on the persistence of L. monocytogenes in different natural and farm environments.

LISTERIA

SPP. IN

SOIL

AND

VEGETATION

L. monocytogenes as well as other Listeria spp. appears to be commonly present in soil and vegetation in the natural environment. Most studies on Listeria in the natural environment conducted to date have almost exclusively focused on farm environments and associated croplands. Interestingly, most studies on Listeria in natural environments not associated with farms indicate that prevalence of L. monocytogenes is lower than that of other Listeria spp. [127,166]. MacGowan et al. [127] specifically reported that L. seeligeri was more frequently isolated from soils than L. innocua or L. monocytogenes. In early work Weiss and Seeliger (1975) isolated Listeria spp. from plant samples collected from cornfields (9.7% of samples positive), grain fields (13.3%), cultivated fields (12.5%), uncultivated fields (44%), meadows and pastures (15.5%), forests (21.3%), and wildlife feeding areas (23.1%) in southern Germany [197]. Soil samples taken at a depth of 10 cm showed a significantly lower Listeria spp. prevalence as compared to surface soil samples. Although the original paper by Weiss and Seeliger (1975) reported L. monocytogenes prevalence, only 37 of 103 Listeria isolates elicited disease consistent with listeriosis in a mouse bioassay. This indicates that as many as 64% of these isolates may have been Listeria spp. other than L. monocytogenes or L. ivanovii, thus suggesting that these isolates represented a variety of Listeria spp. Welshimer and Donker-Voet [199] did not isolate L. monocytogenes from soil or dead vegetation sampled from agricultural sites in early autumn, yet soil and decayed vegetation sampled the following spring were nearly all positive for the organism. Listeria isolates from this study were also evaluated for virulence in mice and not all were found to be virulent [199], again suggesting the occurrence of species other than L. monocytogenes. Clearly, changes in the Listeria taxonomy that have occurred over the years may require careful interpretation of older studies. These studies may have used a broader definition of a L. monocytogenes sensu lato, which might include Listeria spp. other than those currently classified as L. monocytogenes. Only very limited recent data are available on the presence and distribution of L. monocytogenes and other Listeria spp. in soils, limiting our understanding of the soil ecology of these organisms [127]. More recently, Fenlon et al. [63] examined samples of grass, leaves, stems, and roots/stems from two crops of growing sward before harvesting. L. monocytogenes was not detected in any of the samples, but L. innocua and L. seeligeri were isolated from 3 of 10 samples from the root/stem area. L. monocytogenes was detected though in 9 of 10 samples of cut grass from the same crops that had wilted for 24 h before ensiling. Whittenbury [201] had demonstrated the importance of the sheath area as a source of lactic acid bacteria, so Fenlon [63] hypothesized that the higher incidence of L. monocytogenes in harvested (processed) grass compared with other plant products could be attributed to the presence of a sheath of decaying plant material at the base of the plant that might act as an inoculum at harvest. Although this process may contribute to contamination of grass with Listeria during the ensiling process, Listeria contamination of plant-based feeds and foods probably more likely results from direct deposition of animal feces, spreading of animal waste and sewage sludge as fertilizer, or from indirect contamination via feces-contaminated soil.

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TABLE 2.1 Survival of L. monocytogenes in Various Environmental Samples Sample Soil Sterile soil (I) Clay soil (I) Sealed tubes Fertile soil (I) Sealed tubes Cotton-plugged tubes Top soil (I) Exposed to sunlight Not exposed to sunlight Moist soil Dry soil Soil Soil

Storage Temperature (°C)

Survival (days)

Outside: winter/spring 24–26 24–26

154 225 67

24–26 24–26

295 67

NG NG NG NG 4–12 18–20

12 182 ~497 >730 240–311 201–271

5 Outside Outside Outside Summer Winter

182–2190 347 730 242 36 106

Sewage Sewage sludge cake (NC) Surface Interior Sprayed on field

28–32 48–56 Outside

35 49 >56

Water Sterilized pond water (I) Unsterilzed pond water (I) Pond water Pond water Pond water/ice Pond/river water Pond/river water Water Distilled water (I)

Outside Outside 35–37 15–20 2–8 37 2–5 Outside 4

7 <7–63 346 299 790–928 325 750 140–300 <9

Animal feed Silage (NC) Silage (NC) Mixed feed Oats (I) Hay (I) Straw (NC/I) Straw (I) Straw Straw

4 5 Outside Outside Outside ca. 22 Outside Outside: summer Outside: winter

450 180–2190 188–275 150–300 145–189 365 47–207 23 135

Fecal material Cattle feces (NC) Moist horse/sheep feces (I) Dry horse/sheep feces (I) Sheep feces Liquid manure Liquid manure

Notes: (I) = inoculated; NG = not given; (NC) = naturally contaminated. Sources: Adapted from al-Ghazali, M. R. and S. K. al-Azawi, 1988, J. Appl. Bacteriol. 65:203–208 and 209–213; Amtsberg, G., 1979, Dtsch. Tierarztl. Wochenschr. 86:253–257; and Mitscherlich, E. and E. H. Marth, 1984.

29

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Supporting the importance of animals as sources of L. monocytogenes in soil and on plants, Fenlon et al. [63] did not find any L. monocytogenes in the few soils examined that were associated with vegetable crops, but isolated L. monocytogenes from soil collected from fields where cattle or sheep on silage diets had been kept. Although lettuce and vegetables generally show low L. monocytogenes and Listeria spp. prevalence [57,63,77], preharvest contamination of vegetables with L. monocytogenes can provide a source for human listeriosis infections. For example, an outbreak involving 42 human cases in Nova Scotia in 1981 was linked to consumption of coleslaw. This coleslaw was produced from cabbage harvested from fields fertilized with untreated sheep manure—obtained from a farm with a history of ovine listeriosis [168]. Some studies have focused on describing the occurrence of Listeria in the natural environment; others have taken a more experimental approach to evaluating the ability of Listeria to survive in soil. Survival of L. monocytogenes in soil likely depends on a variety of factors, including soil type, moisture content, and water activity. Welshimer [198] used cotton wool-plugged tubes containing clay or fertile soils inoculated with L. monocytogenes and stored for 67 days at 24 to 26°C, to show that cell numbers decreased in both by seven orders of magnitude as the soils dried out. When these experiments were repeated with tubes sealed to prevent water loss, L. monocytogenes numbers in clay soil still decreased by seven orders of magnitude, but numbers in fertile soil decreased by only two orders of magnitude. In a study using autoclaved soils inoculated with L. monocytogenes at approximately 5 × 10–2 CFU/g and stored at ambient winter temperatures ranging from –15 to +18°C, Botzler [25] found an increase in L. monocytogenes numbers to 1 × 107 CFU/g over a 154-day period. It is not known what effect the autoclaving process had on Listeria growth by increasing availability of nutrients and eliminating competing organisms. Watkins and Sleath [195] demonstrated that there was little decrease in L. monocytogenes numbers in soil on land to which sewage sludge was applied; an initial inoculum of 170 CFU/100 g was recovered consistently over an 8-week period. Salmonellae, applied at a similar rate, decreased to undetectable levels in less than 4 weeks. L. monocytogenes appears to be able to survive and possibly even multiply in some or many soil types; damp surface soil and the presence of decaying vegetation appear to provide a particularly suitable environment for L. monocytogenes and Listeria spp. However, there appears to be no convincing evidence that soil or certain soil-associated niches serve as true reservoirs for L. monocytogenes. Similarly, our understanding of the soil ecology of other Listeria spp. is also extremely limited.

LISTERIA

SPP. IN

SEWAGE

Sewage appears to be contaminated commonly with L. monocytogenes and other Listeria spp. For example, Watkins and Sleath [195] reported L. monocytogenes levels >18,000 CFU/L in trade effluents associated with animals and sewage sludge after treatment. Levels of the organism in settled raw sewage from primary tanks ranged from 700 to 18,000 CFU/L. Much higher levels of Listeria spp. were reported [72] in a West German sewage treatment plant; untreated waste and filtered effluent contained 103 to 105 CFU Listeria spp./mL. The fact that there was only a 10-fold reduction in numbers between untreated and treated waste suggests that biological oxidation may not be an effective method for eliminating viable Listeria in sewage. Studies in northeastern Scotland [63] showed that L. monocytogenes numbers ranged from 120 CFU/mL in untreated sewage to 2–21 CFU/mL in treated effluent. Anaerobic digestion reduced numbers to 1.1 CFU/g, and lime-treated sludge had no culturable Listeria species. Al-Ghazali and al-Azawi [3,4] specifically studied survival of L. monocytogenes during sewage treatment and in stored sewage sludge cake in Iraq. A decrease of 85 to 97% in viable Listeria numbers occurred during activation and digestion stages of the sewage treatment process [3] and <3 to 15 L. monocytogenes CFU/mL were recovered from final effluent and sludge cake. These same authors [4] also demonstrated that

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the listeriae were inactivated when sludge was stored in direct sunlight for at least 8 weeks. Inactivation was slower in the interior of the sludge piles. Studies in the United Kingdom showed that when L. monocytogenes–contaminated sewage sludge was spread on land, numbers of the pathogen failed to decrease over an 8-week period [195]. In 2003, Garrec et al. investigated the impact of sludge treatments on five types of sludge in three different treatment plants and found that only liming the sludge reduced Listeria spp. loads to less than detectable levels [71]. They found that 87% of dewatered sludge contained Listeria spp., while 73% contained L. monocytogenes. In addition, 96% of sludge stored in tanks contained Listeria spp. and 80% contained L. monocytogenes. Concentrations of L. monocytogenes found in the dewatered sludge were lower (0.15 to 20 MPN/g dry matter) than concentrations found in sludge stored in tanks (1 to 240 MPN/g dry matter) [71]. In a survey of five types of sludge, Italian investigators also found the highest concentrations of Listeria spp. (L. monocytogenes, L. innocua, L. welshimeri, and L. grayi) in activated sludge (2,743 MPN/g dry matter); the lowest concentration was found in digested sludge (6 MPN/g dry matter) [41]. Both authors note that sludge from these treatment plants is used to fertilize agricultural fields and thus may represent a constant source of Listeria contamination of crops bound for human or animal consumption [41,71]. Because fecal contamination has been linked to one foodborne outbreak of human listeriosis associated with coleslaw [168], caution should thus be taken when applying potentially contaminated sludge and animal wastes on crops that may subsequently be consumed raw. Using isolates from a defined geographic region, Lozniewski et al. used serotyping, phage typing, and PFGE with two enzymes to further probe for links between L. monocytogenes present in sewage sludge and human listeriosis cases [121]. In this study, reported in 2001, all sludge isolates except one were distinguishable from human isolates; two human isolates were found to be indistinguishable from one sludge isolate, although no epidemiological data were available to further evaluate the relevance of these findings. Thus, although it has been shown that sewage used as fertilizer often contains L. monocytogenes and other Listeria spp. and that one human listeriosis outbreak has been linked to contamination by manure application [168], further studies are needed to define the importance of sewage as a source of human and animal listeriosis cases. Optimized sewage treatment processes and appropriate sludge and sewage application guidelines may help to minimize Listeria dissemination into the environment and possibly subsequent introduction into the human or animal food chain.

LISTERIA

SPP. IN

WATER

The ubiquitous nature of L. monocytogenes and Listeria spp., in conjunction with use of surface waterways for discharge of sewage effluents, inevitably results in the presence of these organisms in a wide range of surface waters, including lakes, rivers, and streams. For example, Dijkstra [47] found L. monocytogenes in 21% of the surface water samples in the northern Netherlands. Although widely distributed, L. monocytogenes has been reported to be present in varying numbers in water. Watkins and Sleath isolated L. monocytogenes from seven rivers in the United Kingdom at levels ranging from 3 to >180/L [195]. A study of the course of the River Don in northeastern Scotland [63] showed that between 42 and 53% of sites were positive for L. monocytogenes (with levels ranging from 10 to 350 CFU/L) with no measured factor, seasonal or otherwise, related to presence or numbers of L. monocytogenes. However, a survey of 128 fresh-water samples from four rivers and one lake in Greece showed a rather low prevalence (4%) of L. monocytogenes [12] and a smaller survey of 15 groundwater samples in Belgium yielded only one L. monocytogenes–positive sample [187]. Few reports have examined occurrence of Listeria spp. other than L. monocytogenes in surface waters. One study of surface waters (canals, rivers, ponds, and lakes) in the United Kingdom

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[67] analyzed 30 samples (100 mL) from 21 sites, finding 8 sites positive for L seeligeri (27%), 1 for L. innocua, and 1 for L. welshimeri, and no sites positive for L. monocytogenes. In another study, Bernagozzi et al. [20] cultured 15 samples from two rivers in Italy for presence of Listeria and found that 47% of the samples contained at least one Listeria sp. From their positive cultures they isolated three different species, including six L. monocytogenes, six L. innocua, and two L. grayi [20]. In addition to studies on Listeria spp. in fresh waters, some data are also available on Listeria spp. in marine environments. Soonthoranant and Garland [177] found L. monocytogenes in 35 to 100% of samples collected from discharges of a sewage treatment pond and from fish-processing factory effluents, which also contained sewage. Inshore marine waters, which eventually received these discharges, contained L. monocytogenes in 6.6% of samples. They also found L. monocytogenes in 15% of Pacific oysters and blue mussels, a fact of interest because these are bottom-dwelling filter-feeders that may naturally concentrate bacterial cells as a result of their feeding behavior. Colburn et al. [34] found that 81% of fresh water and 62% of low-salinity waters harbored Listeria spp., including L. monocytogenes. Specifically in bay water, one of three samples was found to contain three Listeria spp. including L. monocytogenes, L. innocua, and L. welshimeri. They also found L. innocua in 1 of 35 oysters sampled [34]. Furthermore, Motes [134] isolated L. monocytogenes from shrimp caught off the U.S. Gulf Coast, and Destro et al. [43] showed that some strains associated with shrimp can persist in processing plants and enter the final product. At present no epidemiological data suggest the occurrence of direct infection of humans through L. monocytogenes–contaminated water. However, Gray et al. [84] successfully infected two sheep, four goats, and one cow with L. monocytogenes–contaminated water. Based on quantitative estimates from natural water samples [20,63,195], it seems unlikely that L. monocytogenes contamination in water could reach sufficient numbers to cause human infections, unless gross fecal contamination occurs. It is conceivable, however, that L. monocytogenes–contaminated water might serve as an indirect source of human infections from marine or freshwater fish or seafood harvested and stored for extended times under refrigeration [98]. Appropriate thermal processing and/or application of listeriocidal treatments can prevent Listeria contamination of ready-to-eat products though, if post-processing contamination is prevented [180].

LISTERIA

SPP. IN

ANIMAL FEEDS

Most formulated animal feeds have low levels of available water, which restricts multiplication of Listeria spp. and L. monocytogenes. The same is true of hay and cereal grains; although L. monocytogenes has been reported in such materials [70], the numbers are unlikely to reach levels that present a serious risk to animals. Many formulated feeds are sold in pellet form and will have received a degree of heat treatment capable of killing a high proportion, if not all, Listeria present. The animal feed most closely linked with animal listeriosis is silage, and this association is well documented. Olafson [145] noted the link between “Listerella” encephalitis in ruminants and silage in 1940, and in 1960, Gray [82] reported isolating L. monocytogenes from the fetus of a pregnant mouse fed poor-quality silage implicated in death and abortion in cattle. Identical serotypes of L. monocytogenes were isolated postmortem from the mice and cattle. Today, numerous reports link the feeding of silage with listeriosis outbreaks in sheep and cows [60,65,74,75,88,120,138,206]. Much of this problem can be attributed to the high numbers of L. monocytogenes present in contaminated silage [59,60] as compared with other animal feeds. In good-quality silage prepared from grass, maize, whole crop cereals, or leguminous plants, which may or may not be wilted (dried to optimum moisture content) in the field before ensiling, the onset of anaerobic conditions stimulates the indigenous or inoculated lactic acid bacteria to multiply quickly. As these bacteria expand to populations as high as 109 CFU/g within 48 h, they convert the plant sugars to lactic acid, causing a rapid decrease in pH [129] (well-preserved silage generally has a pH < 4.5). Such acidic conditions inhibit the growth of spoilage microorganisms and Listeria.

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Higher dry-matter silage tends to have a higher pH, but lower levels of available water in these silages may help minimize Listeria growth. Grass silage in cooler, wetter climates tends to have lower sugar levels and higher moisture contents resulting in a poorer, slower fermentation. These silages may be more susceptible to L. monocytogenes contamination and growth than grass and maize silage in countries with warmer climates. Although Listeria die in well-fermented silage, if the pH increases before all bacterial cells are killed, the surviving Listeria will multiply. Dijkstra [44] showed that L. monocytogenes can survive 4 to 6 years in naturally contaminated silage. Fenlon et al. [63] also noted that L. monocytogenes could survive over 1 year in bags used to wrap big bales and stored for reuse. Fortunately, much of the L. monocytogenes contamination in silage occurs in visibly moldy areas, and if these are removed and discarded before feeding, exposure of animals to high levels of L. monocytogenes can be considerably reduced [61]. In addition to commonly fed types of silages, more obscure feed sources of animal listeriosis have also been reported, including silage made from orange peel and artichokes [191]. Several outbreaks in Canada and the northern United States have also reportedly been caused by cattle feeding on ponderosa pine needles [2]. A variety of studies have confirmed that L. monocytogenes contamination is most frequently associated with poor-quality silage. In 1979, Grønstol [88] analyzed 291 grass silage samples from 113 farms and isolated L. monocytogenes from 22% of samples with a pH <4.0, from 37% of samples with a pH of 4.0 to 5.0, and from 56% of samples with a pH >5.0. In a study of clamp silage implicated in an outbreak of listeriosis in cattle [60], levels of L. monocytogenes in excess of 12,000 CFU/g were found in the surface layer (pH 8.3 to 8.5), whereas no Listeria spp. were detected 15 cm into the silage mass where the pH was 4.5. In another study [64], the extent of Listeria contamination in silage was directly related to its hygienic quality as measured by the numbers of Enterobacteriaceae present. This case indicates that management of postharvest processing of the grass can markedly affect numbers of Listeria present. Gitter [74] noted that from 1975 to 1985, the incidence of listeriosis in sheep in Great Britain increased from less than 50 cases per year to over 250. During the same period, only a small increase in the number of cattle listeriosis cases was observed. In addition, the pattern changed from isolated single cases to much larger flock-wide outbreaks. This was attributed to a change in the conserved forage used to feed sheep. In Great Britain, sheep are mainly kept on upland areas and before this time were traditionally fed hay. The wet climate of these hill farms is not conducive to the making of good-quality hay, and silage was not an economic option before the mid-1970s because it required expensive capital outlay for silos. The invention of the big-baler and the half-ton round bale made silage feeding feasible by baling grass and sealing it in large plastic bags to make silage. Unfortunately, some of the early attempts to make silage in this way were not very successful, and baled silage was of poorer quality than clamp silage [58]. L. monocytogenes appears to be predominantly a surface problem and big bales have a much greater surface area than clamp or silo silage, so the potential for contamination to develop is much greater in incorrectly prepared bale silage and if aerobic deterioration takes place. Fenlon [59] reproduced the problem in 500-g laboratory bales ensiled in plastic bags held for 2 to 3 weeks before testing. L. monocytogenes was detected at levels of ≥1.1 × 106 CFU/g silage in moldy areas near the tie end of the bag where air infiltrated. In the center of the bags pH remained at 3.8, silage appeared to be of good visual quality, and no Listeria were isolated. Donald et al. [50] specifically demonstrated the relationship among oxygen tension, pH, and L. monocytogenes growth by infusing laboratory silos with gas mixtures containing from 0.1 to 5.0% oxygen and showing that the greater the oxygen level was, the more rapidly the pH rose with subsequent multiplication of Listeria. In addition to systemic listeriosis, silage has also been implicated as a cause of inflammation of the iris (iritis) by L. monocytogenes [194], particularly in cattle. This condition appears to occur when cattle and sheep burrow their heads into bale silage in self-feeding systems and the eye

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becomes infected via abrasions caused by stems of grass contaminated with the organism. Modifying feeding practices to prevent eye contact with silage is the best preventative measure [119]. Although only limited studies are available on the molecular ecology of L. monocytogenes in most natural environments, a variety of subtyping studies on L. monocytogenes in silage have been published [17,120,190,203,205,206,208]. Almost all studies were specifically performed to track the sources of animal listeriosis cases, but the data available provide some interesting insight into the molecular ecology of Listeria. Many studies showed that a considerable diversity of L. monocytogenes can be found in an individual silage sample, a single silo, and on a given farm. Wiedmann et al. reported two outbreaks of epizootic listerial encephalitis, one in sheep and one in goats [206]. In both outbreaks, L. monocytogenes was isolated from silage and brain tissue samples and subtyped using random amplified polymorphic DNA analysis. Subtype analysis revealed two distinct L. monocytogenes strains, one that was identical to the sheep brain isolate, in the silage associated with the outbreak in sheep. In the goat outbreak, three brain isolates and one silage isolate all demonstrated different subtypes. Wiedmann et al. further reported an additional analysis of the previous goat and sheep outbreaks in addition to two bovine listeriosis outbreaks using ribotyping [205]. For all but one outbreak, L. monocytogenes strains represented by the same ribotype were found in clinical and silage samples. Additional L. monocytogenes strains with ribotypes different from those of the respective clinical samples were isolated from all silage samples, except for the outbreak in goats. On one farm five different ribotypes were isolated from silage samples collected from a single bunker silo, and on another farm three different L. monocytogenes ribotypes were isolated from a single 25-g silage sample. Similarly, Baxter isolated three different MEE types of L. monocytogenes from silage collected on a single farm with an outbreak of listerial encephalitis; interestingly, all of the silage isolates were distinct from the strain isolated from clinical specimens collected from infected animals on this farm [17]. Low et al. [120] also found six distinct phage types among 45 L. monocytogenes isolates from baled silage. These studies provide clear evidence that a diverse population of L. monocytogenes strains exists in farm environments, of which some may be more likely than others to cause disease [205]. Investigations of an epizootic of listerial encephalitis in a flock of 655 sheep showed that all seven clinical L. monocytogenes isolates had ribotypes identical to those for isolates from farm equipment used to transport silage. Corn silage, which was not fed to the sheep, also contained L. monocytogenes of the same pattern type as defined by ribotyping. L. monocytogenes was not isolated from the stored haylage designated for feeding the sheep (the cut-off point for isolation was <102 CFU/g). Based on ribotype results, it appears that corn silage cross-contaminated the haylage destined for the sheep during handling with a front-end loader, providing possible evidence for environmental transmission. In this case, suspension of silage feeding coincided with cessation of listeriosis cases. In another investigation of listeriosis among goats, Wiedmann et al. (1999) found that during a 16-month period, 10 goats with listeriosis were identified in two herds that shared three bucks, including one that died of listeriosis [208]. Analysis revealed that a single genetically unique L. monocytogenes strain had infected all goats from which isolates were available. Silage was not fed to either herd, and L. monocytogenes was not isolated from vaginal or rectal swab specimens obtained from healthy goats or from samples of feed. Three bucks were the only common elements between the two herds. The authors hypothesized a venereal route of transmission in this outbreak, but environmental contamination other than silage could not be excluded as a source [208]. These case studies further support that the environment can also represent a direct source for animal infection.

REGULATION OF LISTERIA GENE EXPRESSION AND MULTIPLICATION

DURING

ENVIRONMENTAL SURVIVAL

Studies on the presence and persistence of Listeria spp. and L. monocytogenes in different natural environments have significantly advanced our understanding of the ecology of these bacterial

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species. Studies on gene expression by Listeria spp. and L. monocytogenes in the environment outside a host and under conditions typically encountered in the environment may provide critical information on the biology of these organisms. Although the genetics of virulence and regulation of virulence gene expression in L. monocytogenes are covered in more detail in chapter 5, it is important to emphasize that negative regulation of virulence gene expression during survival outside a host may be critical for a saprophytic pathogen such as L. monocytogenes. Expression of virulence genes and synthesis of virulence factors outside the host may represent a burden compromising the ability of L. monocytogenes to survive in the natural environment and limiting its potential for transmission [189]. Evidence is accumulating that L. monocytogenes has evolved signal-sensing and signal-transducing mechanisms to sense environmental signals to adapt virulence gene expression to the particular needs of saprophytic versus parasitic lifestyles [189]. Although temperature appears to be one environmental signal controlling virulence genes, other signals also appear to be contributing to down-regulation of virulence gene expression outside a host [189]. For example, some studies suggest that the main L. monocytogenes virulence gene regulator, prfA, is down-regulated by the presence of the disaccharide cellobiose, a product of plant decomposition found ubiquitously in the environment [146]. In addition, the presence of bioavailable iron also appears to down-regulate virulence gene expression [35]. The availability and application of microarrays for studying global gene expression patterns in L. monocytogenes [133] will provide unique opportunities to further probe and compare Listeria gene expression under different environmental conditions. This will thus help us to improve our understanding of strategies Listeria uses to facilitate environmental survival and transmission.

TRANSMISSION DYNAMICS As outlined earlier, considerable data are available on prevalence of L. monocytogenes in different environments; however, we still lack a good understanding of overall transmission dynamics of L. monocytogenes. Even though L. monocytogenes is widely distributed in the natural environment and humans and animals are likely to come in frequent contact with L. monocytogenes through a variety of sources, food or feedborne exposure, respectively, seem to be the most common sources of infection. Considering the apparently frequent exposure of humans and animals to L. monocytogenes, the rare occurrence of clinical disease raises the intriguing question of the importance of human and animal infections for survival of L. monocytogenes. On the one hand, human and/or animal infections may represent a critical part of L. monocytogenes survival by providing a mechanism for rapid multiplication to high numbers and wide environmental dispersal, e.g., through fecal shedding. On the other hand, L. monocytogenes may be an “accidental” pathogen with humans or farm ruminants representing dead-end hosts that do not critically contribute to survival of the species L. monocytogenes. In the latter case, survival in the natural environment, including possibly some yet undefined host species (e.g., protozoans, lower vertebrates, or mammals other than the apparent human and ruminant hosts), may be critical to the evolutionary success of this bacterial species. Continued research on the ecology and evolution of L. monocytogenes and Listeria spp. will be necessary to define the transmission of this pathogen and to control and reduce human and animal infections. This section reviews our current knowledge on the transmission of L. monocytogenes with a particular focus on the role of the natural environment in the transmission of this pathogen.

NATURAL ENVIRONMENT

AND

TRANSMISSION

OF

HUMAN LISTERIOSIS

Overall, there is strong evidence that most human listeriosis cases are caused by foodborne infections, usually from contaminated ready-to-eat foods [93]. Although it is conceivable that

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L. monocytogenes can enter the food chain at many and possibly even at nearly any point, foodprocessing environments appear to be of particular importance as sources for introduction of L. monocytogenes into the food system [181]. Food contamination incidences can often be tracked back to postprocessing contamination in food-processing plants; however, the contribution of contamination at retail and in-home or at restaurants to human foodborne listeriosis infections has not been clearly defined. The role of contaminated raw animal-based agricultural products (e.g., milk, meat) as a direct source of L. monocytogenes contamination of ready-to-eat food products is likely to be minimal because commercially applied heat treatments generally kill L. monocytogenes effectively enough to provide an appropriate margin of safety. Although infected animals and contaminated agricultural environments rarely appear to be a direct cause of human infections, animal sources can play an important role in animal-derived food products that are not processed before consumption (e.g., raw milk). In addition, manure from infected or shedding animals may represent a source of food contamination, such as in the listeriosis outbreak in Nova Scotia in 1981, which was linked to consumption of coleslaw [168]. Transmission of Listeria between Natural and Food-Processing Environments Considerable numbers of cross-sectional and longitudinal studies on the subtype diversity and ecology of L. monocytogenes in food-processing environments have been performed, but only a few studies have specifically probed the transmission of Listeria between natural and food-processing environments. Longitudinal studies in different types of food-processing facilities have demonstrated that a large diversity of L. monocytogenes strains exists in different foods and food-processing environments. In many food-processing plant environments persistent and transient L. monocytogenes strains appear to be present [97,143,159,173], although some plants appear to contain only transient strains [143]. Persistent strains can be present in processing plants for months and years, up to 7 to 12 years [109]. For example, Harvey and Gilmour [89] used MEE and RFLP analysis to compare L. monocytogenes isolates from four milk-processing centers and two dairy farms in Northern Ireland with food and clinical isolates. They found that recurrent strains, specific to each dairy processor, colonized plants over long periods. When isolates from a poultry-processing environment were examined with RAPD analysis, Lawrence and Gilmour [113] showed that a single RAPD type was predominant in the raw processing environment over the 6-month period of the study and survived the clean-in-place schedules. In a follow-up study 12 months later, the same RAPD type was again isolated from final cooked products. Norton et al. showed that specific L. monocytogenes ribotypes persisted over time in the environments of two of the three smoked-fish processing plants studied [143]. Similar findings were reported by a variety of groups, which also showed the persistence of specific L. monocytogenes subtypes in different food-processing plants, including smoked fish, poultry, meat, and dairy plants [1,13,97,113,137,160,173]. Although it is clear that a large diversity of L. monocytogenes strains exists and persists in food-processing environments, information on how these strains enter that environment is limited. In one of the few studies probing the linkages between L. monocytogenes and Listeria in the natural environment, Arimi et al. used automated ribotyping to probe for links between on-farm sources of Listeria contamination (dairy cattle, raw milk, and silage) and contamination of dairy-processing environments [10]. Among a total of 475 Listeria isolates from 20 different dairy-processing facilities and different farms, 8 L. monocytogenes and 12 non-L. monocytogenes ribotypes were found in dairy-processing and farm environments [10]. Although the study design did not allow for conclusions on the directionality of transmission, these data support that Listeria clonal groups found in farm environments can also be present in dairyprocessing environments.

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Ecology of Human Outbreak-Associated L. monocytogenes Strains Until the 1980s, when several large outbreaks of human listeriosis occurred, listeriosis was considered a rare and sporadic human disease. As outbreak and case investigations implicated contaminated foods as the most likely sources of human L. monocytogenes infections, increased efforts were placed on linking human listeriosis cases to presence of this pathogen in foods and in the environment. Although most human listeriosis cases have been thought to be sporadic, multiple large human listeriosis outbreaks have been reported over the last 20 to 25 years. In addition, recent evidence suggests that a higher number of human listeriosis cases than previously assumed might represent outbreaks or single source clusters [168]. Most large human listeriosis outbreaks have been associated with L. monocytogenes serotype 4b strains, including outbreaks linked to contaminated coleslaw [169], soft cheese [22,104], p aˆ té [73], and pork tongue [103]. Additional outbreaks caused by serotype 1/2b strains have been linked to milk [40], rice salad [164], and imitation crabmeat [56]. Only few outbreaks have been linked to other serotypes, including an outbreak in Finland caused by a serotype 3a strain and linked to contaminated butter [125] as well as a L. monocytogenes serotype 1/2a outbreak in the United States linked to sliced turkey [69]. L. monocytogenes serotype 1/2b and 4b strains as well as 3b and 3c strains have been recognized to form a distinct evolutionary lineage of L. monocytogenes (lineage I), while serotypes 1/2a, 1/2c, and 3a [135] form lineage II. Serotypes 4a and 4c form lineage III; strains classified in this lineage appear to be rarely isolated from foods and human listeriosis cases [106]. Interestingly, subtyping efforts have also shown that the serotype 4b strains responsible for human listeriosis outbreaks can be grouped into three “epidemic clones,” each of which has caused at least two large human listeriosis outbreaks [28,103,109]. The three subtypes associated with multiple human listeriosis outbreaks, include ribotype DUP-1038B, linked to outbreaks in Anjou (France, 1976), Nova Scotia (Canada, 1981), Los Angeles (United States, 1985), and Switzerland [106]; ribotype DUP-1042B, linked to outbreaks in Boston (United States, 1979), Massachusetts (United States, 1983) [106], and ribotype DUP-1044A, linked to two outbreaks in the United States in 1998 to 1999 and in 2002 caused by consumption of contaminated hot dogs [6] and sliced turkey [8], respectively. These epidemic clonal groups have also been confirmed by other subtyping methods [109,148]. With the large diversity of molecular subtypes observed among L. monocytogenes strains from various sources, it is noteworthy that the larger outbreaks have almost all been attributed to lineage I strains and the three clonal groups described in the preceding paragraph. Investigations of many of these outbreaks have included or precipitated investigations to identify the origin and distribution of these outbreak strains. For example, Norton et al. found that the epidemic clones represented by ribotypes DUP-1038B, DUP-1042B, and DUP-1044A were also present in smoked-seafood processing plants not linked to any of the outbreaks caused by these strains [143]. A study using MEE for subtype differentiation showed that the electrophoretic type (ET) of the serotype 4b strain that caused the soft-cheese outbreak in Switzerland was also widely distributed in bovine milk, bovine feces, minced meat, silage, and soil as well as in human and animal clinical cases [24]. Although MEE typing reportedly has good discrimination for serotype 1/2 isolates, MEE may not adequately discriminate between serotype 4b strains [80], such as the strain responsible for the Swiss listeriosis outbreak. For example, Donachie et al. [49] showed that PFGE typing could further differentiate serotype 4 isolates of the same ET obtained from a variety of sources and that all but one of the human isolates could be grouped into exclusive human PFGE types. Thus, the data on prevalence of outbreak-associated clonal groups always need to be evaluated carefully along with the discriminatory power of typing methods used. In a retrospective subtyping study of clinical, environmental, and food-processing plant isolates from the 1985 Mexican-style soft-cheese outbreak in Los Angeles [116], Wesley and Ashton [200] showed that the strain isolated from clinical specimens was also recovered from samples of curd,

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the pasteurizer, cooler water, a floor drain, and insects caught in the factory. The widespread distribution of this strain in the plant environment was linked to poor hygiene in the plant. McLauchlin and Nichols [131] also proposed a relationship between poor hygiene, as measured by total viable bacterial counts and the presence of Listeria spp. in 4,405 samples of seafood. A similar relationship between food-processing hygiene and Listeria was shown when paˆ té samples were tested in an outbreak in the U.K. [73]. Poor processing plant hygiene likely increases the risk of L. monocytogenes persistence and survival in food-processing environments as well as the risk of consistent introduction of L. monocytogenes from natural environments into the processing plant with subsequent possible contamination of the foods produced. In 2000, a human listeriosis outbreak in the United States was linked with consumption of delistyle sliced turkey [7] and provided intriguing information on the ecology of L. monocytogenes. The unique L. monocytogenes subtype responsible for this outbreak was also responsible for a single listeriosis case linked to consumption of hot dogs produced in the same facility in 1988 [14]. This indicates that this subtype persisted for more than 10 years in this facility [109], while maintaining its ability to cause human disease. In conclusion, subtypes and clonal groups linked to human listeriosis outbreaks may be widely distributed in many environments, including food-processing and natural environments (see fig. 2.1). It thus appears that epidemic L. monocytogenes clones do not represent highly host-adapted subtypes, but rather have maintained their ability to survive in various environments. Most human listeriosis outbreaks likely follow one of two scenarios. In scenario 1, errors in food handling (e.g., poor hygiene) lead to high levels and/or high prevalence of contamination of one or a few lots of foods from a given producer, which subsequently cause infections in multiple susceptible individuals. In scenario 2, a highly virulent L. monocytogenes strain, which persists in a foodprocessing facility, contaminates multiple lots, possibly over days, months, and years with possible widespread occurrence of human infections (depending on food distribution) [181]. In scenario 1, the immediate source of L. monocytogenes may include the natural environment or the foodprocessing plant environment; in scenario 2, the food-processing environment will be the immediate source (even though the original source responsible for introduction of a given subtype in a plant is usually not known and may include the natural environment). Although most “outbreak strains” thus appear to have the ability to survive in natural environments, the immediate source of many human listeriosis outbreaks is likely to be the processing plant environment.

Ecology of L. monocytogenes Strains Associated with Sporadic Human Listeriosis Cases Although a number of listeriosis outbreaks have been reported, most human listeriosis cases likely represent sporadic cases (and possibly small outbreaks) caused by a wide variety of L. monocytogenes strains [130,167]. Similar to the findings described for epidemic outbreaks, most sporadic cases appear to be caused by strains representing serotypes 4b and 1/2b, which can be grouped into L. monocytogenes lineage I [202,204]. For example, using L. monocytogenes isolates collected over a 30-year period from human listeriosis cases and foods, McLauchlin showed that the serotype distribution among human listeriosis cases differed from that among food isolates [130]. Characterization of human isolates showed that serotype 4b was most common (60%), and serotypes 1/2a (17%), 1/2b (11%), and 1/2c (4%) were less common. Among food isolates, serotype 1/2a was most common (32%), and serotypes 4b (22%), 1/2b (15%), and 1/2c (21%) were less common. Similarly, Norton et al. found a significantly higher proportion of human isolates (69.1%) than isolates from three smoked-seafood processing plants classified as lineage I; lineage II strains were more common among smoked seafood processing plant isolates [144]. Jeffers et al. [106] also showed that lineage I strains (which include serotypes 4b and 1/2b) were significantly more common among isolates from human clinical cases as compared to isolates from animal cases. In conjunction

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with tissue culture studies, which showed that lineage I strains formed larger plaques as compared to lineage II strains [144,204], these data may indicate that serotype 4b and/or lineage I strains may be characterized by increased human virulence as compared to some or all lineage II and or serotype 1/2a strains. The latter may have increased environmental survival capabilities or may have adapted to survival in foods and the food-processing environment. Although some comparative studies on subtype diversity of environmental and food L. monocytogenes isolates and diversity of human sporadic cases are available, most reports have focused on comparing L. monocytogenes strains associated with human sporadic cases with strains isolated from food samples and food-processing plants [10,24,142,144,182]. For example, Norton et al. [144] specifically compared the frequency of L. monocytogenes strains (as determined by EcoRI ribotyping) isolated from three smoked-seafood processing plants with their frequency among human disease associated isolates. There were significant differences in distribution of ribotypes among human and food isolates; however, 14 ribotypes isolated from samples collected in the three smoked-fish processing plants were also represented among human clinical isolates. Only extremely limited data are available on frequency of sporadic case-associated strains in the natural environment. For example, Arimi et al. [10] reported that human clinically associated ribotypes were also found among samples collected from silage (four ribotypes) and dairy processing plant environments. Although surveys of L. monocytogenes subtypes associated with human and animal clinical cases, foods and food-processing facilities, and natural environments provided information on the associated L. monocytogenes strain diversity, subtyping alone does not provide information on the virulence potential of different L. monocytogenes subtypes and strains. Virulence mechanisms of L. monocytogenes are well described in Chapter 5 of this book; however, much of our understanding comes from the intensive study of relatively few L. monocytogenes strains. Surveys of virulence-associated characteristics of clinical and/or environmental L. monocytogenes strains have been performed using mouse bioassays, a chicken embryo test, and/or characterization in tissue culture assays. Most comparative mouse virulence studies of human clinical and food isolates have not identified consistent differences between human clinical and food isolates, but have shown considerable strain variation in virulence [109]. Some studies have revealed apparent differences between human clinical and food isolates though, including a study using the chick embryo test [141]. Characterization of selected L. monocytogenes isolates from food-processing environments representative of different ribotypes showed that some of these environmental isolates had an impaired ability to invade and/or replicate in tissue culture cells, indicating that at least some L. monocytogenes subtypes present in food-processing environments may have limited human-pathogenic potential. Similarly, other groups have also shown that some food isolates are virulence attenuated in mouse models of infection [27,33,109]. Based on these findings, it appears appropriate to hypothesize that a considerable proportion of L. monocytogenes present in natural environments is likely to have the ability to cause human and/or animal infections, although some strains found in natural environments may be virulence attenuated. Further studies using appropriate animal and tissue culture models to characterize L. monocytogenes isolates are required to understand the virulence potential of the L. monocytogenes strains found in natural environments. Although Chapter 4 summarizes current knowledge on human fecal carriage of L. monocytogenes, it is important to consider human fecal shedding and carriage in the ecology of L. monocytogenes. Early studies reported a relatively high percentage (up to 62%) of positive fecal specimens from human volunteer surveys; however, these studies involved cohorts most likely exposed to higher levels of environmental L. monocytogenes, including slaughterhouse and farm workers. Recent work has shown that carriage rates among healthy humans may be much lower than was previously assumed. Grif et al. show the rate of carriage among randomly selected human volunteers to be very low using culture-based methods (0.2%) [86], although a slightly higher percentage of specimens tested positive using PCR (3.6%). In another study, Grif and colleagues followed three human subjects longitudinally for a 1-year period for L. monocytogenes by PCR detection,

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standardized culture, and IMS culture [87]. In this study they found that each volunteer intermittently shed L. monocytogenes serotypes 1/2a or 1/2b with no coincidental overt clinical disease. The three volunteers in this study averaged two episodes of shedding over a maximum 4-day period [87]. Patients with diarrheal listeriosis manifestations also shed L. monocytogenes [40,69] and there are also some indications that humans with clinical listeriosis symptoms may shed L. monocytogenes in their feces [168]. Although it is likely that fecal shedding in humans with and without clinical symptoms contributes to dispersal of L. monocytogenes, including into the natural environment, the relative importance of this dispersal mechanism for the ecology of this organism remains to be determined. Overall, a variety of studies have established that a diversity of L. monocytogenes subtypes are responsible for sporadic human listeriosis cases. Many or some of the human clinical diseaseassociated subtypes have also been isolated from food-processing and natural environments. Because only very limited subtype studies on L. monocytogenes in the natural environment have been published, we can only extrapolate from studies on L. monocytogenes subtypes in processing plants as to the presence and survival of human disease-associated L. monocytogenes subtypes in the natural environment. It appears likely that subtypes associated with human sporadic cases are present and also survive and multiply in the natural environment. The natural environment may thus serve as a source (although not necessarily a reservoir) of these pathogenic strains, but it is unlikely that the environment serves as a direct source for human infection. Rather, natural and farm environments are likely to serve as sources of L. monocytogenes subtypes introduced into food-processing environments. Subsequent food contamination by L. monocytogenes introduced from environments outside a processing plant and multiplication in contaminated RTE refrigerated foods may then provide L. monocytogenes numbers required for infection. There is evidence that the L. monocytogenes populations causing human disease and the populations present in natural environments overlap; however, some observations also support the hypothesis that specific L. monocytogenes strains may be predominantly environmental [24,63,144,182] and have limited ability to cause human disease.

NATURAL ENVIRONMENT

AND

TRANSMISSION

OF

LISTERIA

IN

ANIMALS

As outlined earlier, listeriosis infections in farm animals and particularly in cattle and other farm ruminants (e.g., goats and sheep) are often linked to consumption of contaminated silage. In addition, animal listeriosis cases sometimes occur in animals that are not fed silage and environmental sources have been speculated to be responsible for at least some of these cases [108,208]. The agricultural environment thus may serve as an important source for contamination of silage and may also be a direct source of animal infection in some cases. As also summarized in one of the previous sections, a variety of studies have found a considerable diversity of L. monocytogenes subtypes in feed (primarily silage) samples as well as in other environmental samples collected on farms with listeriosis cases in the animals kept on a given premise. Linking presence of a specific L. monocytogenes subtype in feeds to animal infections may be challenging due to the possibly long incubation periods for animal listeriosis [120,207]. Nevertheless, in many case and outbreak investigations, identical L. monocytogenes strains have been recovered from animals with clinical disease and from silage fed to them [65,120, 188,190,200,203,205]. However, silage may contain a diverse array of L. monocytogenes strains [63,162,190,205]. Although animals may thus be exposed to multiple L. monocytogenes subtypes, only a single subtype generally appears to be isolated from a given animal with clinical symptoms, even if multiple specimen types are tested (e.g., blood and cerebrospinal fluid). Interestingly, although in some outbreaks multiple distinct L. monocytogenes subtypes were responsible for multiple animal cases that occurred on a single farm at a given time [17,118,205], in others a single subtype was responsible for all cases observed [17,118,203]. In a survey of PFGE types among L. monocytogenes

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isolated from clinical samples and feedstuffs in 21 flocks of sheep, Vela et al. also showed that, in most cases, clinical strains from different animals of the same flock had identical PFGE types and L. monocytogenes strains with PFGE types identical to those of clinical strains were isolated from silage, potatoes, and maize stalks [190]. These studies indicate that listeriosis epizootics may differ with regard to the causative strains and their spread. In some epizootics, a single, possibly highly virulent, strain may be responsible for infection of all involved animals, whereas in others distinctive strains (possibly with lower virulence and/or transmission potential) may be responsible for individual infections [203]. In many of the listeriosis outbreaks in farm animals a considerable proportion, and sometimes all, of the L. monocytogenes subtypes found on a given farm were only isolated from environmental samples and not from infected animals [17,118]. These findings could indicate that some environmental isolates may have a limited ability to cause disease and thus further supports that L. monocytogenes strains may differ in their virulence characteristics. In addition to clinical cases, L. monocytogenes has been found in the feces of a wide variety of healthy animal species. Gray and Killinger [83] listed 37 mammals from whose feces the organism had been isolated. Given that L. monocytogenes is distributed so widely on vegetation in nature, it is not surprising that its presence in fecal materials from grazing animals such as healthy sheep [88,120,172], goats [117,210], and cattle [62,94,210] is well documented. However, presence of L. monocytogenes in fecal materials also has been reported for pigs [52,152], chickens [15,81,136], turkeys [19,90,136], pheasants [171], gulls [58], rooks [58], pigeons [150], fish [11], and crustaceans [11]. L. monocytogenes appears to be a food-related pathogen; naturally occurring listeriosis has been recorded in many other animals, including mice [172], voles [139], rats [172], rabbits [139], guinea pigs [153], chinchillas [66], lemmings [150], hyraxes [150], mink [111], skunks [153], horses [197], dogs [178], cats [139], foxes [139], deer [139], buffalo [52], giraffes [38], bats [96], ducks [150], partridges [150], eagles [150], parrots [150], canaries [150], starlings [111], frogs [26], turtles [26], ticks [5], and flies [5]. Thus, it is reasonable to suspect that these animals also excrete the organism in their feces. Various reports support that the diet of healthy animals may have a considerable effect on excretion of L. monocytogenes. For example, Low et al. [120] reported a low incidence of L. monocytogenes excretion in a flock of 100 grazing sheep. Similar to previous findings by Husu [99] that the tendency is for L. monocytogenes excretion rates to be lower in grazing animals, the prevalence of L. monocytogenes in fecal samples increased significantly (P < 0.00001) to between 10 and 33% once silage feeding commenced. Phage typing of fecal isolates from the 100 sheep fed silage revealed 10 different phage types as compared to 6 phage types identified among the isolates from the silage fed to these animals. Several strains present in feces were absent from silage. Sheep consume large amounts of silage (indeed, the authors noted that a single sheep may consume 100 to 1000 times more silage than the amount of silage tested for Listeria); thus, Listeria strains present in excreta may be more likely to be representative of the total silage population. A report by Fenlon et al. [63] also supports that grazing animals show a lower prevalence of L. monocytogenes in fecal samples as compared to silage-fed animals. While grazing, none of the cattle tested (n = 10 and 13) among two groups of animals excreted L. monocytogenes. When tested after silage feeding commenced, 4 of 14 (28.6%) in one group and 4 of 13 (30.8%) in the other excreted L. monocytogenes. Numbers of Listeria in the excreta were low, ranging from present in 25 g to 11 CFU/g. In the same study, examination of feces of sheep on a hay diet showed no detectable Listeria, presumably because the moisture level in hay is too low to support Listeria growth. Studies on fecal and litter samples from other food animal species, such as chickens and pigs, generally found low prevalence of L. monocytogenes and Listeria spp. [63]. In a larger study on the presence of Listeria spp., Dijkstra [46] examined the intestines of 2,373 broilers from 146 farms and showed that 4.1% were contaminated with Listeria. Husu et al. [100] noted that most 2-day-old chicks dosed orally with L. monocytogenes had eliminated the organism within 9 days, indicating that chickens are unlikely reservoirs of the organism and that any carriage

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is probably transient. Overall, it thus appears that L. monocytogenes can be isolated from many farm animals, although the organism appears most prevalent in silage-fed ruminants. In addition, shedding of L. monocytogenes in ruminant stool can occur in higher numbers when animals are subjected to stressful conditions (see Chapter 4 for a comprehensive discussion). Clearly, L. monocytogenes appears to be widely distributed in agricultural environments and silage, and infection of farm ruminants appears to be more common than infection of many other animal species. Oral infection appears to be the most common route of infection for ruminants, although alternative transmission pathways have sometimes been suggested [194]. Although L. monocytogenes is also often found in fecal material collected from farm ruminants, particularly those fed silage, it is not clear whether farm animals can be true carriers or whether presence in fecal materials generally represents a transient “pass-through” phenomenon. Although we have developed some understanding on the sources of animal listeriosis infections, the overall transmission dynamics of L. monocytogenes in farm environments is not well understood. For example, although it is likely that clinically apparent or unapparent animal infections are critical to L. monocytogenes survival in farm environments, one might also hypothesize that presence of manure (and other nutrient-rich environments) alone, even in the absence of infection, may be sufficient to allow for L. monocytogenes survival and multiplication. Detailed longitudinal studies of on-farm transmission of L. monocytogenes combined with mathematical modeling efforts will be required to truly understand the ecology and transmission of L. monocytogenes in farm animals and farm environments.

TRANSMISSION

OF

LISTERIA

WITHIN

NATURAL ENVIRONMENTS

A variety of efforts have been made to better understand transmission of L. monocytogenes in farm animals and farm environments and from foods to humans; however, only very limited data are available on the transmission and spread of L. monocytogenes in natural environments, including infection of wild mammals and nonmammalian or invertebrate hosts. Based on a variety of case reports (see preceding discussion) that indicate a broad host range for L. monocytogenes among predominantly captive and farm animals, it appears likely that wild animals may at least occasionally experience L. monocytogenes infections and clinical disease. In addition, we are only beginning to understand the complex dynamics of the relationships between microorganisms and between microorganisms and multicellular organisms. Aside from bacteria–bacteria interactions, protozoa play a large role in marshalling bacterial populations through predation and grazing. Because L. monocytogenes is an intracellular pathogen found in many different environments, it has been hypothesized that protozoa may provide a potential host for this pathogen similar to the intracellular human pathogen, Legionella pneumophila, which multiplies intracellularly in Tetrahymena thermophila [110]. Many other bacteria, including other human and animal pathogens, can survive within a variety of protozoan species [13a]. In preliminary studies, Ly and Müller demonstrated that L. monocytogenes may be able to survive and multiply within the protozoans Tetrahymena pyriformis and vegetative Acanthamoeba sp. [123,124]. It is thus possible that protozoa may represent a reservoir of pathogenic Listeria and serve to maintain virulence genes during environmental survival; it may be possible that Listeria-protozoan interactions played a critical role in evolution of Listeria intracellular survival mechanisms and virulence. It has also been hypothesized that protozoans or other alternate nonhuman hosts may serve as a host for L. seeligeri, which carries many of the typical virulence genes found in L. monocytogenes and L. ivanovii [78], but is not able to cause infections in animal or tissue culture models that have been successfully used to study L. monocytogenes pathogenesis [68,128].

DEFINING GLOBAL LISTERIA TRANSMISSION PATHWAYS

AND

RESERVOIRS

The natural environment harbors a highly diverse range of Listeria spp. and L. monocytogenes strains, including some with the potential to cause clinical listeriosis in humans and animals.

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The presence of these organisms in natural environments appears to be characterized generally by low prevalence and low numbers, although a high prevalence of positive samples and the presence of high numbers of organisms have been described in some instances and for some environments and sample types. Although it is apparent that the species within the genus Listeria are all widely distributed in nature and that food- and feedborne transmission is critical for human and animal infections, we have been unable to define the global Listeria transmission pathways and the true reservoirs of pathogenic and nonpathogenic Listeria species. Haydon et al. [91] define a reservoir as “one or more epidemiologically connected populations or environments in which the pathogen can be permanently maintained and from which infection is transmitted to the defined target population.” Although it seems possible that the farm environment (e.g., soil, plant materials, feed) may indeed represent a reservoir for some Listeria subtypes that can cause animal infections, it is unclear whether ruminants represent a true target population or whether Listeria could be permanently maintained in this environment without causing animal infections. It is conceivable that for human infections, the transmission cycle may draw on a wider dynamic, possibly involving farm and food-processing environments. Most evidence indicates that at the processing stage of food and feedstuffs (e.g., silage preparation) amplification of numbers or persistent contamination occurs, leading to potentially serious human and animal health hazards. Stringently enforced good manufacturing and hygiene practices in the food-production and foodprocessing environments are thus critical to prevent introduction of pathogenic Listeria spp. into the feed and food chain from environmental sources. In this context, it is noteworthy that poultry products [132,185] may be more commonly contaminated with L. monocytogenes than beef [55,115,175], even though the environment in which beef cattle are reared appears to show a higher prevalence of this organism than that of the intensively reared broiler chicken [63]. However, in processing, the chicken may be exposed to greater risk of contamination from other carcasses and mechanical equipment than beef carcasses [158]. In conjunction with many other data on L. monocytogenes in the food-processing environment, it thus appears that the environment of food-processing establishments represents a major direct source of L. monocytogenes found in foods destined for human consumption. In addition, some human listeriosis cases may be caused by direct introduction of L. monocytogenes into a food item from nonfarm and nonprocessing environments, but this contamination is likely to be of a low level and sporadic. Direct infection of humans from human environmental sources is rare; however, one listeriosis outbreak report from Costa Rica implicates bathing in mineral oil contaminated with a L. monocytogenes 4b strain as the source of illness in nine newborns [170]. Although some transmission cycles proposed here may seem compelling, very few data could be used to quantify and define the importance of different environments as sources or reservoirs of virulent strains of L. monocytogenes that can enter and pass along the food chain and cause human infections. For example, no quantitative data are available to be used to evaluate the true significance of L. monocytogenes present in farm animals or in farm environments as a direct or indirect source of human infections. Defining the reservoirs and transmission of L. monocytogenes thus represents a formidable challenge, and improved knowledge on the transmission pathways of this organism will be critical to improve our ability to reduce human listeriosis cases and outbreaks. Even more limited than our knowledge of the transmission of L. monocytogenes is our understanding of the ecology of other Listeria spp. This is evidenced by the fact that we have no indications as to the biological role of the virulence genes present in L. seeligeri, which does not cause human or animal infections in any known model systems [78]. Although food microbiologists often use presence of Listeria spp. as an indictor for the presence of L. monocytogenes, it is becoming increasingly clear that the physiology and ecology of L. monocytogenes and other Listeria spp. differ considerably. Further studies and data on the ecology of Listeria spp. other than L. monocytogenes will thus likely provide additional knowledge that will help us to understand the natural history and ecology of L. monocytogenes.

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In addition, development of large standardized repositories of Listeria subtyping data (such as http://www.pathogentracker.net) will allow comparative studies on the genetic diversity of Listeria and L. monocytogenes strains from human and animal disease, food and food-processing environments, and the natural environment. In combination with longitudinal studies and mathematical modeling efforts, these data will likely improve our understanding of the ecology and transmission dynamics of Listeria spp. in general and the human pathogen L. monocytogenes in particular.

ACKNOWLEDGMENTS We thank and acknowledge David Fenlon for allowing adaptation of his previous chapter from the second edition of Listeria, Listeriosis, and Food Safety for this edition. This work was supported in part by National Institutes of Health Award No. R01GM63259 (to M.W.). The Pathogen Tracker 2.0 online public subtyping database is supported by USDA Special Research Grants 2001-3445910296 and 2002-34459-11758 (to M.W.)

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204. Wiedmann, M., J. L. Bruce, C. Keating, A. E. Johnson, P. L. McDonough, and C. A. Batt. 1997. Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential. Infect. Immun. 65:2707–2716. 205. Wiedmann, M., J. L. Bruce, R. Knorr, M. Bodis, E. M. Cole, C. I. McDowell, P. L. McDonough, and C. A. Batt. 1996. Ribotype diversity of Listeria monocytogenes strains associated with outbreaks of listeriosis in ruminants. J. Clin. Microbiol. 34:1086–1090. 206. Wiedmann, M., J. Czajka, N. Bsat, M. Bodis, M. C. Smith, T. J. Divers, and C. A. Batt. 1994. Diagnosis and epidemiological association of Listeria monocytogenes strains in two outbreaks of listerial encephalitis in small ruminants. J. Clin. Microbiol. 32:991–996. 207. Wiedmann, M., and K. E. Evans. 2000. Infectious diseases of dairy animals: Listeriosis. In Encyclopedia of dairy science. London: Academic Press. 208. Wiedmann, M., S. Mobini, J. R. Cole, Jr., C. K. Watson, G. T. Jeffers, and K. J. Boor. 1999. Molecular investigation of a listeriosis outbreak in goats caused by an unusual strain of Listeria monocytogenes. J. Am. Vet. Med. Assoc. 215:369–371, 340. 209. Wilson, I. G. 1995. Occurrence of Listeria species in ready to eat foods. Epidemiol. Infect. 115:519–526. 210. Winkenwerder, W. 1967. The occurrence of Listeria monocytogenes in cattle in Niedersachsen. Berl. Münch. Tierärztl. Wochenschr. 80:445–449. 211. Yu, L. S., and D. Y. Fung. 1993. Five-tube most-probable-number method using the Fung–Yu tube for enumeration of Listeria monocytogenes in restructured meat products during refrigerated storage. Int. J. Food Microbiol. 18:97–106.

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3 Listeriosis in Animals Irene V. Wesley CONTENTS Introduction ......................................................................................................................................55 Incidence ..........................................................................................................................................56 Predisposing Factors ........................................................................................................................56 Transmission to Humans..................................................................................................................57 Transmission to Animals..................................................................................................................57 Sheep.......................................................................................................................................57 Goats .......................................................................................................................................60 Cattle.......................................................................................................................................62 Swine ......................................................................................................................................66 Fowl ........................................................................................................................................67 Minor Species.........................................................................................................................69 Fish and Crustaceans..............................................................................................................71 Treatment..........................................................................................................................................72 References ........................................................................................................................................73

INTRODUCTION Numerous animal species are susceptible to listeric infection, with a large proportion of healthy asymptomatic animals shedding Listeria monocytogenes in their feces. Although most infections are subclinical, listeriosis in animals can occur sporadically or as epidemics and often leads to fatal forms of encephalitis. Clinical listeriosis in livestock presents as encephalitis, septicemia and abortions during the last trimester of gestation. Virtually all domestic animals are susceptible to listeriosis; sheep [7,103,204,206,239, 285], cattle [103,204,208,217,226], goats [166,167,243], and, less frequently, birds [100,199,204,223] succumb to infection. Several comprehensive reviews have detailed the distribution and pathology of L. monocytogenes in food animals [7,56,102, 123,158,173,175,186,219,229]. Not all serotypes are pathogenic to livestock and may be divided into at least three lineages based on DNA profiles. Interestingly, isolates of lineage I predominate in human infections, whereas animal isolates cluster in lineage III [294,295]. L. monocytogenes enters the animal usually through ingestion. Following entry into the intestinal epithelium via M cells in Peyer’s patches or epithelial cells [215,229], a bacteremic or septicemic phase or latent infection may develop, depending on the immune status of the host. L. monocytogenes subsequently colonizes the viscera, gravid uterus, or medulla oblongata [152]. In pregnant animals, the organism can localize in the placentomes and enter the amniotic fluid. The fetus aspirates the pathogen, which multiplies and kills the fetus late in gestation [267]. A single flock may experience abortion, septicemia, and encephalitis [176].

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INCIDENCE Listeriosis in domestic livestock is recognized worldwide despite significant differences in climate and livestock management practices [101,279]. In addition, most infections in livestock are subclinical and therefore go undiagnosed [152]. Because listeriosis is not a reportable disease, the exact incidence of infections in domestic livestock remains unknown, thus precluding comparison of prevalence data between countries [221]. Livestock losses attributed to L. monocytogenes may be substantial. During the early 1970s, the agricultural economies of Australia and Norway were adversely affected by the loss of approximately 1 million and 2,000 to 2,500 sheep, respectively, from listeric infection. Before a vaccine became available and lowered the incidence of listeriosis, infections in Norwegian livestock remained relatively constant with approximately 1,900 to 2,300 sheep herds, 90 to 160 goat herds, and 3 to 17 cattle herds affected during the 10-year period between 1977 and 1986 [304]. In the United Kingdom, approximately 30,000 ovine abortions occur annually with 0.05 to 0.13% attributed to listeriosis [7]. In The Netherlands, between 1970 and 1985, the annual percentage of bovine abortions attributed to L. monocytogenes in cattle ranged between 0.7 and 8.7%, with an average of 3.2%, which represented between 234 and 928 cases [69]. Prevalence studies may be based on microbiological surveys of healthy cattle as well as serosurveys. In a 1995 survey of domestic and companion animals in Germany [288], L. monocytogenes was found in fecal samples of healthy bovines (33%), sheep (8%), birds (8%), pigs (5.9%), horses (4.8%), and dogs (0.9%). In addition to relatively small numbers of acutely infected sheep, goats, and cattle, substantially larger proportions of animals within a herd may be asymptomatic carriers of L. monocytogenes and shed the organism in feces and milk [221,239]. The role of the symptomless carrier was clearly demonstrated in another report in which 30 of 44 listeriosis outbreaks on sheep farms involved introduction of clinically healthy animals from known infected herds [239]. Although the humoral immune response may not be pivotal, serum antibodies are useful for serodiagnosis of Listeria carriage in healthy animals [27,29,192]. Sindoni et al. reported Listeria antibodies in apparently healthy sheep (18.5%), cows (10.4%), goats (17.3%), and swine (13.3%), suggesting previous exposure to L. monocytogenes [247]. Although these data estimate the distribution of L. monocytogenes infections in healthy carrier animals, they should be interpreted with caution because antibodies to a number of Gram-positive microbes may cross-react with Listeria antigens. In contrast, antibody titers to listeriolysin O (LLO) and actA proteins, which are regarded as virulence proteins, are a more specific indicator of infection and do not cross-react with other Listeria species [32]. LLO antibodies have been used to monitor a variety of livestock, including cattle, buffalo, sheep, and goats. To illustrate, in experimentally infected goats, an increase in LLO antibodies was correlated with rapid clearance of L. monocytogenes from the gastrointestinal tract and thus predicted a favorable outcome [193].

PREDISPOSING FACTORS A number of factors contribute to susceptibility to infection: season; immune status, such as pregnancy; on-farm feeding; and management practices, including feeding silage as well as breed of livestock species. Seasonal variation in the number of cases of animal listeriosis has often been observed. To illustrate, in the Northern Hemisphere (England, Bulgaria, Hungary, United States, France, and Germany), cases in domestic animals generally occur from late November to early May with the greatest incidence during February and March [2,102]. Consumption of contaminated silage contributes significantly to infection in livestock. For example, listeriosis cases increased when animals were fed silage during periods of extreme cold, whereas sharp decreases in numbers of reported cases were observed as soon as pasture was available. Data from The Netherlands [69] indicate that most cases of listeric abortion in cattle occurred between December and May. Approximately 40% of these cases were linked to consumption of

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contaminated silage. Recent changes in production methods have reduced levels of L. monocytogenes in silage, which in turn has decreased the incidence of listeriosis in silage-fed animals. Yet an increase in ovine listeriosis with conversion to big bale silage production has been documented in the United Kingdom [299]. Although in Norway listeriosis can be diagnosed year-round in sheep and goats, the illness is more prevalent from October to June and also appears to be influenced by housing and feeding conditions [221].

TRANSMISSION TO HUMANS Transmission of L. monocytogenes from livestock to humans occurs by (1) direct contact with infected animals, especially during calving or lambing; and (2) consumption of contaminated raw milk. To illustrate, primary cutaneous listeriosis is regarded as an occupational disease of veterinarians and farmers who have attended deliveries of stillborn or aborting bovine fetuses [4,44,188,206,210,275]. A case of septicemia and ultimately fatal meningitis was reported in a Dutch farmer who developed cutaneous lesions after assisting in the delivery of a stillborn calf [197]. Apart from human infections acquired as a direct result of contact with infected animals or consumption of foods from an infected animal, the connections, if any, between human and animal listeriosis are unclear. The springtime peaks of animal listeriosis and the autumn seasonality of human cases suggest that cases are not causally related. The source of contamination for human foods and animal feed is usually environmental [173,187]. The rise in cases of human listeriosis may result, in part, from changes in food manufacturing, postprocessing contamination [173], and improved diagnostic methods. The WHO concluded that foodborne listeriosis is predominantly transmitted by nonzoonotic means and that L. monocytogenes is an environmental organism whose primary route of transmission to humans is via foods contaminated during production [293]. More specifically, sensitive molecular tools, such as multilocus enzyme electrophoresis and pulsed field gel electrophoresis, suggest that L. monocytogenes strains isolated from meat or raw milk may have originated mainly from the processing environment rather than animals [31,113]. However, it is possible that live animals brought into slaughter may introduce Listeria into the processing plant. Although an animal origin of contamination was inferred for the three major epidemics occurring in North America, in the absence of documented evidence, the role of direct animal involvement in human foodborne outbreaks remains speculative [290].

TRANSMISSION TO ANIMALS SHEEP Ovine listeriosis is caused by L. monocytogenes serotypes 1/2, 3, and 4 as well as by L. ivanovii [171]. Although “listeric-like” infections were previously observed in sheep [239], Gill is credited with the first isolation of L. monocytogenes from domestic farm animals [94]. In 1929, he observed an illness in sheep in New Zealand that he called “circling disease.” This name is still used today to describe listeric encephalitis, encephalomyelitis, and meningoencephalitis [239]. Attack rates are higher in sheep (up to 30%) than in cattle (up to 15%), suggesting the greater susceptiblity of ovines to listeriosis [173,234]. As in other livestock species, the clinical manifestations of ovine listeriosis are (1) encephalitis; (2) placentitis, with abortions occurring in the last trimester [103,140,178,280]; and (3) gastrointestinal septicemia with hepatitis, splenitis, and pneumonitis [154]. Encephalitis is the most common form diagnosed in sheep [152]. Lambs as young as 5 weeks may develop septicemia with older feedlot lambs (4 to 8 months) manifesting encephalitis. All sheep are probably exposed to the same contaminated feed, indicating a high natural resistance with the total of 5 to 10% of exposed animals exhibiting clinical signs [152]. Meningoencephalitis caused by L. monocytogenes is the most common bacterial infection of the central nervous system of adult sheep [237]. After an incubation period of approximately

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3 weeks, clinical symptoms of ovine encephalitis appear. These include elevated temperature and refusal to eat or drink followed by neurological disturbances, which include grinding of teeth, paralysis of masticatory muscle, and excessive salivation caused by the animal’s inability to swallow resulting from damage to the cranial nerve. At this point the animal moves in circles to the right or left, depending on the direction in which the head is bent. This characteristic movement accounts for the name “circling disease.” In advanced stages, muscular incoordination develops and is followed by inability of the animal to walk. Death usually occurs within 2 to 3 days after onset of clinical symptoms, with the illness seldom lingering beyond 10 days [239]. In the brainstem, Listeria antigens are characteristically variable but always sparse. Perivascular microabscesses with L. monocytogenes and microgranulomas in histopathologic specimens of brain stems are characteristic. T lymphocytes (CD8+ and CD4+) and B lymphocytes contribute to the inflammatory process [157]. Direct entry via abrasions or lesions of the buccal mucosa, lips and nostrils, or conjunctiva may lead to encephalitis. Because entry into dental terminals of the trigeminal nerve in sheep can cause an ascending neuritis and encephalitis [49,50], listeric encephalitis is most common in winter and early spring in sheep that are losing and cutting teeth [17]. L. monocytogenes penetrates the buccal epithelium, accesses endings of the trigeminal (V) and hypoglossal (XII) cranial nerves, and enters the brain stem where replication and dissemination to the medulla and pons occur. In severe cases, respiratory failure and death follow within 1 month of infection [152,171]. Abrasions of the eye by contaminated silage lead to ophthalmitis without any other clinical manifestations [283]. After ingestion and hematogenous spread to the gravid uterus, L. monocytogenes appears within 48 h in the amniotic fluid and fetus [171]. Listeric infections in pregnant sheep often result in premature birth and infectious abortions [103,178,280]. This seldom occurs concurrently with encephalitis [131]. Initially, pregnant ewes contract purulent metritis, from which most recover. Intrauterine transmission of L. monocytogenes results in septic infection of the fetus, which in turn gives rise to abortion or premature delivery; most fatalities occur as stillbirths. Clinical symptoms are resolved following expulsion of the fetus, after which the ewes recover. Morbidity in ewes ranges from 1 to 20% [272] with mortality of lambs usually high [154]. Experimental injections of pregnant ewes with L. monocytogenes led to abortion in 10% of inoculated animals and retardation of bone growth in lambs [98]. Septicemia is most frequent in neonates and lambs and follows 2 to 3 days after oral infection, though congenital and navel infection can also occur [171]. Septicemia is characterized by an elevated temperature, loss of appetite, and diarrhea. Although death may eventually occur as a result of extensive liver damage and focal pneumonia, the mortality rate is much lower for the septicemic than for the encephalitic form of listeriosis. Most importantly, silage feeding, followed by season, breed, and on-farm management practices, and stress caused by overcrowding, contributes to ovine listeriosis. The quality of silage may influence the incidence of ovine listeriosis [82]. In an outbreak of ovine listeriosis in the United Kingdom, the D value of silage, which measures digestibility, was below the optimum of 65 to 70% [299]. In Scotland, listeriosis caused by serotype 1/2 occurred in sheep that were reluctant to eat poor-quality silage [176]. Silage analysis indicated pH > 4.0 and a high ash content reflecting soil contamination. Despite antibiotic treatment, 19 ewes died; more than 60 developed vaginal discharges and at lambing 94 were barren [176]. A British study linked silage feeding and development of ovine listeric encephalitis (relative risk of 3.8). The excretion of L. monocytogenes by sheep was linked to diet: animals fed entirely on hay or manufactured diets did not excrete detectable levels of L. monocytogenes; animals fed on silage commonly excreted the organism [297]. An outbreak in a flock consisting of 450 sheep (attack rate 11.8%) with a case fatality rate of 94.3% was traced to feeding poor-quality silage (pH 7.8) that was highly contaminated (106 L. monocytogenes/g) [272]. The serovar and phagovar of the L. monocytogenes strains isolated from two silage samples and the brains of 3 of the 53 affected sheep were indistinguishable [272]. Another outbreak, with a mortality of 2%, in a flock

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of 650 sheep was traced to contaminated silage. The rise and fall of cases coincided with the silage feeding practices [297]. Whereas the incidence of L. monocytogenes in sheep and cattle has declined in The Netherlands (attributed in part to the change of silage production), in Great Britain modifications in silage production may have led to an increase in ovine listeriosis [96]. To illustrate, in 1975, listeriosis was reported in a modest number of cattle (n = 37) and sheep (n = 44). By 1984, sheep listeriosis cases had increased (n = 269) with little change in the number of bovine listeriosis cases [96]. In parallel with the rise in the incidence in the United Kingdom there was also a change in the disease pattern, with encephalitis and abortion occurring in the same flock [96,176]. Ovine listeriosis occurs most frequently in late autumn, winter, and early spring. Stress factors, such as abrupt changes in feed, introduction of new animals into the flock and resultant overcrowding, concurrent disease, changes in dentition, and physical or viral damage to the epithelial lining of the digestive tract may predispose to infection [151,152]. Climatic changes, such as heavy rains [303], especially following a drought, which may spoil feed [228], or periods of wintry cold weather, which may cause animals to be housed indoors [190,285], are also determinants. In Saudi Arabia, an outbreak occurred in a flock of 2,100 Naimi sheep during the winter rainy season with a resultant morbidity rate of 7.1% and mortality rate of 2.4%. Encephalitis was observed in pregnant ewes, but no abortions were seen. Sheep were grazed during the day and housed in the evening with access to grass rolls, which were most likely the source of infection [2]. A prospective study conducted on early lambing flocks in southwest England (1989–) monitored 4,413 lambs from birth to slaughter for listerial meningoencephalitis. Two of three flocks developed clinical disease attributed to serotype 1/2. Six weeks before lambing, ewes on the two affected premises were bedded in straw; ewes on the farm without clinical listeriosis were on softwood slats [104]. In the single flock with no cases, preventive measures after lambing included replacing bedding and silage daily and regular cleaning of the silage feeders, which were on concrete floors. In the affected flocks, silage was replaced on alternate days and the silage feeders were on soilbased floors and thus easily contaminated. Before weaning, the lambs were eating more silage because ewes were producing less milk. Although all lambs were exposed to these risk factors, only an estimated 1.3% developed clinical infection and death occurred in 0.56% (21/4413) of lambs from 4 to 32 days after weaning [103]. Multiple strains of the same or different serotypes may be isolated in an outbreak in the same flock. When isolates of the same serovar are recovered from a single outbreak, they may be further differentiated by DNA fingerprinting, phage typing [10], or pyrolysis mass spectrometry [172]. To illustrate, DNA fingerprinting by random amplified polymorphic DNA (RAPD) analysis and ribotyping affirmed the identity of L. monocytogenes strains from silage, farm equipment, and sheep brain [296,298]. In contrast, examination of outbreaks of ovine listeriosis in Scotland indicated multiple strains of L. monocytogenes serovar 1/2 recovered from the silage incriminated in the outbreak. By multilocus enzyme electrophoresis (MEE), the L. monocytogenes strains for each of the affected animals in an outbreak were identical. Yet by MEE, none of the strains from the silage matched those recovered from brains. This could reflect bias during sampling from the bales, thus missing a small virulent population of L. monocytogenes in vegetation which, upon entry and replication into the ovine host, became the dominant strain [21]. Reports on the incidence of L. monocytogenes in ewes’ milk are limited. In Spain, L. monocytogenes was present in 2.2% of 1,052 ewes’ milk samples representing 283 farms. Yet L. monocytogenes was recovered from 18% of milk tanker samples in Spain. Farm ewes’ milk samples indicated contamination by L. monocytogenes was significantly higher on premises where cows were also reared than on farms where only ewes were maintained [230]. The data suggest environmental contamination on farms from L. monocytogenes excretion in cows’ feces or common exposure to contaminated ensilage on the premises shared by sheep and cattle [230]. Interestingly, no seasonal variation in milk contamination was evident in that study.

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Although not widely practiced, most likely because of limited cost benefits, vaccination with live attenuated strains of L. monocytogenes may effectively reduce ovine listeriosis [109,110, 165,201,282]. In Norway, immunization of sheep with a commercially available live attenuated vaccine of serovars 1/2a and 4b lowered the incidence of listeriosis and abortions when compared with unvaccinated control farms [109,110]. In this study, half of the sheep in 70 flocks (total of 3,130 sheep) with a history of listeriosis received two attenuated strains of L. monocytogenes serotypes 1/2 and 4b, whereas the remaining half of the sheep served as unvaccinated controls [109]. Both groups of animals were then housed together in the same pens. Results of this study showed listeriosis incidences of 1 and 3% in vaccinated and unvaccinated sheep, respectively. The incidence of abortions was 0.7% in vaccinated compared to 1.1% in unvaccinated flocks [110]. In another study, an experimental polyvalent vaccine, consisting of serovars 1/2a and 4b, which were attenuated via metabolic drift mutations, was tested in sheep, lambs, and ewes [165]. The results of field tests indicated that vaccinated ewes delivered more lambs free of listeriosis (93.4 vs. 69.7%) and of higher birth weight (2.2 vs. 1.8 kg) than lambs from control unvaccinated ewes. In addition, L. monocytogenes was not isolated from milk samples of vaccinated ewes, in contrast to controls in which 32% of milk samples yielded Listeria [165]. Although vaccination produced few adverse side effects, economic constraints suggest that it should be restricted to flocks to achieve maximum cost benefit. Preferential breed susceptibility may occur [201]. In a survey conducted in the Midwest in which consumption of contaminated silage was a key factor, unvaccinated Rambouillet ewes were more at risk (odds ratio 4.6) than other ewes; yearling ewes were more at risk than older animals (odds ratio 4.1). Interestingly, although use of a bacterin did not decrease the risk of L. monocytogenes in Rambouillet ewes (odds ratio 0.8), it did among ewes of other breeds (odds radio 0.1). This indicates that the inherent susceptibility of Rambouillet ewes to L. monocytogenes cannot be modified by vaccination [201]. As in other livestock species, serosurveys have been used to estimate the occurrence of listeriosis in sheep [250]. However, antibodies to other Gram-positive microbes cross-react with Listeria antigens, thus giving rise to false positive results. Berche and coworkers [26] developed a highly specific test for L. monocytogenes based on detection of antibodies to listeriolysin O (LLO). Antibodies to LLO have been detected in lambs experimentally infected with L. monocytogenes [13,163,174]. Listeria ivanovii was first described in association with ovine abortion and accounts for up to 8% of all animal listeriosis cases [10,240]. Listeria ivanovii is a recognized cause of ovine abortions, stillbirths, and unthrifty lambs [36,62,126,133,177,178,186]. Interestingly, goats may be resistant to L. ivanovii. To illustrate, L. ivanovii and L. monocytogenes were reported in two migratory flocks of sheep and goats in Himachal Radesh, India. Whereas L. monocytogenes was isolated from sheep and goats, L. ivanovii was cultured only from sheep [244]. Factors that predispose livestock to L. ivanovii abortions are similar to those described for L. monocytogenes: lowering of ewes’ resistance to infection following general immune suppression as well as to nutritional stress, periods of cold and wet weather, feeding of poor-quality silage, and exposure to carrier animals [73,95,219]. Outbreaks of L. ivanovii have been reported to affect up to 45% of pregnant ewes [133]. One outbreak in New South Wales involved a total of 110 animals that aborted or died shortly after birth. Heavy grazing by sheep (120 sheep per hectare for 18 days) on pastures cut for hay that had not been baled and had spoiled because of heavy rains was presumed to be the source of initial contamination of L. ivanovii [242]. Multiple hepatic foci were seen in aborted lambs. In addition, L. ivanovii was cultured from fetal liver, lung, and stomach contents [242]. A report of bovine abortions caused by L. ivanovii associated the grazing of cattle on pastures previously used by sheep [3].

GOATS Clinical listeriosis in goats as in sheep presents as encephalitis, septicemia, and abortion. [37]. As is true for other livestock species, asymptomatic infections also have been noted in goats [239].

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After ingestion, L. monocytogenes penetrates the intestinal tract, sets up a transient bacteremia and spreads to the central nervous system, viscera, or placenta. Depression, loss of appetite, a drop in milk yield, and elevated body temperature (up to 41°C) are the first indications of septicemia. The animals may have diarrhea [107]. In the pregnant doe, L. monocytogenes may penetrate the placenta and enter the fetus, where it replicates and causes late-term abortion. In experimental studies in pregnant goats, localization of L. monocytogenes in the placenta led to an elevation in prostaglandin F2 and a decline in progesterone levels. A slight decrease in secretion of estrone sulfate by the fetal-placental unit prompted myometrial contraction and abortion [75]. As with other species of livestock, goats excrete Listeria in feces and milk during and after septicemia and may contaminate the environment. Thus, newborn kids housed with does may be infected through the navel or through sucking on soiled teats [108]. Meningoencephalitis is the most frequently reported form of listeriosis in goats with fatalities achieving 60% [107]. Early signs of listerial encephalitis mirror lesions to the respective cranial nerves: drooping ears, marked drooling, tongue protrusion, and cud retention because of difficulty in swallowing [37]. Goats may be more susceptible than sheep to listeric encephalitis based on the severity of brain damage in goats [16]. In an outbreak of encephalitis and abortion in a mixed herd of goats and sheep in Iraq, the morbidity (30 vs. 16.7%) and mortality (21 vs. 15%) was higher in goats than in sheep [303]. Heavy rains, cold weather, and susceptibility of pregnant animals may have contributed to high numbers of encephalitis cases [303]. More frequent recovery of L. monocytogenes serotype 1/2b from goats than from sheep has also been documented in Sudan [246]. Listeriosis is linked to the practice of feeding silage [167,171]. However, in a study of 355 goat herds in Missouri, encephalitic listeriosis was correlated with browsing on woody plants and location of the herd in areas with a preponderance of alkaline soil [141]. Heavy browse consumption may have abraded oral tissues followed by penetration by L. monocytogenes into the dental pulp or buccal cavity, which may have led to encephalitis [141]. Although it is not widely practiced, vaccination has limited the number of goat listeriosis cases [65,87,201]. Before vaccination of goats in Norway with a live attenuated strain, the abortion rate in the test herd was 20 to 25%. After vaccination, the incidence rate of abortions decreased to 3% [155]. The optimal time for vaccination of goats as well as sheep may be shortly before mating season [155]. Few studies describe distribution of L. monocytogenes in raw goats’ milk [1]. Gaya et al. cultured L. monocytogenes from 2.6% of 1,445 samples from 405 Spanish goat milk farms located in mountain and plateau regions [92]. Overall, recoveries were higher in the autumn (5.7%) than in the winter (4%) or in spring (0.3%). Interestingly, the higher recovery was traced to farms located on the plateau where goats graze on stubble and germinating vegetation and thus consume more soil in autumn. A seasonal trend was not apparent in goats on mountain farms where vegetation was available throughout the year, thus minimizing soil consumption [92]. In one report [105], L. monocytogenes was detected in 3.6% of raw milk from cows with considerably lower recovery of L. monocytogenes from raw milk from goats (1%) and ewes (2%). Yet in a comparable study of 450 raw goat-milk samples from 39 goat farms in the United States, the 3.8% Listeria isolation rate paralleled earlier data for cows’ milk [1]. In addition, a seasonal trend was noted with isolations higher during winter (14.3%) and spring (10.4%) versus autumn (5.3%) and summer (0.9%) [2]. In Scandinavia, summer visits to local farms resulted in a small cluster of human listeriosis cases resulting in febrile gastroenteritis linked to consumption of cheese [46]. Listeria concentrations in cheeses produced from goat’s milk (~106 CFU/g) and blended milk (~106 CFU/g) were notably higher than in cheeses manufactured from cow’s milk (5 × 102 CFU/g). This may represent the higher concentrations shed in goats versus cows or the result of bacterial replication during refrigeration of the cheese. Goats have been linked to human listeriosis based on DNA fingerprinting. Specifically, an isolate of L. monocytogenes serovar 1/2b from the brain of a goat with listeriosis exhibited the identical DNA profile by ribotyping as an isolate from cheese made from that goat’s milk and an

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isolate from the refrigerator in which the cheese was stored. This suggested that the cheese made from the infected goat’s milk may have contaminated refrigerator shelves, thus serving as a reservoir for L. monocytogenes [72]. In contrast, a human endocarditis fatality with a recent history of exposure to goats showed that the isolates from humans and animals were of the same serogroup. However, the goat and human strains were clearly different via DNA analysis, thus reducing the likelihood of zoonotic transmission [58].

CATTLE The distribution of L. monocytogenes undoubtedly reflects the available pool of susceptible animals. In North America, most listeriosis cases occur in cattle (82%); a smaller percentage is found in sheep (17%) and an even lower percentage in pigs [22]. From 1993 to 2000, listeriosis cases (n = 346) submitted to the Iowa State University Veterinary Diagnostic Laboratory [292] were from cattle (85.8%), sheep (9.5%), goats (3.8%), a llama (0.3%), and two horses (0.6% each). In Missouri [143], from 1986 through 1994, encephalitic listeriosis cases were from cattle (67%), goats (30%), and sheep (13%). In Iowa, 88% of listeriosis cases were diagnosed as encephalitis [292]. In marked contrast, from 1975 to 1984, listeriosis cases in Great Britain were from sheep (63%) and cattle (32%), with pigs, goats, fowls, and other species constituting less than 1% each of the total submissions [299]. The symptoms of bovine listeriosis include encephalitis, abortion, and septicemia with miliary abscesses [144]. In 1928, Matthews detailed an outbreak of encephalitis of unknown origin in cattle which, in retrospect, was probably bovine listeriosis [181]. Since then, listeric encephalitis has been well documented [60,66,143,221,226,239]. However, even in acute outbreaks, generally no more than 8 to 10% of a herd succumbs to infection. In Switzerland, bovine listeriosis is the most frequently diagnosed neurological disease [120] and thus may be the most common cause of bacterial infection of the central nervous system in adult cattle [226]. Foodborne transmission is the main mode of infection in naturally occurring listeriosis in cattle with silage most frequently implicated. As in sheep, following ingestion, Listeria is disseminated via hematogenous spread to the viscera, brain, and gravid uterus. In addition, because L. monocytogenes is present in soil, fecal material, or other vegetation, it may enter via abrasions of the nostrils or the conjunctiva while grazing or via the teat of a lactating cow [139]. Direct injection of the conjunctiva, resulting in keratoconjunctivitis [214], has occurred as a result of contaminated silage particles abrading the faces of browsing cattle [194]. By travel along peripheral nerves, especially the hypoglossal (XII) and trigeminal (V) cranial nerves innervating the buccal cavity, L. monocytogenes enters the central nervous system and localizes in the pons and medulla. Damage to the cranial nerves (indicated by Roman numerals in parentheses) underlies the clinical presentation. For example, lesions of the fifth (V) cranial and mandibular nerves lead to inability to eat or drink or retain food in the mouth. Excessive salivation because of difficulty in swallowing (IX and X); protrusion of the tongue (XII); ataxia or circling (VIII); facial paralysis, including unilateral drooping of the lip, ear, and eyelid (VII); and strabismus (VI), reflect damage to the respective cranial nerve [225]. In the advanced stage, as vision and locomotion are impaired and the animal becomes increasingly irritable, the illness may be confused with rabies or lead poisoning. Finally, the animal lapses into a coma and generally dies within 1 to 2 days [239]. Histopathologic lesions of the brainstem consist of foci of necrosis infiltrated with neutrophils, macrophages, and bacteria [138]. Perivascular cuffing with mononuclear cells is evident [267]. Unlike listeric encephalitis in sheep and goats, most cattle survive at least 4 to 14 days after the initial onset of symptoms, with a few reports of spontaneous recovery [221]. Listeriosis in cattle is associated with abortion [99,208,221,239] during the last trimester of pregnancy. However, as demonstrated by Dora, an 11-year-old cow with atypical mastitis, healthy calves are born to chronic carriers that shed the pathogen in milk [71]. As was true for

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sheep, L. monocytogenes is transmitted to the placenta and enters the fetus. Meningitis in neonates may follow intrauterine infection with the septicemic young animals dying shortly after birth [241]. In the U.S. Midwest, L. monocytogenes is the third most frequently encountered bacterium from bovine late trimester abortion cases. L. monocytogenes accounted for 1.35% of bovine abortions and stillbirth submissions received by the South Dakota Animal Disease Research and Diagnostic Laboratory from 1980 to 1989 [153]. Ten years earlier, 2.2% of 2,544 bovine abortion cases examined in the Northern Plains region of the United States were attributed to listeriosis [154]. Similarly, in Germany from 1984 to 1989, L. monocytogenes serotype 1/2 was recovered in 1.2% (122/9931) and 1.8% (122/9931) of bovine abortions [30]. L. ivanovii is most often associated with sheep [21,38,62,126,133,177,178,242] and is sometimes reported from cattle [3,95]. In California, during a 3-year period, five cases of listeric bovine abortions of a total of 243 fetuses submitted for evaluation were diagnosed. L. ivanovii was recovered from four cases and L. monocytogenes was isolated from only a single bovine abortion. The pathologic findings in these five listeriosis cases were similar [3]. L. monocytogenes may enter a herd by contaminated feeds, introduction of new stock, and rodents. Bovine abortions and stillbirths occur shortly after contaminated silage is eaten [5]. Improvement in silage production and hay making reduced (from 8.7 to 1.2%) the number of listeric bovine abortion cases in The Netherlands [63]. A change in silage production also decreased the percentage of carrier animals (from 15 to 0.8%) based on fecal sampling [68]. As expected, the number of healthy carriers is lower on premises without overt disease than on premises with clinical listeriosis. To illustrate, L. monocytogenes was cultured from 2.0% of cows on farms without L. monocytogenes and in 6.7% of healthy animals on farms on which listeriosis had occurred [63]. This may indicate exposure to a common source of infection. Rodents are known carriers of L. monocytogenes and fecal contamination of animal feed is a potential source of contamination [5,101,151]. Normal healthy cattle may intermittently shed Listeria in their feces; prevalence rates range from a few percent up to 52%, with some seasonal differences [81,127,271,288]. Fecal shedding may reflect levels of L. monocytogenes in feed. Shedding of L. monocytogenes (52%) was related to feeding wet feed (e.g., silage of beet tops, oat, and pea straw), 67% of which was contaminated [252]. L. monocytogenes was isolated from the feces of 8.7% of nearly 4,000 randomly selected dairy cows in Finland over a 2-year interval [129]. Again, L. monocytogenes was recovered more frequently from bovine feces on farms where L. monocytogenes was in feed than on premises where feed (silage or pasture grass) was negative for L. monocytogenes [127,129]. Switching cattle from grazing to a diet of silage increased fecal shedding of L. monocytogenes. Distribution was seasonal: L. monocytogenes was recovered from feces more frequently during the indoor season (9.2%) than when animals were on pasture (3.1%). As expected, the seasonal occurrence of L. monocytogenes in milk reflected the frequency of L. monocytogenes in feces but not in grass silage and pasture grass; thus, fecal contamination during milking could be inferred [127]. Although not particularly common, generalized listeric infections can give rise to mastitis [41,60,71,97,145,216,258,274,281]. Beginning in 1938, Schmidt and Nyfeldt postulated that a small outbreak of human listeriosis in Denmark may have been caused by drinking milk from mastitic cows. However, the role of Listeria in mastitic infections was not clearly identified until 1944 when Wramby [302] isolated L. monocytogenes from milk and udders of mastitic cows in Sweden. In 1956, de Vries and Strikwerda [60] described another case of bovine mastitis in which a penicillinresistant strain of L. monocytogenes was cultured from one quarter of a 6-year-old dairy cow. Following acute onset, the condition soon became chronic with shedding of L. monocytogenes in the milk for 3 months. Prolonged excretion of L. monocytogenes in milk [64,65,131, 208,209, 221], apparently normal appearance of the milk, and consumption of raw milk on farms could be important in transmission and epidemiology of Listeria infection [103].

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L. monocytogenes strain Scott A (serotype 4b), a clinical isolate recovered from the New England outbreak, has produced experimental mastitis in dairy cows [39,291]. Following repeated intramammary inoculation of 34 Holstein cows with 103 to 107 L. monocytogenes cells, 75% of the animals became chronically infected and shed Listeria in milk (ca. 103 to 105 L. monocytogenes CFU/mL of milk) intermittently for up to 8 months. The intramammary route of inoculation simulated infection from contaminated bedding directly into the teat canal [291]. Interestingly, one of these experimentally infected cows delivered a normal, healthy bull calf. Bourry et al. [34] induced mastitis also via intramammary inoculation with a single dose of 300 L. monocytogenes serotypes 4b and 1/2. L. monocytogenes was recovered from the supramammary lymph node, but not from the spleen or liver. DNA profiles of isolates recovered throughout experimental infection confirmed persistence of the inoculated strain [34]. As with sheep [7] and goats [166], L. monocytogenes also is shed in milk by healthy dairy cattle with no indication of mastitis [63,80,97,123,130,131,198,278]. Schultz [235] collected milk samples from 1,004 cows and isolated L. monocytogenes from the milk of 10 animals (0.1%), 7 of which appeared perfectly healthy. Shedding of Listeria in milk from these animals was intermittent, but continued for as long as 12 months. Examination of dairy herds in Yugoslavia [247] also has demonstrated that clinically healthy cows are asymptomatic carriers of L. monocytogenes and secrete the organism in their milk for months over several lactation periods. In one such survey, L. monocytogenes was detected in milk from 3.2% of 845 clinically normal cows on seven farms in which listeriosis had been previously diagnosed [156]. Earlier, Kampelmacher [148] reported that dairy cattle shed L. monocytogenes at levels of 10,000 to 20,000 CFU/mL of milk. Surveys of the raw milk supply prompted by dairy-related outbreaks of human listeriosis in 1983, 1985, and 1987 confirmed that asymptomatic cattle are carriers for L. monocytogenes. L. monocytogenes has been reported in raw cow’s milk with a distribution generally ranging from 0.1 to 45% [22,70,80,81,88,105,118,170,254,260,265]. In the United States and Canada, the incidence is reported to be no more than ~5% [135], although in the northeastern United States, Hayes et al. [118] reported L. monocytogenes in 12% of raw milk samples. L. monocytogenes was isolated from 4.1% of raw milk samples from Tennessee [231]. Interestingly, in that U.S. study, consumption of raw bulk milk was reported by 35% of dairy producers [231]. Jayaro et al. also reported that nearly 60% of dairy farmers consumed raw milk with few reporting clinical illness [135]. The recoveries varied depending on whether individual cows, bulk tanks maintained on the farm, or milk tankers serving multiple premises were sampled [114,230]. A 23-year survey involving 36,200 dairy herds in Denmark indicated that the incidence of L. monocytogenes-infected cows varied from 0.01 to 0.1% and of herds with an infected cow ranging from 0.2 to 4.2%. Yet, 8.5% of bulk milk samples (n = 445) were contaminated with L. monocytogenes [6]. Differences in recovery may also result from variations in farm management practices and regional, seasonal differences, as well as variations in isolation protocols. Indirect contamination of bulk milk occurs under unhygienic milking conditions, if L. monocytogenes is present in feed, feces, udder surface, or bedding [81], or if an animal is recovering from a recent infection [98,192]. On-farm risk management factors have been reported for New York dairy farms [15]. Specifically, the observed risk (OR) was lower for farms that used milking parlors (OR = 0.21, P = 0.01) than for premises using a bucket system. Premilking teat disinfection (OR = 0.26, P = 0.001), premilking examination for abnormal milk (OR = 0.4, P = 0.01), and dry-cow therapy for >6 months (OR = 0.34, P = 0.04) were protective factors. In contrast, use of Escherichia coli J5 vaccine (OR = 3.3, P = 0.03) was linked to higher incidence. Regional differences have been documented despite relatively low recovery rates (~5%) of L. monocytogenes in milk. Yet Dominguez-Rodriguez et al. [70] found L. monocytogenes in 45.3% of 95 raw milk samples from a single bulk tank (80,000-liter capacity). The dairy received raw milk from a number of small farms in western and central Spain over a 16-month interval. Seasonal distribution of L. monocytogenes in raw milk, mirroring numerous determinants including a change

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of diet or weather-related stress, has been observed. Lovett et al. [170] surveyed raw milk at various times from three different regions in the United States and found L. monocytogenes in 4.2% of the overall samples. However, the recovery of L. monocytogenes from Massachusetts samples was seasonal with the incidence highest during cooler months and lowest in hot-weather months. A more recent survey of 404 New York dairy farms revealed higher recoveries (odds ratio = 1.8) in spring (18%) than other seasons [115]. In the Pacific Northwest, prevalence varied from 4.7% in November to 7% in June. In that study, PFGE profiles suggested relatively stable clones, which may reflect selection and survival of strains that have adapted to the bovine host [198]. Interestingly, no seasonal trend was exhibited by samples recovered from Ohio, Kentucky, and Indiana [173]. A study of L. monocytogenes in raw milk in Nebraska, a state that experiences severe climatic changes, indicated a seasonal distribution with 6% of raw milk samples harboring L. monocytogenes in February and 2% of samples positive in July [164]. Seasonal variation may be related to silage feeding during the winter. L. monocytogenes was cultured from 4.9% of raw milk samples collected from 70 Irish farms with the incidence higher in the winter when cows were housed indoors than in the summer [224]. In Scotland, a seasonal distribution was indicated for L. monocytogenes, which was present overall on 25 premises of the 160 farms surveyed (16%). Contamination was sporadic, with bacterial titers generally less than one L. monocytogenes per milliliter. Although more raw milk samples were positive for L. monocytogenes in January than at other sampling dates throughout the year, the authors caution that no link to farm management practices was evident [84]. In Ontario, Canada, raw milk recoveries indicated seasonal and geographical differences, with the incidence higher in the eastern region [85,254]. Serosurveys have monitored distribution of L. monocytogenes in dairy cows [71,198]. Infection in cattle can be estimated by measuring antibodies to whole cells and antibodies specifically targeting antibodies to listeriolysin (LLO) [12,32,33] as well as internalin A [32]. Because up to 33% of dairy cattle continued to shed L. monocytogenes despite high serological titers, antibody levels do not appear to modulate recovery of L. monocytogenes from milk [71,257,276]. As in humans [26], sheep [13,163,174], and goats [193], antibodies to listeriolysin O (LLO) have confirmed previous or current infection with L. monocytogenes in cattle [12,32,33]. When antibody titers to LLO and internalin A were correlated with dairy farm practices, a positive correlation was found between feeding corn silage (OR = 6.5). In contrast, use of rubber bedding mats (OR = 0.2) and an electrical device to train cows to defecate and urinate off the bedding area (OR = 0.36) were correlated with low LLO and internalin A titers, suggesting low exposure to L. monocytogenes. Serum levels also imply differences in breed susceptibility [32]. Stress-related immunosuppression associated with change of diet, weather, transportation [83], pregnancy, parturition, and lactation may lower resistance to bovine listeriosis [239]. Dexamethasone mimics the stress-related release of glucocorticoids. In cattle, dexamethasone elevates total white blood neutrophil counts and decreases eosinophil and lymphocyte populations. When administered to cows experimentally infected with L. monocytogenes, dexamethasone increased the shedding by up to 100-fold of L. monocytogenes in milk [291]. The increased levels of L. monocytogenes in the milk may reflect impairment of cell-mediated immune mechanisms and phagocytic cell functions that underlie listerial immunity [269]. Likewise, transport of live animals over long distances may increase the level of fecal excretion of L. monocytogenes. A major concern of bovine listeriosis is the potential risk posed to humans. In Denmark, a case-control study indicated that human listeriosis was frequently linked to consumption of unpasteurized milk (risk factor of 8.6), although other factors, such as immunosuppression and underlying diseases, were regarded as more significant [137]. Furthermore, a comparison of 33 isolates from bovine mastitis and 27 human clinical isolates in Denmark recovered during 1993 was made by sero- and ribotyping. Serotyping showed that all bovine and 63% of human isolates belonged to serogroup 1, whereas 37% of the human isolates were of serogroup 4. DNA fingerprinting by ribotyping indicated that a low but constant percentage of Danish dairy herds have cows infected

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with L. monocytogenes strains similar to human clinical strains [141]. L. monocytogenes ribotypes common to dairy processing and the farm environment (dairy cattle, raw milk, silage) were also reported in the United States. This indicated that the farm may serve as a reservoir for L. monocytogenes strains capable of entering the dairy-processing facility [8]. Alternatively, it verifies the ubiquitous distribution of the pathogen. Although foodborne listeriosis in humans is more frequently linked to consumption of contaminated dairy products than to beef consumption, L. monocytogenes was recovered from 3% of composite fecal samples representing 224 feedlot beef cattle [249]. In a limited study of experimentally infected Holstein cows (n = 4), L. monocytogenes was cultured from muscle, organ, and lymphoid tissues at 2 days after infection; none was recovered at 6 or 54 days after inoculation [142]. This indicates that cattle may be an insignificant source of L. monocytogenes contamination in meat [168]. Epizootics have been observed in feedlot cattle and in beef cattle herds [5,301]. Transport of cattle over long distances increased the level of fecal excretion of L. monocytogenes, but not contamination of carcasses [83]. Yet in this study L. monocytogenes was detected in 91% (21 of 23) of minced beef samples, again demonstrating that postharvest processing significantly increases the level of contamination compared to that of the whole carcass [83]. This hypothesis is further strengthened by tracking L. monocytogenes strains by multilocus enzyme electrophoresis. L. monocytogenes strains of electrophoretic type (ET) I have been implicated in major human foodborne epidemics and are found coincidentally in livestock. L. monocytogenes strains of ET I predominate in cattle at the beginning of slaughter, but are not detected on the carcasses at the end of processing or in the environment of the abattoir [31]. In contrast, environmental strains, such as ET 19, contaminate the carcass during processing. Likewise, ET 19 strains were found on the carcasses of pigs at the end of processing in two slaughterhouses, but not on live animals or at the beginning of slaughter. Taken together, these studies indicate that contamination of meat during processing occurs from L. monocytogenes strains that reside in the packing plant rather than from strains indigenous to animals [31].

SWINE Porcine listeriosis manifests primarily as septicemia. Encephalitis is reported less frequently and abortions are rare [29]. Clinical septicemia is usually observed in the neonate where hepatic necrosis may be a characteristic feature [112,191]. Unlike its frequent occurrence in ruminants (cattle, sheep, and goats), listeriosis is rare in monogastric swine. Slabospits’Kii [253] first reported Listeria infection in young swine raised on a Russian farm and designated the organism as L. suis [29]. The first description of porcine listeriosis in the United States occurred when Biester and Schwarte [28] reported it in Iowa in swine with encephalitis. Later, Kerlin and Graham [150] recovered Listeria from the liver of a pig with no clinical signs of encephalitis. In Norway, Hessen [121] reported listeric septicemia in piglets raised on a farm where sheep had died of listeriosis several weeks earlier. Whether transmission was from the sheep to the pigs or the result of common exposure is unknown. In natural and experimental infections, listeriosis is more severe in young animals [30,42,148]. Piglets succumb to infection whereas adults generally survive. In the neonate, L. monocytogenes may originate from the tonsils of the sow, penetrate the intestinal tract of the piglet, and become systemic [267]. Neonatal listeriosis may be seasonal with cases peaking in early winter [169] and spring. Listeric encephalitis seldom occurs in pigs. Symptoms of central nervous system disturbance, including incoordination and progressive weakness followed by death, are characteristic of listeriosis of the younger animal. Meningoencephalitis in swine begins with a sudden refusal to eat and is typically followed by various neurological disorders including trembling, partial paralysis, incoordination, circling movements, and convulsions. Histopathological findings from meningoencephalitis

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include severe monocytic infiltration. Numerous blood vessels, particularly those in the pons, reveal perivascular cuffing [29,102,219,239]. Although listerial meningoencephalitis in swine is infrequent, several such outbreaks have been reported, including one in India in which 27 of 75 pigs died [179,220]. In England, the Veterinary Investigation Center reported only 14 listeriosis cases in swine between 1975 and 1982 as compared to 666 cases in sheep and 472 cases in cattle [96]. Listeriosis in pigs is also reported to be uncommon in The Netherlands [200]. Porcine listeriosis comprised 1% of listeriosis cases in Western Canada [22]. In Iowa, a major hog-producing state, from 1993 to 2000, of a total of 253 listeriosis submissions to the state veterinary diagnostic laboratory, none were from pigs. In that same interval, 87% of listeriosis cases in Iowa were from cattle [292]. Earlier, Blenden reported that cattle and sheep accounted for 274 of 281, or 98%, of listeriosis cases submitted to the Missouri Diagnostic Laboratory; only 1 case was from pigs [29]. Missouri, like Iowa, is a major hog-producing state. Because few surveys are available describing the prevalence of L. monocytogenes in healthy pigs, its distribution (0 to 16%) may be estimated from surveys of fecal excretors and recoveries from tonsils and carcass swabs collected at slaughter [86,125,203,277]. In a study in the United States of 300 market-weight hogs, L. monocytogenes was recovered overall from 2.4% of hog tissues, including tonsils (7.0%) as well as thoracic (3.5%) and superficial inguinal (1.9%) lymph nodes [146]. For cull sows (n = 181)—animals raised primarily for reproductive purposes and under less dense animal housing conditions—L. monocytogenes was cultured from a single tonsil sample (0.6%) and from none of the rectal content samples (Wesley, unpublished). In a limited study of 131 wild boars in Japan, L. monocytogenes serotype 4b was cultured from 2% of fecal samples. The lower frequency of recovery may be attributed to low population density in the wild. No attempts were made to recover the pathogen from other sites, including tonsils [117]. Asymptomatic carriers of Listeria may be more prevalent in Eastern Europe [221,222]. Ralovich [221] reviewed studies in which fecal recovery rates of 47% in individual animals and in 11 of 12 farms were described. A high infection rate in pigs has led to speculation that swine may be important reservoirs of L. monocytogenes in Eastern Europe [102]. Husbandry practices such as feeding pigs dry feed or silage, rearing in closed houses, maintaining specific-pathogen-free (SPF) herds as well as differences in sampling sites (tonsils vs. feces) and seasonality may account for variation in the incidence of healthy porcine carriers reported. To illustrate, in Yugoslavia, L. monocytogenes was recovered more frequently from tonsils of pigs raised on silage (61%) than animals raised on dry feed (29%). Recoveries from meat generally exceed isolation from live animals, suggesting postslaughter contamination during processing. Skovgaard et al. [252] reported that although only 1.7% of pig fecal samples yielded L. monocytogenes, the pathogen was detected in 12% of ground pork samples in Denmark, indicating dissemination of Listeria during processing—an observation made by other investigators [40]. In France, an outbreak involving 279 human cases incriminated pickled pork tongue as a major vehicle of transmission, although other highly processed, ready-to-eat delicatessen items subject to environmental contamination were also implicated [59,134]. Live hogs were thought to have introduced contamination into the processing plant. Although limited epidemiological data are provided, two cases of human neonatal listeriosis may have been linked indirectly to contact with pigs [256]. Alternatively, they may reflect exposure to a common source of contamination.

FOWL Avian listeriosis was first described in 1935 [238], 3 years after TenBroeck isolated L. monocytogenes (then Bacterium monocytogenes) from diseased chickens. Wild [35,227,288] and domestic avians, including turkeys [24,116,204], ducks [100,239], geese [100,239], and pheasants [100], may be asymptomatic carriers. Up to 33% of all healthy chickens may asymptomatically shed

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L. monocytogenes in fecal material [66,67,252]. Birds most likely become infected by pecking Listeria-contaminated soil, feces, or dead animals; however, contaminated fecal material also may pose a hazard to other livestock. Bovine encephalitic listeriosis developed in four cows housed in stables where chicken litter was used as flooring. L. monocytogenes serogroup 4b was recovered from the bovine brains, litter, and the intestinal contents of 4.1% of the donor birds (n = 2.3 million) [68]. In another study, the increased incidence of L. monocytogenes in rooks coincided with nesting season and the peak of ovine listeriosis, which in turn was linked to consumption of contaminated silage [82]. Despite the many sporadic cases of avian listeriosis that have been documented over the last 60 years, this disease is far less common in birds than in sheep, goats, and cattle [100–102, 199,211,236]. Listeriosis in birds may be a secondary infection associated with viral infections [57] as well as salmonellosis, Newcastle disease, fowl pest, coryza, coccidiosis, worm infestations, mites, enteritis, lymphomatosis, ovarian tumors, and other immunocompromising conditions [102]. In 1988, an encephalitic form of listeriosis was reported in broiler chickens in California [54]. Predisposing conditions that may have precipitated the outbreak included recent debeaking and vaccination with a modified live viral arthritis vaccine given subcutaneously in the neck. L. monocytogenes serotype 4b was recovered from a liver and multiple brain samples. Three years later, a second outbreak occurred in breeder replacement birds, affecting 0.3% of the 54,000 birds in the flock. L. monocytogenes was recovered from soil samples collected near an adjacent dairy, but not from other sites on the premises. The stress associated with the unusually cold climate described in the report coupled with vaccine stress of the 7- to 10-day-old chicks may have led to this outbreak [54]. Septicemia, the most frequent manifestation of listeriosis in domestic fowl, is characterized by focal necrosis within the viscera, particularly the liver and spleen [102]. Although not present in all cases [199], cardiac lesions frequently develop and, in turn, lead to engorgement of cardiac vessels, pericarditis, and increased amounts of pericardial fluid [102]. Other conditions produced by the septicemic form of avian listeriosis have included splenomeglia, nephritis, peritonitis, enteritis, ulcers in the ileum and ceca, necrosis of the oviduct, generalized or pulmonary edema, inflammation of the air sacs, and conjunctivitis. In acute cases, lesions resulting from these conditions may be partially obscured by congestion and hemorrhages throughout the viscera [102]. Unfortunately, domestic fowl that suffer from listeric septicemia normally exhibit few overt signs of disease other than progressive emaciation and usually die 5 to 9 days after infection. Although far less common than the septicemic form of listeriosis, L. monocytogenes also can produce meningoencephalitis in domestic fowl. Domestic birds suffering from listeric meningoencephalitis exhibit several striking behavioral changes, including incoordination, tremors, torticollis, unilateral/bilateral toe paralysis, and dropped wings, all of which directly relate to disturbances of the central nervous system [18]. Such infections are virtually always fatal. Postmortem examination often reveals congestion and necrotic foci in the brain along with many of the aforementioned conditions that are characteristic of listeric septicemia. Microscopically, gliosis and satellitosis in the cerebellum and microabscesses containing Gram-positive bacteria are found in the midbrain and medulla of birds with encephalitic listeriosis [55]. L. monocytogenes colonizes chick embryos and young birds; older birds appear to be more resistant [11,61,102,111,266]. Following oral challenge of chickens with 102 or 106 L. monocytogenes cells, Bailey et al. [15] detected the pathogen more frequently in ceca, spleen, liver, and cloacal swab samples from 1- rather than 14- or 35-day-old chickens. Diarrhea and emaciation have been noted in experimental infection, thus facilitating spread via feces and nasal secretions. In a later study, 2-day-old chicks were experimentally infected with L. monocytogenes. Although most of the inoculated chicks appeared healthy, depression, ruffled feathers, dullness, and diarrhea followed by death were noted 2 to 5 days after inoculation. Milder symptoms such as anorexia and drowsiness were also observed in a few of the animals. At 5 days after infection, 100% of the cecal samples

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yielded L. monocytogenes. However, the percentage of L. monocytogenes-positive birds decreased and by day 28, L. monocytogenes was recovered from the caeca of only 10% of experimentally infected birds [128]. In another report using a smaller number of birds, L. monocytogenes was only found on the first day following infection in 15% of fecal samples [183]. Taken together, these data suggest that L. monocytogenes is cleared rapidly, indicating that birds are at best transiently infected and not likely reservoirs of L. monocytogenes [305]. Interestingly, following artificial infection, Pustovaia [218] found that Columbiformes, Passeriformes (perching birds), and Galliformes were susceptible, while Falconiformes and Strigiformes (owls) were resistant [124]. L. monocytogenes has been used to investigate macrophage function in retrovirus infection and the cell-mediated immune response in susceptible and resistant chickens exposed to Marek’s disease virus [45,57]. Viral infection depressed the resistance of 10-day-old chickens to experimental infection with an avian osteopetrosis virus [57]. When compared to virus-free chickens, the dually infected birds were less efficient in clearing L. monocytogenes from their spleens [57]. L. monocytogenes is present in healthy birds from nondetectable levels to up to 33% [66,67,93,123,132,288]. Yet contamination in retail poultry ranges from 17 to 70%, again suggesting postharvest contamination [14,31,86,93,183,207]. A link between transport stress and fecal shedding of L. monocytogenes has been suggested. In one study L. monocytogenes was found in 33% of pooled fecal samples collected from cages, suggesting recrudescence of L. monocytogenes because of transport stress. But no data on the L. monocytogenes status of these birds before shipment are provided [252]. In an effort to trace the source of L. monocytogenes present in retail poultry, low levels of natural carriage (5%) were reported in cecal samples of parent flocks providing broilers. Yet, L. monocytogenes was not cultured from cecal samples from over 2,000 broilers from 90 flocks in Denmark [208]. However, L. monocytogenes was found in processed poultry. Comparison of DNA fingerprinting patterns by pulsed-field gel electrophoresis (PFGE) indicated that live birds contributed little to the total contamination of the product [207,232].

MINOR SPECIES L. monocytogenes has been diagnosed in a number of minor wildlife and livestock species, such as horses, llamas, animals raised commercially for pelts, companion animals, deer, and primates [195]. The routes of transmission and symptoms parallel those of cattle, sheep, and goats. As with other livestock species, Listeria infection in horses can cause abortion [289], septicemia [25,51,74,106], and encephalitis as well as keratoconjunctivitis [233]. In contrast to cattle and sheep, few cases of equine listeriosis are reported [160,182,185,239,263,264,268]. A survey of fecal samples of 400 German horses indicated a carrier rate of 4.8% for L. monocytogenes, 6% for L. innocua, and 1.5% for L. seeligeri; less than 1% harbor L. welshimeri [288]. The results of a limited number of surveys describing L. monocytogenes-seropositive horses should be interpreted cautiously in light of possible cross-reactivity of antibodies of other bacterial species to Listeria. Previous history of contact with cattle and feeding on silage may explain sporadic cases of equine listeriosis [184]. L. monocytogenes was reported from four Welsh and two Shetland ponies housed together with cattle, one of which was diagnosed with listeriosis, and given poor-quality silage [74]. At necropsy, L. monocytogenes was cultured from the equine liver, spleen, heart, kidneys, and lungs [74]. In Tasmania, abortions occurred in two mares allowed to graze on pasture that had previously been a sheep farm but had most recently served as a dairy farm. L. monocytogenes serogroup 1 was cultured from the lung and stomach of one fetus. Following antibiotic therapy, the mare was bred and later gave birth to a normal live foal, indicating again that L. monocytogenes infection may not impair fertility [180]. Equine abortion, preceded by mild respiratory tract infection, caused by L. monocytogenes serotype 4 was reported in a mare that had wintered with cattle and been on ensilage [289].

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L. monocytogenes was cultured from the equine fetal liver, lung, spleen, and stomach. Neonatal septicemia was documented in a 3-day-old foal whose mare was housed indoors and fed poorquality, contaminated hay [122]. As is true in other species, the origin of infection in equine listeriosis cases may be unknown [233]. To illustrate, L. monocytogenes was recovered from the brainstem of a 16-year-old Welsh pony gelding with signs of ataxia, weakness, and deficits of cranial nerves. No immunological deficit was detected and there was no history of contact with ruminants or access to silage. A 21-day-old Appaloosa filly was examined because of diarrhea of 2 weeks’ duration [284]. Septicemia was diagnosed based on presence of L. monocytogenes cultured from blood. No sources of infection were evident, thus leading to speculation that L. monocytogenes was transmitted to a foal via contaminated mare’s milk. As described in humans and livestock, listeriosis occurs in immunocompromised hosts with defects in the humoral and cell-mediated immune components [269]. Listeriosis has been described in an Arabian foal with combined immunodeficiency [51]. The 1-month-old foal was ataxic, lethargic, failed to nurse, and spent most of the time with its head down. Most strikingly, hoofs were dragged when the animal was exercised. At necropsy widespread lesions were present in the viscera and central nervous system. In the llama, listeriosis occurs as a septicemia with meningitis in neonates, but more commonly causes asymmetric vestibular disease in adults [19]. Most animals affected are weaned and grazing or consuming roughage, but are not on silage. Multifocal suppurative encephalitic listeriosis was diagnosed in two adult llamas that were pregnant. L. monocytogenes was cultured from one of two animals and was visualized in the brainstem lesions of both llamas by fluorescein-conjugated antibody to L. monocytogenes [43]. In another report, L. monocytogenes caused fatal meningo-encephalomyelitis in a 3- to 5-month-old llama. The animal displayed unilateral peripheral disease progressing to encephalitis [270]. The source of infection for cases detailed in these two reports is unknown [19,43]. Listeriosis may have an economic impact in commercial pelt farms. An outbreak of disseminated visceral listeriosis in chinchillas in Nova Scotia [89] incriminated consumption of contaminated sugar beet pulp, although L. monocytogenes was not isolated from the feed. The outbreak occurred in a colony with 23% mortality of breeding chinchillas [300]. Approximately 4 days before death, animals were anorexic and hunched and some had torticollis (twisted necks). However, many animals were found dead without clinical signs [300]. Hay contaminated with rodent, bird, or ruminant feces has been implicated in previous outbreaks of chinchilla listeriosis and removal of contaminated feed often interrupts the cycle of transmission [47,89]. L. monocytogenes could have been transmitted by coprophagia because animals defecated in dust bath pans and the pans were transferred from cage to cage [298]. In an enzootic of listeriosis in a rabbitry, L. monocytogenes 1/2a was cultured from feed samples and from a doe that had died of septic metritis [213]. The early literature describes L. monocytogenes in nondomesticated ruminants, including reindeer, roe deer, antelope, and a Grant’s gazelle with previous contact with listeric sheep [20,23,76, 78,149,204,286]. L. monocytogenes has been recovered from ruminants housed in zoological parks [9,76,78,179,286]. Meningoencephalitis occurred in 42 deer in a flock of 1,800 head in a park during the winter and early spring in Denmark. This was preceded in the previous spring by the death of six deer that exhibited circling and appeared to be blind. The following year, the first sign of illness was a drooping ear, due to paralysis of the facial nerve, and a slight inability to follow the herd. No external source of L. monocytogenes was evident and stress because of a poor beech-mast crop, increased stocking rate of animals resulting in overcrowding with possible introduction of healthy carrier animals, and sudden change in weather were all potential contributing factors [76]. Listeriosis is rarely reported in dogs and has caused encephalitis, including circling, and abortion [261]. In a survey of domestic animals, L. monocytogenes was detected in 1.3% of dog (n = 300) and 0.4% of cat (n = 275) fecal samples [288]. The low recovery may indicate that companion

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animals are not important in the epidemiology of listeriosis in humans [287] or that shedding is sporadic. In contrast, serosurveys indicating that up to 90% of dogs may be seropositive [251] should be interpreted cautiously because of the cross-reactivity inherent in agglutination tests. A single report on the possible transmission to L. monocytogenes from humans to dogs [262] warrants re-evaluation because the isolates were later identified as L. innocua. Nevertheless, recovery of a single species of Listeria from humans and dogs in close proximity could reflect substantial environmental contamination rather than human-to-dog passage. Listeriosis occurs in nonhuman primates, where it manifests as septicemia [48,303], meningoencephalitis [61], and stillbirths [185,273], as documented in a large outdoor breeding colony in California [212]. Transmission may occur by consumption of contaminated foods [273]. Experimental infections were attempted by exposure to aerosols [147] as well as by feeding [79,255]. Cynomolgus monkeys (Macaca fascicularis) shed L. monocytogenes in their feces for up to 21 days after receiving an oral dose of 109 Listeria. Neither septicemia nor encephalitis was reported, indicating that the normal healthy primate is resistant to L. monocytogenes. In contrast, abortion and stillbirths were reported in rhesus monkeys fed 108 CFUs in a high-fat vehicle [255].

FISH

AND

CRUSTACEANS

Consumption of seafood is growing in popularity, and the aquaculture industry is responding by providing fish raised in controlled fish farms rather than depending on the availability of fresh caught products. In 1980 in New Zealand, Lennon et al. [160] reported a cluster of 22 perinatal human listeriosis cases. A weak association between these cases and the consumption of contaminated raw fish and shellfish was established. DNA fingerprinting determined that fish in the patient’s refrigerator were identical. The first report of the occurrence of listeriosis in fish was in 1957 from Romania [259]. In that study, L. monocytogenes was isolated from the viscera of pond-reared rainbow trout that presumably became infected after consuming contaminated donkey meat. Results of current surveys indicate that Listeria is absent from live seawater fish, but is present in live freshwater species. In contrast, L. monocytogenes was recovered from two of five intestinal tracts of market-purchased fresh water fish in India [119]. Channel catfish (Ictalurus punctatus) is the most widely cultured species in the United States; most commercial ponds are located in the southeastern region of the country. A study of catfish, water, and feed collected from university ponds in Alabama indicated presence of L. monocytogenes on the skin and viscera (mean presumptive count = 1.99 log CFU/wet weight). L. monocytogenes was not detected in water or feed. Unfortunately, the authors provide no data on the percentage of L. monocytogenes–positive fish, but conclude that the bacterial concentrations in the viscera suggest that cross-contamination is possible during evisceration [162]. A survey of three rainbow-trout farms in Switzerland showed that L. monocytogenes was present in the feces (40%) and on the skin (33%, 5/15) of fish from one of the farms. Yet L. monocytogenes was detected on the finished product in only 6% of the fish from this farm. In contrast, L. monocytogenes was not found in feces, skin of fish, or the finished product of two of the farms where fish were raised in concrete ponds and starved 3 to 7 days before harvest [136]. Similarly, L. monocytogenes was not recovered from the skin, gills, intestines, tank water, or diet of striped bass grown in recirculating water tanks [202]. Experimental infection of zebrafish (Brachydanio rerio) indicated the LD50 was higher in fish than in mice and that L. monocytogenes did not multiply in fish [189]. Experimental infection stimulated an increase in granulocyte and monocytes. In contrast to L. monocytogenes, strains of L. welshimeri, L. innocua, and L. seeligeri killed more than 50% of fish 7 days postinfection [189]. Brackett [36] proposed that contamination of fish and shellfish through their ambient waters may influence distribution of L. monocytogenes. Surface waters, sewage effluents, and agricultural

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runoff all may potentially contribute Listeria spp. to the aquatic environment. In addition, presence of L. monocytogenes in sea gulls may be another source of shellfish contamination [82]. In 1959, L. monocytogenes was detected in crustaceans gathered from a Russian stream [248]. A more recent survey conducted on the Gulf Coast of the United States examined shrimp, oysters, and estuarine waters for L. monocytogenes [196]. L. monocytogenes was detected in 11% of unprocessed shrimp (n = 74), but not in oysters (n = 75), although some of the oysters were harvested from prohibited shellfish-growing sites. In a parallel study conducted in fresh water tributaries off the Humboldt–Arcata Bay in Northern California, L. monocytogenes was detected in the freshwater samples (61%), some of which received runoff from nearby farms. However, L. monocytogenes was not found in oysters in that study [52] or in oysters kept in live holding tanks in seafood markets in Seattle [53]. Taken together, these data indicate that live fish and shellfish are not likely carriers of L. monocytogenes. PCR tests have been used to detect L. monocytogenes in experimentally contaminated marinated rainbow trout [77]. With the increased interest in L. monocytogenes in fish and seafood [77], this sensitive technique may be useful for the rapid screening of live shellfish and fish for L. monocytogenes. Nevertheless, the paucity of reports documenting L. monocytogenes in live freshwater fish and shellfish suggests that Listeria spp. cultured from the retail product are most likely from postharvest contamination.

TREATMENT Poor husbandry, consumption of contaminated feed, and stress are important in initiating the disease. Thus, identifying and eliminating these factors are critical to preventing recurrences. In general, because antemortem diagnosis is rarely made, treatment is seldom attempted. Because listeric encephalitis is a rapidly debilitating disease in ruminants, treatment must be initiated early during the course of infection if there is to be any reasonable hope of survival. L. monocytogenes is resistant to many drugs but is sensitive to chlortetracycline. The intravenous injection of chlortetracycline (10 mg/kg body weight per day for 5 days) is effective in meningoencephalitis of cattle but less so in sheep [219]. If penicillin is used, high doses are required because of the difficulty of maintaining therapeutic levels in the brain. Penicillin G should be given at 44,000 U/kg body weight, intramuscularly daily for 1 to 2 weeks [90]. If signs of encephalitis are severe, death usually occurs in spite of treatment. Supportive therapy, which is usually reserved for valuable animals, includes fluid and electrolyte replacement and is indicated for animals having difficulty eating and drinking as a result of neural damage. Excessive salivation leads to acidosis, which is remedied by intravenous replacement of bicarbonate ions. Permanent neurological damage often occurs in ruminants, despite proper therapy. In view of the severe economic losses from listeric encephalitis in sheep, it may be prudent to consider vaccinating animals against listeriosis, particularly if they are raised in areas prone to listeric infection [202]. In birds, tetracyclines (5 to 10 mg/kg body weight daily for 1 week) are efficacious in acute and subacute forms. Treatment of the chronic form is unsuccessful. As with other livestock species, rigid sanitation and disinfection procedures with culling and isolation of affected birds may be helpful [91]. A benefit of early treatment of animals with listeriosis has been demonstrated. However, timeliness is most important and recognition of the disease depends upon observation of clinical signs. In cattle and sheep the appearance of clinical signs is an indication of neurological damage and thus of a guarded prognosis for treatment. In all cases, the economics of the attempted treatment must be considered along with the alternative of humane euthanasia.

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4 Listeriosis in Humans John Painter and Laurence Slutsker CONTENTS Introduction ......................................................................................................................................85 Human Infection and Clinical Manifestations of Listeriosis ..........................................................87 Listeriosis during Pregnancy ..................................................................................................87 Neonatal Disease: Early Onset...............................................................................................87 Neonatal Disease: Late Onset ................................................................................................88 Invasive Disease in Nonpregnant Adults .........................................................................................88 Noninvasive Disease: Gastrointestinal Illness .................................................................................90 Asymptomatic Carriage..........................................................................................................91 Epidemiological Patterns of Listeriosis...........................................................................................94 Epidemic Listeriosis ...............................................................................................................94 Sporadic Disease—Incidence.................................................................................................97 Sporadic Disease—Dietary Risk Factors...............................................................................98 Sporadic Disease—Possible Other Sources.........................................................................100 Diagnosis ........................................................................................................................................100 Treatment........................................................................................................................................101 Prevention.......................................................................................................................................101 References ......................................................................................................................................102

INTRODUCTION Listeria monocytogenes has been recognized as a human pathogen since 1929 [114], but the route of transmission was unclear until the 1980s when a series of outbreaks indicated that L. monocytogenes was transmitted by food [13,43,59,70,89,102,123,130]. It is now recognized that nearly all cases of human listeriosis are foodborne [1,104,112,132,136]. Although listeriosis is a very small fraction of all illness due to known foodborne pathogens, it is an important cause of severe illness, accounting for 3.8% of foodborne disease hospitalizations and 27.6% of foodborne disease deaths [104]. Development of improved laboratory techniques to detect and subtype L. monocytogenes has also contributed to an improved understanding of human listeriosis [9,11,14,125,126]. Listeria monocytogenes is found in multiple ecological sites throughout the environment, including soil [152], water, and decaying vegetation [151,153]. Control of human listeriosis, therefore, relies on improving the understanding of how to control Listeria contamination of food. Human disease caused by L. monocytogenes occurs most frequently in women of childbearing age, infants, and the elderly (Figure 4.1). The risk of listeriosis is greatest among certain welldefined high-risk groups, including pregnant women, neonates, and immunocompromised adults but may occasionally occur in persons who have no predisposing underlying condition (Table 4.1). The ongoing epidemic of acquired immunodeficiency syndrome (AIDS), as well as widespread use of immunosuppressive medications for treatment of malignancy and management of organ transplantation, has expanded the immunocompromised population at increased risk of listeriosis.

85

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35%

Percent of Total

30% 25% FEMALE

20% MALE

15% 10% 5% 0% <1

>=1-2

>=2-5

>=5-15

>=15-25 >=25-35 >=35-45 >=45-55 >=55-65 >=65-75

>=75

age group

FIGURE 4.1 Percent of reported laboratory-confirmed listeriosis within age groups for males and females, United States, 2000 to 2004. Isolation of Listeria monocytogenes reported to CDC through the Public Health Laboratory Information System (PHLIS).

Unlike infection with other common foodborne pathogens such as Salmonella, which rarely result in fatalities, listeriosis is associated with a mortality rate of approximately 20% [53]. The high case-fatality rate, growing awareness of listeriosis as a foodborne disease, and increasing clinical concern about illness in the expanding population of highly susceptible persons have resulted in increased attention to the importance of L. monocytogenes as a human pathogen. In this chapter, we will consider various aspects of listeriosis in humans, including infection and clinical manifestations of disease, epidemiological patterns of disease, diagnosis, treatment, and prevention. Information on the microbiology, ecology, pathogenesis, detection, subtyping, manifestations of infection in other animals, and occurrence of L. monocytogenes in various foods is presented elsewhere in this book. Although some foodborne outbreaks of listeriosis will be discussed here, a more exhaustive treatment of foodborne listeriosis outbreaks can be found in Chapter 10.

TABLE 4.1 Clinical Syndromes Associated with Infection with Listeria monocytogenes Population

Predisposing Condition or Circumstances

Clinical Presentation

Diagnosis

Fever ± myalgia ± diarrhea Preterm delivery Abortion Stillbirth

Blood culture ± amniotic fluid culture

Sepsis, pneumonia Meningitis, sepsis

Blood culture Cerebrospinal fluid culture

Prematurity

Nonpregnant adults

Sepsis, meningitis, focal infections

Culture of blood, cerebrospinal fluid, or other normally sterile site

Immunosuppresion, advanced age

Healthy adults

Diarrhea and fever

Stool culture in selective enrichment broth

Possibly large innoculum

Pregnant women

Newborns: <7 days old ≥7 days old

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HUMAN INFECTION AND CLINICAL MANIFESTATIONS OF LISTERIOSIS LISTERIOSIS

DURING

PREGNANCY

Infection with L. monocytogenes in pregnant women may result in fetal loss, stillbirth, premature delivery, or neonatal infection. Listeriosis may occur at any time during pregnancy, but is most frequently documented during the third trimester [18,109]. Because bacterial cultures are not routinely obtained from spontaneously aborted fetuses or stillborn neonates, it is difficult to estimate accurately the proportion of fetal loss that may be attributable to infection caused by L. monocytogenes during pregnancy. In one study, L. monocytogenes was cultured from placental and fetal samples in 1.6% of spontaneously aborted pregnancies [55]; other estimates have varied [4]. Among pregnant women diagnosed with listeriosis, the rate of fetal mortality is between 16 and 45% [54,100,138]. In a recent review of the literature, 20% of listeriosis cases during pregnancy resulted in spontaneous abortion or stillbirth; of the remaining pregnancies, 63% resulted in neonatal infection [109]. Preterm delivery resulted from 6 (55%) of 11 listeriosis cases identified at four hospitals in the United States during a 10-year period [109] and 4 (36%) of 11 at two hospitals in Israel during a similar 10-year period [12]. Most cases of listeriosis during pregnancy occur in otherwise healthy women. Among cases identified by literature review, only 4% had a possible predisposing condition (corticosteroid use, diabetes mellitus, systemic lupus erythematosus, or HIV infection) [109]. Women pregnant with multiple gestations may be at increased risk for listeriosis compared with singleton pregnancies. In Los Angeles from 1985 through 1992, rates of listeriosis were approximately four times higher among women with multiple rather than singleton pregnancies [98]. Although most cases of neonatal listeriosis present with severe illness, symptoms in women with listeriosis during pregnancy may be nonspecific, and they often manifest as a mild illness. In a case-control study of sporadic listeriosis, women who delivered infants with listeriosis were significantly more likely than controls to report histories of fever, headache, myalgia, or gastrointestinal symptoms [134]. Approximately two thirds of pregnant women with listeriosis report fever or flu-like illness lasting approximately 6 days (range: 1 to 21 days) [18,103,109]. These symptoms are associated with the bacteremic phase of infection and represent the optimal time to obtain diagnostic blood cultures. In a recent case-series of pregnant women with listeriosis, none of whom had evidence of severe systemic illness, 37% yielded a positive blood culture for Listeria [109]. Infection of the fetus with L. monocytogenes is thought to result from transplacental transmission following maternal bacteremia, although some infections could also occur through ascending spread from vaginal colonization. Intrauterine infection may result in preterm labor, amnionitis, spontaneous abortion, stillbirth, or early-onset neonatal infection. In one case-series, there was evidence of chorioamnionitis in all cases of early-onset neonatal infection [109]. Neonatal infection can be prevented by antibiotic treatment during pregnancy. Case reports suggest that fetal- or early-onset neonatal infection does not always follow maternal listeriosis for which treatment has been delayed or not given [49,78,89]. However, given the potentially adverse consequences of maternal listeriosis and the availability of effective treatment, it is prudent to evaluate all febrile episodes during pregnancy with blood cultures. It is not recommended that women with a history of pregnancy-associated listeriosis undergo routine microbiological screening or antimicrobial prophylaxis during subsequent pregnancies. However, dietary counseling on avoiding high-risk foods should be given.

NEONATAL DISEASE: EARLY ONSET In contrast to the mild clinical illness seen in maternal listeriosis, neonatal infection caused by L. monocytogenes is a serious and often fatal disease. Neonatal listeriosis is divided into two clinical forms: early onset and late onset. These clinical forms parallel the pattern of the disease seen in

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neonates with infection from group B streptococci and as such suggest different modes of transmission for the two forms. Early-onset neonatal listeriosis occurs in infants infected in utero, and it results in illness at birth or shortly thereafter, usually defined as occurring within the first week of life. Between 45 and 70% of neonatal listeriosis is of early onset [48,103]; this disease often presents with sepsis rather than meningitis [54]. Signs and symptoms include respiratory distress, fever, and neurologic abnormalities. Listeria monocytogenes may be isolated from blood, cerebrospinal fluid, oropharyngeal secretions, placenta, amniotic fluid, urine, or external sites including conjuctiva, ear, nose, or throat [109]. Less frequently, infants with early-onset disease may present with granulomatosis infantiseptica, a syndrome characterized by disseminated abscesses or granulomas in multiple internal organs, including the liver, spleen, lungs, kidney, and brain [64]. In this syndrome, evidence of amnionitis or meconium-stained fluid may be present, and the infant may appear obviously ill; in some instances, however, the infant may merely appear weak and may develop respiratory or circulatory insufficiency. Early-onset disease in the neonate may be complicated by aspiration of meconium fluid with resultant respiratory complications, including cyanosis, apnea, and pneumonia.

NEONATAL DISEASE: LATE ONSET In contrast to early-onset disease, the late-onset type may occur from one to several weeks after birth [54,148]. Infants are usually born healthy and full term to mothers who have had uncomplicated pregnancies. Similarly to late-onset neonatal disease caused by infection with group B streptococci, listeriosis in these neonates presents as meningitis more frequently than in early-onset disease. In a review of neonatal listeriosis cases from 1967 to 1985 in Britain, 39 of 42 (93%) infants with late-onset disease had meningitis [103]. Listeria monocytogenes accounted for nearly 7% of all neonatal meningitis in England and Wales during a similar time-period [142]. Active surveillance for listeriosis in the United States in 1986 documented meningitis as the presenting syndrome in 88% of late-onset cases [54]. Mortality rates for early- and late-onset disease are usually 20 to 30% [19,91,103]. Although transplacental transmission of L. monocytogenes is the presumed source of infection in early-onset disease, the route of infection in late-onset neonatal disease is not well understood. Acquisition of infection during passage through the birth canal is likely, although cases of lateonset disease have been reported following cesarean delivery. Because most listeriosis is foodborne, contaminated foods likely contribute to transmission through direct or indirect contact with highrisk foods. Clusters of late-onset disease have been identified in newborn nurseries, suggesting that some nosocomial transmission also occurs [81,87,110,139]. In one outbreak of late-onset disease in Costa Rica, infection was linked to contaminated mineral oil used to bathe newborns [133].

INVASIVE DISEASE IN NONPREGNANT ADULTS Listeria is an important cause of bacterial meningitis in nonpregnant adults and the case-fatality rate is approximately 30% [108]. In a recent review of 103 cases of acute bacterial meningitis in adults, Listeria monocytogenes was the second most frequently isolated bacteria, accounting for 10% of cases [79]. In an older study in the United States, Listeria was the fifth most common cause of meningitis in 1986 [154]. Listeria may be an increasing cause of meningitis in adults as a result of improved vaccination for pneumococcal and meningococcal disease and should be considered when adults present with acute meningitis. Nonpregnant adults with listeriosis most frequently present with sepsis, meningitis, or meningoencephalitis. Presenting symptoms in nonpregnant adults with central nervous system listeriosis may include fever, malaise, ataxia, seizures, and altered mental status. Interestingly, in contrast with meningitis due to other pathogens, approximately one third of patients with listerial meningitis

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initially present without meningeal signs [108]. Fever is generally present in patients with bacteremia; other nonspecific symptoms such as malaise, fatigue, and abdominal pain may also occur. The cerebrospinal fluid may exhibit a pleocytosis; the Gram stain may show Gram-positive bacilli but is often unrevealing. Because the spinal fluid white cell count and differential, glucose, and protein levels can vary widely, the spinal fluid profile cannot be used to differentiate listerial meningitis from meningitis caused by other bacteria. Difficulties in diagnosing listerial meningitis were highlighted in a review of bacterial meningitis in adults [79]. Most patients with listerial meningitis had negative CSF Gram stains and normal CSF glucose. CSF Listeria antigen detection was positive in only a third of cases. However, 80% of cases had a positive blood culture. CT scan or MRI of the brain may reveal focal lesions and/or hydrocephalus in half of Listeria cases with clinical evidence of central nervous system (CNS) abnormalities [108]. Listerial brain stem encephalitis (rhombencephalitis) occurs infrequently and is characterized by asymmetrical cranial nerve deficits, cerebellar signs, and hemiparesis or hemisensory deficits [5,140,145]. In addition to cases with central nervous system listeriosis, many listeriosis patients present with bacteremia without evidence of meningitis. In the United States, active surveillance in an aggregate population of 34 million persons in 1986 found that 66% of 179 nonpregnant adults had bacteremia without meningitis, 19% had meningitis with concurrent bacteremia, and 12% had meningitis without documented bacteremia [54]. Focal infections are rare and usually result from seeding during a preceding bacteremic phase. Endocarditis from L. monocytogenes occurs primarily in patients with an underlying cardiac lesion, including prosthetic or porcine valves, and is clinically indistinguishable from other causes of endocarditis [10,52,96]. Other sites of involvement have been reported, including endophthalmitis [8,90], septic arthritis [34,111], osteomyelitis [76], pleural infection [101], and peritonitis [112]. Cutaneous infections without bacteremia have been reported in persons handling infected animals [118] and in accidentally exposed laboratory workers [3]. Various clinical conditions are associated with listeriosis in nonpregnant persons; these include malignancy, organ transplants, immunosuppressive therapy, infection with the human immunodeficiency virus (HIV), and advanced age [6,54,119,131]. A frequent feature of these conditions is a decreased level of cell-meditated immunity. Cancer, especially lymphoreticular malignancy, has been associated with listeriosis in several reviews [92,103,108,113]. In a review of 458 patients with listerial CNS infections between 1966 and 1997, the most frequent underlying conditions were [108] malignancy (24%, of which 67% of known malignancies were hematologic) transplants (21%, of which 90% were solid organ and 10% bone marrow) alcoholism and/or liver disease (13%) miscellaneous immunosuppressive therapy (11%) HIV/AIDS (8%) diabetes (7%) autoimmune disorder (4%) hemochromatosis (1%) Cases of listeriosis associated with transplantation [56,75,122,124] and other conditions [53,67,85,106,112,113,131] are well documented. Despite the strong association between decreased immunity and invasive listeriosis in nonpregnant adults, many cases do not involve a predisposing condition. In one report, 30% of patients with meningitis and 11% of those with bacteremia caused by L. monocytogenes had no recognized predisposing condition [112]. In another, between 24 and 37% of patients with Listeria infections of the CNS had no known preexisting condition [108]. In these studies, however, cases were largely identified through a literature review and thus may not be comparable to those identified through population-based surveillance.

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In a study of sporadic listeriosis conducted from 1988 to 1990 in diverse geographical populations in the United States, 98% of 98 cases that were not associated with pregnancy occurred in persons with at least one underlying illness, although some had conditions such as heart disease that are not traditionally considered immunosuppressive [132]. The most frequently identified diseases or conditions were heart disease (33%), corticosteroid therapy (31%), cancer (29%), renal disease (24%), and diabetes (24%); several patients had more than one underlying condition. Malignancy, corticosteroid use, and HIV infection or AIDS were the most common immunosuppressive conditions, and at least one of these three conditions was present in 69% of nonpregnant adult patients. There have been many reports of patients with HIV infection or AIDS who contracted listerial meningitis or bacteremia [15,36,38,68,99]. Although listeriosis does not appear to be a common opportunistic infection among persons with HIV infection or AIDS, it nonetheless occurs far more frequently among these individuals than among the general population. In 1989, in a prospective population-based 2-year study in San Francisco, the incidence of listeriosis among AIDS patients was estimated to be 280 times the baseline incidence of listeriosis in the general population [132]. Using similar methodology in the early 1990s, studies in Atlanta [83] and Los Angeles [41] estimated that incidence of listeriosis was 9 to 62 times higher for those with HIV infection and 62 to 145 times higher for those with AIDS. Recent articles have advanced the idea that listeriosis is associated with inflammatory bowel disease (IBD) [20]. The “cold-chain” hypothesis suggests that the incidence of Crohn’s disease parallels the availability of refrigerated foods, and that ingestion of psychrophilic bacteria such as Listeria and Yersinia may lead to an abnormal immunologic response in susceptible persons [77]. Investigators have documented increased serologic response among IBD patients to several enteric pathogens including Listeria [16]; however, attempts to identify L. monocytogenes DNA from enteric biopsies have not demonstrated a significant difference between cases and controls [31,32]. Published experiments have yet to demonstrate a causal role for Listeria in IBD.

NONINVASIVE DISEASE: GASTROINTESTINAL ILLNESS Information from several outbreak investigations suggests that L. monocytogenes causes a febrile gastroenteritis in normal hosts [129]. The first such outbreak of gastroenteritis and fever was linked to consumption of chocolate milk served at a picnic [37]. Picnic attendees drank chocolate milk that was later found to be heavily contaminated with L. monocytogenes (109 CFU/mL); 79% of 58 persons who consumed implicated milk from the picnic reported diarrhea and 72% reported fever. The median incubation period was 20 hours (range: 9 to 32 hours). The same subtype of L. monocytogenes was isolated from the stools of ill persons and the chocolate milk. Ill persons were more likely than well persons to have elevated antilisteriolysin O levels and to have stool cultures that yielded L. monocytogenes. None of the individuals involved in this outbreak had a chronic illness or immunodeficiency and none had evidence of invasive listeriosis. Similar outbreaks of febrile gastroenteritis due to delicatessen meats [50], cheese [22], smoked fish [105], corn [7], and rice salad [128] have been described. A study of seven febrile gastroenteritis outbreaks caused by L. monocytogenes since 1989 suggests a consistent syndrome among otherwise healthy persons [117]. The incubation period is 1 day (range 6 hours to 10 days). Fever is the most frequently reported symptom. Diarrhea (median of 12 stools per 24 hours of maximal diarrhea) with nonbloody stool is the most frequently reported gastrointestinal sign. One characteristic of febrile gastroenteritis outbreaks is unusual compared with other foodborne outbreaks: over half of patients reported somnolence and three quarters reported fatigue. The factors that lead to febrile gastroenteritis versus invasive illness are unclear. Susceptible persons are at higher risk for invasive listeriosis but no host risk factors have been associated with noninvasive listeriosis. Strain difference could account for different syndromes, but no strain

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differences have been observed to date. Strains causing noninvasive gastroenteritis also cause invasive illness. In the chocolate milk outbreak, three cases of sporadic invasive listeriosis were linked to consumption of the implicated chocolate milk; the same subtype of L. monocytogenes as the outbreak strain was isolated from these patients [37]. In studies of the pathogenic characteristics of Listeria strains, no differences have been found between strains linked to invasive illness and those linked to gastroenteritis [47]. Because there is no indication of a Listeria entertoxin, it is hypothesized that febrile gastroenteritis is due to limited invasion of the gut mucosa. The infectious dose of L. monocytogenes necessary to cause listeriosis or febrile gastroenteritis is not fully understood. Quantitatively cultured foods recovered from Listeria outbreaks provide some evidence but the number of organisms ingested is difficult to determine. The density of Listeria in foods implicated in febrile gastroenteritis outbreaks has ranged from 3 × 101 CFU/g to 1.6 × 109 CFU/g with a median of 105 CFU/g. The dose of Listeria sufficient to cause invasive illness in susceptible persons is also poorly understood but is thought to be much lower [95]. The incidence of febrile gastroenteritis caused by L. monocytogenes is difficult to establish because stool specimens from persons with diarrhea are rarely cultured for L. monocytogenes. Clinicians and public health officials should consider examining stool cultures for L. monocytogenes in outbreaks of illness characterized by fever, diarrhea, headaches, and myalgia if stool cultures for other more common enteric pathogens have been negative. When such an outbreak is suspected, care should be taken to notify the laboratory that L. monocytogenes is suspected so that appropriate special culture media are used. The organism is frequently cultured from food, but because the organism is commonly found in many foods, recovery of L. monocytogenes from foods is seldom sufficient to verify the source of infection unless a matching strain is identified from ill persons epidemiologically linked to the food.

ASYMPTOMATIC CARRIAGE L. monocytogenes is distributed throughout the environment and can be frequently recovered from a broad spectrum of foods [42,120] and sometimes from the gastrointestinal tracts of healthy people. Numerous fecal carriage studies of L. monocytogenes among different populations have been done with widely varying rates reported (Table 4.2). Some of this variation may be attributed to differences in populations studied, culture techniques, numbers of specimens obtained from each individual, specimen handling, and whether results were reported as a point or cumulative prevalence. Among surveys reviewed, point prevalence for healthy persons with unknown exposure to Listeria is between 0.6 and 3.4%. A large stool survey conducted in Germany found that fecal specimens yielded L. monocytogenes at a similar rate specimen yield from persons with diarrhea (0.6%) and from healthy food handlers (0.8%) [107]. Among studies of persons exposed to Listeria, fecal carriage rates are much higher. In Denmark, fecal specimens from 26% of 34 household contacts of persons with listeriosis yielded L. monocytogenes [17]. In this study, among 14 households sampled, 5 (36%) had at least one household member with a positive stool culture; however, only two family members had the same serotype of L. monocytogenes as the patient. Up to eight specimens were collected from each household contact, suggesting that the carriage rate in this study may not be directly comparable to that in others. In the United States, 82 household contacts of 28 patients with invasive listeriosis were identified through active surveillance and investigated [135]. Twenty-one percent of these individuals (and households) were positive for L. monocytogenes; 88% of 17 isolates were of the same serotype and enzyme type as the strain from the index patient. The rate of carriage was significantly higher among persons less than 30 years of age than among older persons. In Austria, Grif et al. found that 0.2% of healthy volunteers in a 1-year study were shedding L. monocytogenes by conventional culture; up to 3.6% were positive by PCR techniques [66]. In a related study, three healthy adults provided stool samples over a 1-year period [66]. Stool culture

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TABLE 4.2 Reported Fecal Carriage Rates of Listeria monocytogenes Year

Population

No. Studied

% with L. monocytogenes

Comment

Ref.

Point Prevalence Studies among: Persons with Unknown Exposure to Listeria 1986 Healthy nonpregnant women Healthy pregnant women 1990 Healthy food handlers Persons with diarrhea 1991 Home hemodialysis patients Outpatients with gastroenteritis 2001 Healthy volunteers

Persons Likely Exposed to Listeria 1972 Slaughterhouse workers Hospitalized adults Hospitalized children Hospitalized adults with diarrhea 1992 Pregnant women with listeriosis Household contacts of pregnant women with listeriosis Age-, race-, and hospitalmatched controls Cheese plant employees Household contacts of cheese plant employees 1993 Household contacts of patients with listeriosis (persons) Households of patients with listeriosis with at least one carrier 2003 Healthy adults

59 51 2,000 1,000 80 171 505

3.4 2.0 0.8 0.6 2.5 1.8 0.2 3.6

1,147 1,034 195 595 18 60

4.8 1.2 0 1.0 5.6 8.3

7

0

31 94

9.7 10.6

82

21

28

21

3

1.2

86 107 94 Traditional culture PCR

65

Cold enrichment culture

17

99

133

One-year period; between 242 and 363 samples per person; average of two episodes/ person/year

66

8-week period; up to eight cultures/person Mean 2.5 specimens/patient Mean 2.4 specimens/patient

84

Cumulative Prevalence Studies among: Persons with Unknown Exposure to Listeria 1972 Office workers with no L. monocytogenes contact 1991 Renal transplant patients

26

62.0

177

5.6

1993

147

2.7

Healthy pregnant women

94 63

(continued)

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TABLE 4.2 (CONTINUED) Reported Fecal Carriage Rates of Listeria monocytogenes No. Studied

% with L. monocytogenes

Persons Likely Exposed to Listeria 1972 Household contacts of persons with listeriosis

341

26.0

1972

26

77.0

Year

Population

Laboratory workers handling L. monocytogenes

Comment

6-month period; up to eight cultures/person 8-week period; up to eight cultures/person

Ref.

17

84

yielded L. monocytogenes in 10 (1.2%) of 868 stool specimens. Based on subtyping and the dates of collection, there were five independent exposures to L. monocytogenes—an average of two per person per year; none of the episodes caused illness. All isolates were serotypes 1/2a and 1/2b. Elevated rates of fecal carriage have been demonstrated among those with underlying medical conditions. In a survey of renal transplant recipients, 5.7% of 177 had a positive stool culture over a 1-year period and this was associated with ranitidine use or consumption of three or more types of cheese since the beginning of the year [94]. The same investigators reported fecal carriage rates of 1.8% among 171 patients with gastroenteritis attending a general practice and 2.5% of 80 home hemodialysis patients. Among all three patient groups, Listeria isolations were highest during the months of July and August. Although pregnancy is a strong risk factor for clinical illness due to L. monocytogenes, fecal carriage rates do not appear to be higher among pregnant compared with nonpregnant women. Lamont and Postlethwaite reported a fecal carriage rate of 2% among 51 women early in pregnancy (10 to 16 weeks), similar to the 3.4% rate observed among 59 nonpregnant women attending the same clinic [86]. During a large foodborne listeriosis epidemic in Los Angeles, Mascola et al. compared fecal carriage rates in pregnant women with listeriosis to age-, sex-, and hospital-matched controls; carriage rates were not significantly different between the two groups (1 of 18 versus 0 of 7, respectively, p-value = NS) [97]. Timing of carriage was examined among 147 women attending an antenatal clinic in the United Kingdom [63]. One fecal specimen was obtained during each trimester. Among the four (2.7%) women whose fecal specimens yielded L. monocytogenes, one was in the first, two in the second, and one in the third trimester. The prevalence rate among 60 household contacts of 18 pregnant women with listeriosis in Los Angeles was 8.3%, whereas no Listeria were isolated from 30 household contacts of age-, sex-, and hospital-matched controls [97]. Elevated rates of fecal carriage have also been demonstrated among those with occupational exposure. Kampelmacher et al. also reported high rates of fecal carriage among laboratory workers having daily contact with L. monocytogenes (77% of 26), but rates were also high among office workers who had no contact with the organism (62% of 26). Because stool cultures were collected weekly for 8 weeks, figures from this study represent cumulative rather than point prevalence estimates [84]. Subjects had L. monocytogenes isolated an average of 1.3 times out of the eight serial specimens collected. Investigators in Denmark determined that 4.8% of 1,147 healthy slaughterhouse workers had stool cultures yielding L. monocytogenes [17]. Similar surveys by the same researchers documented fecal carriage rates of 1.2% among 1,034 hospitalized adults, none of 195 hospitalized children, and 1% of 595 hospitalized adults with diarrhea.

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Listeria, Listeriosis, and Food Safety

EPIDEMIOLOGICAL PATTERNS OF LISTERIOSIS Our understanding of listeriosis as a foodborne disease and risk factors for illness caused by infection with L. monocytogenes have increased greatly over the last 25 years through epidemiological studies of epidemic (Table 4.3) and sporadic illness. Compared with other bacterial foodborne illnesses, several characteristics of listeriosis, including the relatively low incidence and the long incubation period, make identifying outbreaks difficult. Improved laboratory-based surveillance is helping to overcome those difficulties. Beginning in 1998, PulseNet [62] has performed molecular subtyping of L. monocytogenes isolates by pulsed-field gel electrophoresis (PFGE) on a routine basis for isolates submitted to public health laboratories. The results have enhanced the ability of public health officials to detect and investigate outbreaks of listeriosis. In the 20 years before PulseNet, five Listeria outbreaks were identified in the United States; 12 Listeria outbreaks have been identified since, despite a decreasing incidence of listeriosis overall (Figure 4.1). Of those outbreaks recognized through PulseNet surveillance, four were multistate outbreaks caused by widely distributed foods, demonstrating the importance of linking geographically dispersed illnesses. Ready-to-eat meats [24,25,116] and unpasteurized cheese [26,93] have been the most frequently identified causes of Listeria outbreaks in the United States from 1998 to 2006.

EPIDEMIC LISTERIOSIS The first convincing evidence that listeriosis can be a foodborne disease came from a 1981 outbreak in Nova Scotia [130]. Thirty-four pregnancy-associated cases and seven cases in nonpregnant adults occurred over a 6-month period in the Maritime Provinces. Of the infants born alive, 27% died. No patient had evidence of underlying immunosuppression. Case-patients were significantly more likely than controls to have consumed locally produced coleslaw in the 3 months before illness onset. The epidemic strain was subsequently isolated from coleslaw in the refrigerator of one patient and later from two unopened packages of the product. Review of the production process determined that cabbage used in the coleslaw came from a farm where cases of listeriosis in sheep had occurred and that the cabbage fields had been fertilized with raw sheep manure. Harvested cabbage was stored over the winter and spring in an unheated shed, potentially enhancing growth of L. monocytogenes. As well as establishing listeriosis as a foodborne disease, this outbreak also highlighted the potential for uncooked vegetables to be a source of infection. Another outbreak of listeriosis, in Massachusetts in 1979, may also have involved raw produce [70]. Twenty patients with serotype 4b infection were hospitalized during a 2-month period during 1979; only nine cases had been detected in the previous 26 months. Ten of the patients were immunosuppressed adults and five died. Fifteen patients were thought to have acquired their infection in the hospital. Patients were more likely than controls to have consumed tuna fish, chicken salad, or cheese, but no single brand was implicated. It was postulated that raw celery and lettuce, served as a garnish with these foods, may have been the vehicle of infection. Although a definitive source of infection was not identified in this outbreak, information suggested that gastrointestinal tract conditions might be important in acquiring infection. Case-patients were significantly more likely than controls to have taken cimetidine or antacids, raising the possibility that, like salmonellosis, decreased gastric acidity might increase the chance that L. monocytogenes could survive passage through the stomach. Pasteurized milk was identified as the most likely vehicle of infection in another large outbreak of listeriosis in Massachusetts in 1983 [43]. Over a 2-month period, 49 cases occurred; 42 were in immunosuppressed adults and 7 in pregnant women. The overall case-fatality rate was 29%. Casepatients were more likely than neighborhood-matched controls to have consumed a specific brand of 2% fat pasteurized milk; other evidence supporting this milk as the vehicle of infection included

a

No. of Cases (deaths) 20 (5) 41 (18) 49 (14) 142 (48) 122 (34) — 10 (0) 279 (85) 18 (0) 48a (0) 105 2 11 5 4 6 30 13 6 54 12 3

Place

Massachusetts, U.S.A. Nova Scotia, Canada Massachusetts, U.S.A. California, U.S.A. Switzerland United Kingdom Connecticut, U.S.A. France Italy Illinois, U.S.A. Multiple states, U.S.A. Florida, U.S.A. Multiple states, U.S.A. Minnesota, U.S.A. New York, U.S.A. New York, U.S.A. Multiple states, U.S.A. North Carolina, U.S.A. California, U.S.A. Multiple states, U.S.A. Texas, U.S.A. New York, U.S.A.

Includes 45 cases of diarrhea and 3 cases of invasive disease.

1979 1981 1983 1985 1983–1987 1988–1989 1989 1992 1993 1994 1998 1999 1999 1999 1999 1999 2000 2000 2001 2002 2003 2003

Year

TABLE 4.3 Selected Foodborne Outbreaks of Invasive Listeriosis

(Raw vegetables) Coleslaw Pasteurized milk Mexican-style cheese Soft cheese P aˆ té (Shrimp) Pork tongue in jelly Rice salad Pasteurized chocolate milk Hot dog Turkey, ham, and roast beef deli meat Pate Deli meats Hot dog — Turkey deli meat Queso fresco Deli sandwich Turkey deli meat Queso fresco, unpasteurized

Implicated (or Likely) Vehicle 0 83 14 66 53 — 33 0 0 0 — — — — — — 27 92 — — — —

% Perinatal Cases

4b 4b

1/2a 4b

1/2a

4b 4b 4b 4b 4b 4b 4b 4b 1/2b 1/2b 4b

L. monocytogenes Serotype

44 85 27 56 10 66 80 38 84 23 26 26 26 26 26 26 116 93 26 25 26 26

Ref.

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a dose–response effect, a protective effect of drinking low-fat milk (1% or skim), an association between the implicated brand of milk and cases of listeriosis in another state, and the linking of a specific phage type of L. monocytogenes with infection in the 2% milk drinkers. The 2% milk came from farms where cows were known to have had listeriosis, and multiple serotypes (but not the epidemic strain) of L. monocytogenes were isolated from raw milk at the implicated dairy. No defects in the pasteurization process were noted at the dairy. Although this outbreak initially raised the question of whether pasteurization was adequate to eliminate L. monocytogenes from milk, subsequent investigations have shown that L. monocytogenes is inactivated by proper pasteurization [27]. In this outbreak, contamination likely occurred during postpasteurization handling. The largest North American outbreak of listeriosis occurred in Los Angeles County, California, in 1985; over an 8-month period, 142 cases were detected [89]. Pregnant women accounted for 93 cases and nonpregnant adults for 49 cases; 48 of the nonpregnant adult cases had predisposing conditions for listeriosis. Among the pregnancy-associated cases, 87% occurred in Hispanic women. The case-fatality rate was 32% among the perinatal cases (all were fetal or neonatal deaths) and 37% in the nonpregnant adults. A case-control study implicated a particular brand of Mexican-style soft cheese produced locally in California as the vehicle, and the epidemic serotypes and phage type were isolated from unopened packages of this product. Inadequate pasteurization and mixing of pasteurized and unpasteurized milk likely contributed to the contamination. This outbreak provided valuable data on the incubation period of invasive listeriosis because food histories were available for four patients who had a single known exposure to the implicated cheese. These patients’ median incubation period of 31 days (range: 11 to 70 days) is far longer than that observed for most common foodborne pathogens, and it highlights the difficulty in obtaining a relevant food history when investigating cases of sporadic listeriosis. This outbreak was detected quickly through public health surveillance because many infections occurred in an ethnic minority group that sought care primarily at one medical facility. However, it is likely that the outbreak would not have been as readily detected had the product been distributed over a larger geographical area or eaten by a more diverse group of consumers. Detection of outbreaks can be improved by active surveillance and timely reporting and by serotyping and molecular subtyping L. monocytogenes isolates. Another soft cheese-related outbreak occurred in Switzerland during 1983 to 1987, with 122 cases of listeriosis affecting 65 pregnant women (and their infants) and 57 nonpregnant adults [13]. Over half of the nonpregnant adult cases had no underlying predisposing condition; the case-fatality rate for nonpregnant patients was 32% [21]. Increasing age and clinical presentation with meningoencephalitis were independently associated with an increased risk of death. Neurological sequelae were present in 30% of survivors at follow-up 5 months to 3 years later. Although early case-control studies failed to incriminate a particular food, in 1987 investigators implicated a locally produced soft cheese as the vehicle of infection. Two epidemic strains of L. monocytogenes serotype 4b with a particular phage type were isolated from the product and led to an international recall. Unpasteurized cheese continues to be a source of outbreaks. In one outbreak in 2000, 13 cases of listeriosis were attributed to consumption of Mexican-style cheese made from contaminated raw milk [93]. Eleven cases involved pregnant women, in whom infection resulted in five stillbirths, three premature deliveries, and three infected neonates. Investigators isolated L. monocytogenes serotype 4b from patients, locally available fresh cheese, and raw milk from a local dairy; all isolates were indistinguishable on PFGE. This outbreak renewed attention on raw milk Mexicanstyle cheese (e.g., Queso fresco) intended for sale to Latin American consumers in the United States, increasing concern that consumption of such cheese may account for the higher proportion of listeriosis among Hispanic women [88]. Outbreaks associated with ready-to-eat meats have been identified in Europe and North America. In the United States, deli meats or hot dogs were implicated in seven Listeria outbreaks reported to CDC (Table 4.3). In one example of a multistate outbreak involving 30 patients in 11 states over

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an 8-month period, delicatessen turkey meat was implicated [116]. Among those ill, 22 (73%) were men or nonpregnant women; eight (27%) were pregnant women or mother–infant pairs. Infection resulted in deaths of four persons, two miscarriages, and one stillbirth. L. monocytogenes was isolated from product samples that matched those isolated from cases. Subsequent tracing back of contaminated delicatessen turkey meat led to two plants in different states. However, one plant served as a copacker for the other plant, and it was determined that all of the implicated meat could have come from one of the plants. Interestingly, meat (turkey franks) from the same plant was identified as a source of listeriosis in 1989 [28,155]. Molecular subtyping indicated that isolates from 1989 and 2000 were indistinguishable. This outbreak indicates that Listeria may persist in a meat plant and could intermittently contaminate food. The source of most infections is not identified; this plant may have been a source of human illness for over 12 years. In England, Wales, and Northern Ireland, the annual total of listeriosis cases approximately doubled from 1987 to 1989 compared with the annual totals for the 3 previous years. Of the 823 isolates reported during 1987 to 1989, 30 to 54% were of two subtypes of L. monocytogenes serotype 4b (4bX and 4b phage type 6, 7) that had occurred less commonly before and after this period [102]. A microbiological survey showed that L. monocytogenes contamination in meat pâté from one manufacturer was more frequent (48% of 107 samples of pâté from the implicated manufacturer versus 4% of 781 samples of pâté from other manufacturers) and heavy (11% of samples of pâté from manufacturer A with ≥1000 organisms/g versus 0.6% of samples of pâté from other manufacturers with ≥1000 organisms/g). Of pâté isolates from manufacturer A, 96% were 4bX or 4b phage type 6, 7, compared with 19% of isolates from other manufacturers. Patients infected with either of these two subtypes were significantly more likely to have eaten pâté than patients infected with other strains. Warning about pâté consumption and removal of manufacturer A’s pâté from sale in late 1989 resulted in a dramatic decrease in the incidence of listeriosis. This investigation illustrated how subtyping can help clarify surveillance data and ultimately lead to public health action. In 1992, a different ready-to-eat meat product was implicated in an outbreak of 279 cases of listeriosis in France [59]. Ninety-two cases (33%) were pregnancy related and 187 occurred in nonpregnant adults; 73 (39%) of the nonpregnant adults had no known predisposing condition for listeriosis. A case-control study implicated one brand of pork tongue in jelly as the major vehicle; however, other ready-to-eat meats, cross-contaminated by the implicated meat at retail stores where the implicated brand of pork tongue in jelly was sold, were thought to have been responsible for some infections. The epidemic strain was isolated from samples of food, and subtyping analysis helped to confirm the findings of the epidemiological investigation that implicated the pork tongue in jelly [80]. Heightened surveillance efforts in France have also led to detection of two smaller outbreaks of listeriosis. In 1993, an outbreak of 39 cases was associated with rilletes (pork pâté) [60]. In 1995, 20 cases were traced to Brie de Meaux cheese made from raw milk. Implicated cheese was removed from sale based on results of the epidemiological investigation [60].

SPORADIC DISEASE—INCIDENCE Although much has been learned about epidemic listeriosis, most cases of human listeriosis occur sporadically. The incidence of listeriosis is difficult to estimate and estimates may vary considerably depending on the methods used to identify sporadic cases. In the United States, voluntary disease reporting and hospital discharge data have been used to estimate the number of sporadic listeriosis cases [35]. However, such methods are generally insensitive and result in underestimates of the true incidence of listeriosis in the population. In 1997, only 373 cases were reported through passive surveillance to U.S. public health officials, but estimates from active surveillance at sentinel sites indicated that 1,259 cases occurred [104].

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Mead et al. assumed the true number of listeriosis cases to be two times the number of reported cases: 2,518 estimated cases per year (9.4 cases per million persons per year) resulting in 2,322 hospitalizations and 504 deaths [104]. Adak estimated 194 cases per year of listeriosis occurred in England and Wales (3.7 cases per million persons per year), resulting in 194 hospitalizations and 68 deaths per year in 2000 [1]. In France, the incidence of listeriosis was estimated from three different sources by Goulet et al. to be between 6.3 and 16.7 per million in 1987 and between 4.1 and 4.4 per million in 1997 [58]. In Denmark, an estimated six to seven cases per million per year occurred during the 1980s [82]. Japan has estimated an average incidence of 0.65 per million between 1996 and 2002 [115]. Differences in national rates should be interpreted in the context of the methods used for case ascertainment and reporting in each country. Without a consistent methodology to estimate incidence, it is difficult to evaluate trends or compare rates between countries. Efforts have been under way in the United States to establish annual incidence estimates for listeriosis. In 1986, active surveillance for listeriosis was initiated by the Centers for Disease Control and Prevention (CDC). Surveillance officers systematically contacted infection control practitioners at all acute-care hospitals and clinical microbiology laboratories in the study areas to collect information on all patients from whom L. monocytogenes was isolated from a normally sterile site [54,132]. In 1986, the annual incidence of listeriosis was 7 cases per million; in 1989, 7.9 per million; and in 1993, 4.4 per million. The declining incidence since 1989 was distributed uniformly throughout the different geographical areas and was attributed to regulatory and educational efforts [143]. Beginning in 1996, the Foodborne Disease Active Surveillance Network (FoodNet) of the CDC’s Emerging Infections Program collected data from several well-defined populations in the United States in representing different geographical areas. To identify cases, FoodNet contacted clinical laboratories weekly or monthly depending on lab size. The incidence of listeriosis was five per million persons in 1996 and 1997 [149]. Comparing 2004 to the average between 1996 and 1998, the incidence of listeriosis declined 40% to 2.7 per million persons [30], which suggests a real and continued reduction in illness (see section on prevention). Based on these data, the projected number of deaths per year due to listeriosis would have declined from 504 in 1997 to 302 in 2004. In the United States, rates have varied by geographical site. Rates in San Francisco (9.3 per million) were twice those in Oklahoma (4.8 per million) [132]. In 1986, perinatal listeriosis rates were highest in Los Angeles (24.3 per 100,000 live births), whereas nonperinatal rates did not vary significantly among the study sites. No clear seasonal trends were noted [54]. Of the 246 cases reported in 1986, 67 (27%) were perinatal and 179 (73%) were nonperinatal. Between 1989 and 1993, pregnancy-associated cases accounted for about one third of all cases each year [143]. Serotypes 4b (43%), 1/2b (35%), and 1/2a (20%) accounted for almost all infections. Surveillance data reported to the CDC from state public health laboratories from 2000 to 2004 indicate the distribution of illness by age group, sex, and ethnicity (Figure 4.2 and Figure 4.3) [40]. The greatest number of cases occurs among infants and the elderly (Figure 4.2). For adults age 15 to 35 years, listeriosis occurs in a higher percentage of women than men of the same age group. The excess numbers of cases in women of childbearing age compared with men, combined with the number of infant cases, represent the burden of pregnancy and perinatal listeriosis. From 2000 to 2004, a far greater percentage of listeriosis occurred in Hispanics from 15 to 35 years of age than in non-Hispanics (Figure 4.3). This difference is largely due to pregnancy and perinatal infections in Hispanic women, who represent an important target group for prevention programs.

SPORADIC DISEASE—DIETARY RISK FACTORS Dietary risk factors for sporadic listeriosis were assessed through case-control studies conducted in the CDC active surveillance project. Patients identified through surveillance were enrolled and matched by age, underlying disease, and healthcare provider (as an indirect measure of geographical location and socioeconomic status). In the 1986 to 1987 study, 82 patients and 239 controls were enrolled.

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10

Single state outbreak Multistate outbreak

9 8 7 6 5 4 3 2 1 0 1978

1980

1982

1984

1986

1988

1990

1992

1994

1996

1998

2000

2002

2004

FIGURE 4.2 Incidence of reported cases (per one million persons; data from active surveillance systems [2004 data are preliminary]) (From Centers for Disease Control and Prevention. 2005. Morbidity Mortality Wkly. Rep. 54:352–356; Gellin, B. G. et al. 1991. Am. J. Epidemiol. 133:392–401; Tappero, J. W. et al. 1995. JAMA 273:1118–1122) and outbreaks of Listeria (outbreaks of confirmed Listeria monocytogenes reported to CDC’s Foodborne Outbreak Reporting System. From Centers for Disease Control and Prevention. 2005. U.S. Foodborne Disease Outbreaks. Published on the World Wide Web at http://www.cdc.gov/foodborneoutbreaks/us_outb.htm), United States, 1973 to 2004.)

Case-patients were significantly more likely than controls to have eaten uncooked or nonreheated hot dogs (frankfurters) or undercooked chicken. An estimated 20% of the overall risk of listeriosis was thought to be attributable to consumption of these foods [136]. The first human case of listeriosis that was microbiologically linked to consumption of ready-to-eat poultry products occurred in 1989. A strain of L. monocytogenes serotype 1/2a with an identical multilocus enzyme elctrophoretic type was isolated from the blood of a patient with cancer, an open package of turkey frankfurters and other opened foods in the patient’s refrigerator, and unopened packages of turkey frankfurters at a retail store [28]. Subsequent investigation of the

35%

Percent of Total

30% HISPANIC (N=228)

25% 20%

NON-HISP (N=918)

15% 10% 5% 0% <1

>=1-2

>=2-5

>=5-15

>=15-25 >=25-35 >=35-45 >=45-55 >=55-65 >=65-75

>=75

Age Group

FIGURE 4.3 Percent of reported laboratory-confirmed listeriosis by age groups for Hispanic and non-Hispanic ethnicity, United States, 2000 to 2004. Isolation of Listeria monocytogenes reported to the CDC through the Public Health Laboratory Information System (PHLIS).

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turkey frankfurter production facility found that cultures from a conveyor belt transporting finished frankfurters yielded the case strain of L. monocytogenes [155]. Systematic culturing at various points in the production process identified likely points where L. monocytogenes was introduced into the product and suggested appropriate control points for reducing contamination in such foodprocessing facilities. From 1988 to 1990, a larger case-control study of 165 patients and 376 controls was conducted that included microbiological assessment of foods eaten by patients [132]. Case-patients were significantly more likely than controls to have eaten soft cheeses or delicatessen-counter foods. In a separate analysis examining dietary risks among a subset of patients defined as highly immunosuppressed (persons with malignancy, AIDS, or organ transplants or who had received corticosteroids or chemotherapy), consumption of undercooked chicken was associated with a threefold increased risk of listeriosis. Other exposures associated with an increased risk of sporadic disease included recent use of antacids, laxatives, or H2-blocking agents. In the microbiological component of this study, foods were collected from the refrigerators of 123 patients [120]. L. monocytogenes was isolated from at least one food in the refrigerators of 64% of patients. Highest contamination rates among the 2,013 food specimens were seen in beef (36%) and poultry (31%) with 7.6% of ready-to-eat foods (processed meats, raw vegetables, leftovers, and cheeses) also yielding L. monocytogenes. One third of refrigerators contained food isolates of L. monocytogenes that were the same multilocus enzyme elctrophoretic type as those isolated from the patient. In multivariate analysis, foods that were ready to eat, foods that contained high numbers of L. monocytogenes, and foods that yielded serovar 4b were associated with disease. Dietary risk factors for sporadic listeriosis were also examined in a recent study in Denmark; drinking unpasteurized milk or eating pâté were the only risk factors identified [82]. However, one third of cases reported during the study period could not be included in the risk analysis for sporadic disease because the ill persons were infected with an outbreak strain epidemiologically linked to Danish blue-mold cheeses.

SPORADIC DISEASE—POSSIBLE OTHER SOURCES Transmission by routes other than food may play a role in a few cases of sporadic listeriosis. Sexual transmission of L. monocytogenes has been hypothesized as a possible route in perinatal listeriosis; however, there is no evidence to support this [121]. Because L. monocytogenes can cause asymptomatic bacteremia and survives refrigeration, it is theoretically possible that transmission through donated blood could occur. Such transmission has been documented for Yersinia enterocolitica but has not yet been described for L. monocytogenes [144]. Nosocomial transmission of L. monocytogenes has been documented in a variety of settings [34,61,70,73,74,81] in addition to the transmission among newborns in a nursery mentioned earlier.

DIAGNOSIS Diagnosis of invasive listeriosis depends on isolation of L. monocytogenes from a normally sterile site such as blood or cerebrospinal fluid. Because the organism may be mistaken for a diphtheroid contaminant on Gram stain, complete bacteriological evaluation should be done. Recovery of the organism from stool samples is helpful when febrile gastroenteritis is suspected, but isolation from stool by itself is not diagnostic because asymptomatic carriage occurs. To document an outbreak of febrile gastroenteritis, the isolation rate among symptomatic persons should be significantly higher than among asymptomatic persons. L. monocytogenes strains isolated from sterile-site specimens usually grow well in routinely used media. The specimen is directly plated on tryptic soy agar containing 5% sheep, horse, or rabbit blood. The organism is usually identified within 36 hours. Isolation of the organism from other sources such as stool specimens that contain large numbers of competing microorganisms is

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more difficult; these specimens should be selectively enriched for Listeria spp. before being plated on Listeria-selective media. Identification of L. monocytogenes by use of fluorescent antibody methods or approaches that use DNA probes coupled with PCR technology may prove useful for some specimens. Experimental assays for antibody to listeriolysin O have been useful in some epidemiological investigations [37] and have been used to support the diagnosis in culture-negative listeriosis of the central nervous system [51].

TREATMENT Controlled trials to determine the optimal antibiotic therapy for listeriosis have not been done. The treatment of choice is high doses of amoxicillin I.V. (2 to 3 g three to four times a day), with an additional dose of gentamicin (360 mg once a day) for nonpregnant adults [72]. The role of aminoglycosides is poorly understood because they penetrate cells poorly and may be ineffective in the living host. L. monocytogenes has grown in cells despite high extracelluar concentrations of aminoglycosides [69]. However, the majority of Listeria may be extracellular in cases of meningitis [71]. Trimethoprim-sulfamethoxazole readily enters cells and kills L. monocytogenes. This drug combination has proved effective in patients with listeriosis who have hypersensitivity to penicillin [5]. Compared with the aminopenicillins, newer quinolone antimicrobials may have increased bacteridicidal activity, cross the blood–brain barrier into the CNS, and accumulate within host cells, but clinical studies have not been done [72]. The ability of L. monocytogenes to grow and survive within cells probably explains the poor response to bacteriostatic drugs and the slow response to penicillin [141]. Bacteriostatic drugs such as chloramphenicol or tetracycline have been associated with high treatment failure rates, so they are not recommended [141]. Cephalosporins are not recommended for treatment because they have a low affinity for the penicillin-binding protein of Listeria [147]. Relapses have been reported in immunosuppressed patients after 2 weeks of penicillin therapy [150]. Because many immunosuppressed patients have a decreased ability to clear infected cells, antibiotic treatment for 3 to 6 weeks may be prudent [6]. Optimal length of therapy for other groups of patients has not been established. A prudent treatment course may be 2 weeks for listeriosis in pregnancy, 2 to 3 weeks for neonatal listeriosis, 2 to 4 weeks for nonimmunosuppressed adults with meningitis and bacteremia, and longer for complicated infections such as endocarditis.

PREVENTION Recognition that most human listeriosis is foodborne has led to control measures that have reduced the incidence of listeriosis. Healthy People 2010 goals established for the United States called for a reduction of foodborne listeriosis by 50% by the end of the year 2005; by 2004, those goals were nearly met [29]. The observed decrease in perinatal and nonperinatal cases since 1989 is likely the result of enhanced listeriosis prevention efforts by the U.S. food industry, including enforcement by regulatory agencies of a zero-tolerance policy for processed meat and intensified clean-up programs in meat-processing facilities. In 2001 and 2003, the Food and Drug Administration (FDA), CDC, and the U.S. Department of Agriculture (USDA) released a national Listeria Action Plan to help guide control efforts by industry, regulators, and public health officials [45,46]. Those plans called for multiple points of action, including increased regulatory guidance over the manufacture of ready-to-eat foods. Also in 2003, following a large outbreak linked to deli turkey meat, the USDA issued new regulations aimed at further reducing L. monocytogenes contamination of ready-to-eat meat and poultry products [39]. Published dietary recommendations for consumers may also have contributed to the decreased disease incidence [29,44,137]. Noncommercial sources of food, such as raw milk cheese, continue to be sources of listeriosis, especially among Latin American women [33,146].

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For persons who are at increased risk for listeriosis, including those who are pregnant or immunocompromised, specific dietary measures can be taken to decrease risk [23]. Such persons should avoid high-risk foods such as hot dogs; deli meats or luncheon meats (particularly deli sliced), unless they are reheated until steaming hot; soft cheeses (such as feta, Brie, and Camembert, blue-veined cheeses, or Mexican-style cheeses such as queso blanco, queso fresco, and Panela), unless they have labels that clearly state they are made from pasteurized milk; unpasteurized (raw) milk; refrigerated p aˆ tés or meat spreads (canned or shelf-stable p aˆ tés and meat spreads may be eaten). In addition, they should avoid getting fluid from hot dog or other deli meat packages on other foods or food preparation surfaces and wash hands after handling hot dogs and deli meats. Dietary and food preparation measures have been recommended to the general public; these should decrease the risk not only of listeriosis but also of other common foodborne diseases, such as salmonellosis and campylobacteriosis. These measures include thorough cooking of raw food from animal sources; washing raw vegetables thoroughly before eating; keeping uncooked meats separate from vegetables, cooked foods, and ready-to-eat foods; avoiding raw (unpasteurized) milk or foods made from raw milk; and washing hands, knives, and cutting boards after handling uncooked foods [29]. In addition to individual advice for consumers, control of listeriosis requires action from public health agencies and the food industry. Important control strategies from public health agencies include developing and maintaining timely and effective disease surveillance programs, promptly investigating clusters of listeriosis cases, and enforcing current regulations designed to minimize L. monocytogenes in foods consumed without further cooking. A survey in the United States found that 1.8% of ready-to-eat foods were contaminated with L. monocytogenes [57]. For foods such as ready-to-eat salad vegetables, only rare servings may be contaminated but the level of contamination may be high [127]. It is unlikely that such contamination would be found during routine product testing. It is imperative therefore that the food industry develop an understanding of how contamination occurs and then implement hazard analysis critical control point (HACCP) programs to minimize the presence of L. monocytogenes at important points in the processing, distribution, and marketing of processed foods [2].

REFERENCES 1. Adak, G. K., S. M. Long, and S. J. O’Brien. 2002. Trends in indigenous foodborne disease and deaths, England and Wales: 1992 to 2000. Gut 51:832–841. 2. Anonymous. 1991. Listeria monocytogenes: recommendations by the National Advisory Committee on microbiological criteria for foods. Int. J. Food Microbiol. 14:185–246. 3. Anspacher, R., K. A. Borchardt, M. W. Hannegan, and W. A. Boyson. 1966. Clinical investigation of Listeria monocytogenes as a possible cause of human fetal wastage. Am. J. Obstet. Gynecol. 94:386–390. 4. Anton, W. 1934. Kritisch-experimentaller Beitrag zur Biologie des Bakterium monocytogenes. Zentralb. Bakteriol. Mikrobiol. Hyg. A. 131:89–103. 5. Armstrong, D. 1995. Listeria monocytogenes. New York: Churchill Livingstone. 6. Armstrong, R. W., and P. C. Fung. 1993. Brainstem encephalitis (rhombencephalitis) due to Listeria monocytogenes: Case report and review. Clin. Infect. Dis. 16:689–702. 7. Aureli, P., G. C. Fiorucci, D. Caroli, G. Marchiaro, O. Novara, L. Leone, and S. Salmaso. 2000. An outbreak of febrile gastroenteritis associated with corn contaminated by Listeria monocytogenes. New Engl. J. Med. 342:1236–1241. 8. Ballen, P. H., F. R. Loffredo, and B. Painter. 1979. Listeria endophthalmitis. Arch. Ophthalmol. 97:101–102. 9. Baloga, A. O., and S. K. Harlander. 1991. Comparison of methods for discrimination between strains of Listeria monocytogenes from epidemiological surveys. Appl. Environ. Microbiol. 57:2324–2331. 10. Bassan, R. 1986. Bacterial endocarditis produced by Listeria monocytogenes: Case presentation and review of the literature. Am. J. Clin. Pathol. 63:522–527.

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129. Schlech, W. F. 1997. Listeria gastroenteritis—old syndrome, new pathogen. New Engl. J. Med. 336:130–132. 130. Schlech, W. F., P. M. Lavigne, R. A. Bortolussi, A. C. Allen, E. V. Haldane, A. J. Wort, A. W. Hightower, S. E. Johnson, S. H. King, E. S. Nicholls, and C. V. Broome. 1983. Epidemic listeriosis—evidence for transmission by food. New Engl. J. Med. 308:203–206. 131. Schuchat, A. 1997. Listeriosis and pregnancy: Food for thought. Obstet. Gynecol. Surv. 52:721–722. 132. Schuchat, A., K. Deaver, J. D. Wenger, B. D. Plikaytis, L. Mascola, R. W. Pinner, A. L. Reingold, C. V. Broome, and T. L. S. Group. 1992. Role of foods in sporadic listeriosis. I: Case-control study of dietary risk factors. JAMA 267:2041–2045. 133. Schuchat, A., K. A. Deaver, P. S. Hayes, L. Graves, L. Mascola, and J. D. Wenger. 1993. Gastrointestinal carriage of Listeria monocytogenes in household contacts of patients with listeriosis. J. Infect. Dis. 167:1261–1262. 134. Schuchat, A., C. Lizano, C. V. Broome, B. Swaminathan, C. Kim, and K. Winn. 1991. Outbreak of neonatal listeriosis associated with mineral oil. Pediatr. Infect. Dis. 10:183–189. 135. Schuchat, A., B. Swaminathan, and C. V. Broome. 1991. Epidemiology of human listeriosis. Clin. Microbiol. Rev. 4:169–183. 136. Schwartz, B., C. A. Ciesielski, C. V. Broome, S. Gaventa, G. R. Brown, B. G. Gellin, A. W. Hightower, L. Mascola, and T. L. S. Group. 1988. Association of sporadic listeriosis with consumption of uncooked hotdogs and undercooked chicken. Lancet 2:779–782. 137. F.S.A.I. Service. 1992. Backgrounder: Listeria monocytogenes. Washington, D.C.: U.S. Department of Agriculture. 138. Siegman-Igra, Y., R. Levin, M. Weinberger, Y. Golan, D. Schwartz, Z. Samra, H. Konigsberger, A. Yinnon, G. Rahav, N. Keller, N. Bisharat, J. Karpuch, R. Finkelstein, M. Alkan, Z. Landau, J. Novikov, D. Hassin, C. Rudnicki, R. Kitzes, S. Ovadia, Z. Shimoni, R. Lang, and T. Shohat. 2002. Listeria monocytogenes infection in Israel and review of cases worldwide. Emerg. Infect. Dis. 8:305–10. 139. Simmons, M. D., P. M. Cockroft, and O. A. Okubadejo. 1986. Neonatal listeriosis due to crossinfection in an obstetric theatre. J. Infect. 13:235–239. 140. Soo, M. S., R. D. Tien, L. Gray, P. I. Andrews, and H. Friedman. 1993. Mesenrhombencephalitis: MR findings in nine patients. Am. J. Radiol. 160:1089–1093. 141. Southwick, F. S., and D. L. Purich. 1996. Intracellular pathogenesis of listeriosis. New Engl. J. Med. 334:770–776. 142. Synnott, M. B., D. L. Morse, and S. M. Hall. 1994. Neonatal meningitis in England and Wales: A review of routine national data. Arch. Dis. Child. 71:F75–80. 143. Tappero, J. W., A. Schuchat, K. A. Deaver, L. Mascola, J. D. Wenger, and T. L. S. Group. 1995. Reduction in the incidence of human listeriosis in the United States: Effectiveness of prevention efforts? JAMA 273:1118–1122. 144. Tipple, M. A., L. A. Bland, J. J. Murphy, M. J. Arduino, A. L. Panlilio, J. J. Farmer, M. A. Touralt, C. R. McPherson, J. E. Menitove, A. J. Grindon, P. S. Johnson, R. G. Strauss, J. A. Bufill, P. S. Ritch, J. R. Archer, O. C. Tablan, and W. R. Jarvis. 1990. Sepsis associated with transfusion of red cells contaminated with Yersinia enterocolitica. Transfusion 30:207–213. 145. Uldry, P. A., T. Kuntzer, J. Bogousslavsky, F. Regli, J. Miklossy, J. Bille, P. Francioli, and R. Janzer. 1993. Early symptoms and outcome of Listeria monocytogenes rhombencephalitis: 14 adult cases. J. Neurol. 240:235–242. 146. Van Kessel, J. S., J. S. Karns, L. Gorski, B. J. McCluskey, and M. L. Perdue. 2004. Prevalence of Salmonellae, Listeria monocytogenes, and fecal coliforms in bulk tank milk on U.S. dairies. J. Dairy Sci. 87:2822–2830. 147. Vicente, M. F., J. Berenguer, M. A. de Pedro, J. C. Perez-Diaz, and F. Baquero. 1990. Penicillin binding proteins in Listeria monocytogenes. Acta Microbiol. Hung. 37:227–231. 148. Visintine, A. M., J. M. Oleska, and A. J. Nahmis. 1977. Listeria monocytogenes infection in infants and children. Am. J. Dis. Child. 131:393–397. 149. Wallace, D. J., T. Van Gilder, S. Shallow, T. Fiorentino, S. D. Segler, K. E. Smith, B. Shiferaw, R. Etzel, W. E. Garthright, and F. J. Angulo. 2000. Incidence of foodborne illnesses reported by the foodborne diseases active surveillance network (FoodNet)—1997. FoodNet Working Group. J. Food Prot. 63:807–809.

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150. Watson, G. W., T. J. Fuller, J. Elms, and R. M. Kluge. 1978. Listeria cerebritis: Relapse of infection in renal transplant patients. Arch. Intern. Med. 138:83–87. 151. Weis, J. 1975. The incidence of L. monocytogenes on plants and in soil. In Problems of listeriosis, ed. M. Woodbine. Leicester, U.K.: Leicester University Press, pp. 61–65. 152. Welshimer, H. J. 1960. Survival of Listeria monocytogenes in soil. J. Bacteriol. 80:316–320. 153. Welshimer, H. J. 1968. Isolation of L. monocytogenes from vegetation. J. Bacteriol. 95:300–303. 154. Wenger, J. D., A. W. Hightower, R. R. Facklam, S. Gaventa, and C. V. Broome. 1990. Bacterial meningitis in the United States, 1986: Report of a multistate surveillance study. The Bacterial Meningitis Study Group. J. Infect. Dis. 162:1316–1323. 155. Wenger, J. D., B. Swaminathan, P. S. Hayes, S. S. Green, M. Pratt, R. W. Pinner, A. Schuchat, and C. V. Broome. 1990. Listeria monocytogenes contamination of turkey franks: Evaluation of a production facility. J. Food Prot. 53:1015–1019.

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Virulence 5 Molecular Determinants of Listeria monocytogenes Michael Kuhn and Werner Goebel CONTENTS Introduction ....................................................................................................................................111 Molecular Aspects of the Invasion of Mammalian Cells..............................................................113 Invasion of Nonprofessional Phagocytic Cells ....................................................................114 Cellular Adhesion .................................................................................................................118 Uptake by Macrophages and Dendritic Cells......................................................................118 Escape from the Phagocytic Vacuole.............................................................................................120 Growth in the Host Cell Cytoplasm ..............................................................................................125 Intracellular Motility and Cell-to-Cell Spread ..............................................................................126 Bile Salt Hydrolase—A Novel Virulence Factor ..........................................................................129 Accessory Virulence Factors..........................................................................................................130 p60, Product of the iap Gene...............................................................................................130 Superoxide Dismutase and Catalase ....................................................................................131 Stress Response Mediators...................................................................................................131 Iron Uptake Systems ............................................................................................................132 PrfA and Regulation of Virulence Gene Expression in L. monocytogenes ..................................132 The Positive Regulatory Factor A (PrfA) ............................................................................132 PrfA-Dependent Promoters, Transcripts, and Mechanism of Temperature-Dependent Virulence Gene Expression......................................................134 Environmental Signals Affecting Virulence Gene Expression ............................................136 Two-Component Systems and Regulation of Virulence Gene Expression .........................137 Lessons Learned from Genome Sequence of L. monocytogenes .................................................137 Evolutionary Aspects .....................................................................................................................138 Open Questions ..............................................................................................................................139 Acknowledgments ..........................................................................................................................139 References ......................................................................................................................................139

INTRODUCTION Studies that aimed to unravel the mechanisms of Listeria monocytogenes pathogenicity and its interaction with hosts on the cellular, molecular, and genetic levels were initiated about two decades ago. The early studies used transposon mutagenesis and infection of primary and established cell lines to obtain insights into the interaction of L. monocytogenes with eucaryotic host cells. The recent sequencing of the genomes of L. monocytogenes and Listeria innocua together with development of genetic tools now allows manipulation of L. monocytogenes, which has, together with cell culture 111

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and transgenic animal models, greatly broadened our understanding of the molecular and cell biology of L. monocytogenes infections. Most of the early studies on the cell biology of L. monocytogenes infections used epithelialike and macrophage-like cell lines [103,229,315]. Macrophages actively ingest L. monocytogenes, but internalization of the bacterium by normally nonphagocytic cells is triggered by L. monocytogenes -specific products. Aside from the internalization step, the intracellular life cycle of the bacteria in phagocytes or normally nonphagocytic mammalian cells is, however, very similar. The pathogen first appears in a vacuole, which is subsequently lysed by some of the ingested bacteria allowing L. monocytogenes to escape into the cytoplasm. Whereas most of the bacteria begin to replicate in the cytoplasm, those remaining in the phagosome are killed and digested. Concomitant with the onset of intracellular replication, L. monocytogenes induces nucleation of host actin filaments arranged to a polar tail. Formation of a tail at one pole of the bacterial cell produces a propulsive force that moves the bacteria through the cytoplasm. Bacteria that reach the surface of the infected host cell induce the formation of pseudopode-like structures with the bacterium at the tip and the actin tail behind it. These pseudopods are taken up by neighboring cells. The bacteria thus entering the neighboring cells are within a vacuole surrounded by a double membrane, which is subsequently lysed to release the bacteria into the cytoplasm of the newly infected host cell. The line drawing shown in Figure 5.1 summarizes this intracellular life cycle. The different steps and the listerial virulence factors involved in this life cycle are discussed in detail later.

FIGURE 5.1 Schematic drawing of the intracellular life cycle of Listeria monocytogenes (A) and representative electron micrographs showing adhesion (B), entry (C), bacteria inside vacuoles (D), bacteria free in the cytoplasm (E), moving bacteria in protrusions (F and G), and bacteria in a double membrane vacuole formed during cell-to-cell spread (H). (Reprinted with permission from Vazquez-Boland, J. A. et al. 2001. Clin. Microbiol. Rev. 14:584–640.)

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FIGURE 5.2 Organization of the central virulence gene cluster of L. monocytogenes and structure of the locus in other Listeria species. Genes belonging to the virulence gene cluster are in gray, and the flanking loci are in black. The virulence gene cluster is inserted in a chromosomal region delimited by the prs and ldh genes. In the plcB-ldh intergenic region, two ORFs, orfA and orfB, are found in all Listeria species, indicating that the insertion point of the cluster is between the prs and orfB loci. In the plcB–orfB intergenic region of L. monocytogenes, there are two small ORFs, orfX and orfZ (stippled), which delimit the putative excision point of the cluster in L. innocua. In the plcB–orfB intergenic region of L. ivanovii, two small ORFs, orfX (encoding a homologue of the orfX product from L. monocytogenes) and orfL (stippled), are also present, which, like those in L. monocytogenes, delimit the deletion point of the cluster in the nonpathogenic species L. welshimeri. The additional ORFs found in the L. seeligeri virulence gene cluster are hatched. (Reprinted with permission from Vazquez-Boland, J. A. et al. 2001. Clin. Microbiol. Rev. 14:584–640.)

Some of the known virulence genes whose products are involved in the intracellular life cycle of L. monocytogenes are clustered on the chromosome in the so-called PrfA-dependent virulence gene cluster. The cluster comprises six well-characterized genes, prfA, plcA, hly, mpl, actA, and plcB (Figure 5.2) along with four small open reading frames (ORFs) of unknown functions downstream of plcB, called orf X, Z, B, and A. The ends of the gene cluster are defined by genes coding for housekeeping enzymes. Distal from prfA, defining the “left” border of the gene cluster, the prs gene is located, encoding a phosphoribosyl-pyrophosphate synthetase [117,127,185]. The ldh gene coding for lactate dehydrogenase together with the orfs A and B [45,117,127,319] mark the “right” border of the gene cluster downstream from plcB and the small orfs X and Z. The products of these virulence genes are listeriolysin (LLO, encoded by hly), a phosphatidylinositolspecific phospholipase C (PI-PLC, encoded by plcA), a phosphatidylcholine-specific phospholipase C (PC-PLC, encoded by plcB), a metalloprotease (Mpl, encoded by mpl), ActA, a protein involved in actin polymerization (encoded by actA), and the positive regulatory factor PrfA (encoded by prfA). The well-studied internalins internalin A (InlA) and InlB are encoded by the inlAB operon [102]. Many other internalin-like genes are found dispersed around the L. monocytogenes chromosome [38,117]. Several other genes suggested to play a role in virulence are located outside the virulence gene cluster [320]. Some of them are, however, connected to the virulence cluster genes because they are also regulated by the transcriptional activator PrfA (discussed later).

MOLECULAR ASPECTS OF THE INVASION OF MAMMALIAN CELLS Uptake of L. monocytogenes by macrophages of different origin is well documented [183,208,253]. Invasion of L. monocytogenes into different, normally nonphagocytic mammalian cell types, including murine and human fibroblasts [87,142,183,253], murine and human epithelial cells

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[8,87,103,253], murine hepatocytes [76,332], human endothelial cells [82,130,131,293,294], and mouse and human dendritic cells [135,173,174,247], has also been described.

INVASION

OF

NONPROFESSIONAL PHAGOCYTIC CELLS

Transposon mutagenesis and an appropriate in vitro invasion assay using Caco-2 epithelial cells resulted in the identification of internalin (InlA), a surface protein of L. monocytogenes, to mediate bacterial invasion into epithelial cells [102]. The mutants identified exhibit a lower invasive capacity than the wild-type strain when tested on different cells. Transposon insertions occur in a chromosomal region, which represents an operon consisting of the inlA and inlB genes. Expression of inlA in L. innocua, a noninvasive Listeria species closely related to L. monocytogenes, renders this species invasive (at least to some extent). This experiment shows that the inlA gene product is necessary and sufficient to mediate invasion. A large number of internalin homologues have since been identified in L. monocytogenes [38,78,89,117,257]. Common to all internalins is an element of several leucine-rich repeats with leucine residues at a fixed position in a typical 22 amino acid (aa) unit. Internalin is an acidic protein of 800 amino acids [77,102] that possesses two extended repeat domains. Domain A consists of 15 leucine-rich repeats, whereas domain B consists of 2.5 repeats of about 70 amino acids (aa) each. The InlA protein has a typical N-terminal transport signal sequence and a cell wall anchor in the C-terminal part comprising the sorting motif LPXTG followed by a hydrophobic membrane-spanning region of 20 aa and a few positively charged aa (Figure 5.3) [77]. This distal LPXTG motif, like similar motifs in other surface proteins covalently linked to the peptidoglycan in Gram-positive bacteria, is responsible for the attachment of InlA to the bacterial cell envelope in a process mediated by the enzyme sortase [21,68,109]. InlB, a 630-amino acid protein also carries an N-terminal transport signal sequence, eight leucine-rich repeats and three C-terminal GW modules; however, in contrast to InlA, it has no LPXTG motif and no cell wall-spanning region (Figure 5.3) [77]. Nevertheless, InlB is a listerial surface protein targeted to the bacterial surface via the interaction of the GW modules with lipoteichoic acid in the listerial cell wall. This type of association appears to be relatively weak because significant amounts of InlB are found in the supernatant fluid [27,158]. To get more insight into the molecular details of InlA- and InlB-mediated cellular invasion, three-dimensional structures of important parts of both proteins were solved at the atomic level [211,284,285]. In InlA and InlB (and also InlH [284]), three N-terminal parts in each protein are combined to form a contiguous internalin domain. In this internalin domain, a central LRR region is flanked contiguously by a truncated EF-hand-like cap and an immunoglobulin-like fold (Figure 5.3). The extended beta-sheet, resulting from the distinctive fusion of the LRR and the immunoglobulin-like folds, constitutes an adaptable concave interaction surface proposed to interact with the respective mammalian receptor molecules during infection. In the case of InlB it was shown that four surface-exposed aromatic amino acids along its concave face are essential for host cell invasion and binding to its receptor Met (discussed later) [206]. Four eucaryotic receptors for internalin and InlB were recently identified [28,159,219,290]. Human E-cadherin was identified as the internalin receptor by a biochemical approach using matrixbound purified InlA to isolate the internalin ligand from epithelial membrane proteins [219]. A member of the cadherin family, E-cadherin is mainly expressed at the basolateral site of enterocytes and is a major constituent of adherence junctions, where it connects adjacent cells by homophilic interactions of its extracellular domains [232]. It binds internalin directly and its location on the basolateral membrane of epithelial cells is in line with previous observations suggesting the basolateral membrane as the entry site for L. monocytogenes [311]. Antibodies directed against the leucine-rich repeat region of internalin block entry of L. monocytogenes into cells expressing E-cadherin, thereby underlining the importance of the repeat regions of internalin for its function as an invasin [219].

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(A)

(B)

FIGURE 5.3 A: Structure of the members of the internalin multigene family present in L. monocytogenes strain EGD-e. S: signal peptide; B: B-repeats; C: Csa domain repeats C-repeat; D: D-repeats. See text for details. (Reprinted with permission from Vazquez-Boland, J. A. et al. 2001. Clin. Microbiol. Rev. 14:584–640.) B: Crystal structure of the N-terminal part of InlA and the distal domain of its receptor E-cadherin. (Reprinted with permission from Heesemann, J. et al. 2003. Biospektrum 9:486–489.)

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The first InlB receptor to be isolated also by a biochemical approach was the complement receptor for the globular part of the C1q-fragment (gC1q-R). Direct interaction of the ubiquitously expressed gC1q-R and InlB was demonstrated and it could be shown that soluble C1q or antibodies against gC1q-R block InlB-mediated entry of L. monocytogenes [28]. The receptor tyrosine kinase Met was recently identified as a second InlB receptor required for InlB-dependent entry of L. monocytogenes into various cells [290]. Treatment of mammalian cells with InlB protein or infection with L. monocytogenes induces rapid tyrosine phosphorylation of Met, a receptor tyrosine kinase for which the only other known ligand is hepatocyte growth factor (HGF). Like HGF, InlB binds to the extracellular domain of Met and induces “scattering” of epithelial cells. Finally, glucosaminoglycans (GAGs) were identified as a third type of InlB ligand [159]. GAGs are present on the surface of mammalian cells, where they decorate the proteoglycans; they promote the oligomerization of growth factors such as HGF. InlB binds to GAGs through its C-terminal GW repeats, which anchor the protein to the bacterial cell surface. GAGs are hence believed to detach InlB from the bacterial surface, thus allowing its interaction with the Met receptor at the contact site of L. monocytogenes on the host cell surface [210]. L. monocytogenes uptake by macrophages and other mammalian cells depends on functional actin microfilaments because invasion requires membrane extensions formed by rearrangement of actin filaments and is hence blocked by treatment with actin depolymerizing drugs such as cytochalasins [103,183]. L. monocytogenes is taken up in a way described as a zipper mechanism, which means that the host cell membrane is in close contact with the bacterium without surface changes in the vicinity. Entry can also be blocked by tyrosine kinase inhibitors such as genistein [272,310,322] and the tyrosin phosphatase inhibitor vanadate [180], which shows that signal transduction events involving various cellular kinases and phosphatases are needed. Epithelial cell invasion by L. monocytogenes is initiated by binding of InlA to its receptor E-cadherin. The structural basis for this highly specific interaction became obvious recently, when the crystal structure of the functional InlA domain, bound to the N-terminal domain of its receptor E-cadherin, was solved [285]. The concave interaction domain formed mainly by the LRR surrounds and specifically recognizes human E-cadherin. Individual amino acid residues in InlA were probed for their role in the InlA-E-cadherin interaction. These studies explained the tremendous specificity of InlA for human E-cadherin [191] because the aa proline at position 16 in human E-cadherin (which is changed to glutamic acid in the mouse homologue) represents a very exposed aa in the molecule responsible for the intimate contact with InlA [285]. The cytoplasmic domain of Ecadherin directly interacts with β-catenin, which in turn recruits α-catenin [193]. α-Catenin directly binds to actin filaments and hence completes the direct link of the listerial receptor to the host cell cytoskeleton. Other proteins normally present in the adherence junction complex are also recruited to the site of L. monocytogenes entry [54], but their precise role in the uptake process, and the rearrangement of the actin network particularly, is unknown. Originally proposed to be a specific factor for hepatocyte invasion [76], InlB was recently shown to be the only relevant internalin for endothelial cell invasion [17,129,130,244]. InlB-specific invasion of human brain microvascular endothelial cells is very sensitive to the presence of adult human serum, which regularly harbors anti-Listeria antibodies [145]. InlB is also involved in epithelial cell invasion [17,203], but InlA and InlB play no role in fibroblast invasion [203]. As mentioned before, three mammalian receptors for InlB have been characterized and a complex series of intracellular signal transduction events are triggered by the interaction of InlB with Met (reviewed in detail by Bierne and Cossart [19] and Cossart et al. [54]), the only one of the three receptors that has a cytoplasmic domain. The PI-3 kinase is in the center of the signal transduction pathway leading to actin rearrangement [150] and is most likely activated upon interaction with adaptor proteins recruited to the phosphorylated cytoplasmic domain of Met [151,290]. The generation of PIP3 by the PI-3 kinase [150] then leads to an activation of the downstream kinases Rac, PAK, and the LIM-kinase [20]. It is currently believed that these events finally lead to the activation of the Arp2/3 complex (see later discussion),

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which is necessary to induce the actin polymerization involved in rearrangement of the actin cytoskeleton during the uptake process [54]. Additionally, the InlB-Met interaction triggers signals leading to activation of the Ras-mitogen-activated protein kinase pathway [52], the activation of phospholipase Cγ, protein kinase C, and Akt, which in turn activate the central transcription factor NF-κB [19]. For a long time, the significance of the listerial surface proteins InlA and InlB in in vivo host cell invasion was less clear. An inlAB mutant is only slightly impaired in virulence in the mouse model upon intravenous infection and the mutant was only transiently impaired in persistence in the liver and behaved like the wild-type in spleens and lymph nodes of infected mice [76]. In a different study inlAB mutants were only rarely found inside hepatocytes compared with the wildtype strain, indicating a role for the inlAB locus in hepatocyte invasion in vivo [105]. Most surprisingly, penetration of the intestinal barrier in orally infected mice was very inefficient and totally independent of the presence of InlA or InlB [191]. This finding is in sharp contrast to the unequivocally proven role of InlA in epithelial cell invasion in vitro. The identification of a single nucleotide exchange in mouse E-cadherin compared to human E-cadherin (Pro16Glu), which renders the mouse homologue unable to bind InlA [191], gave a first hint to clarify the unexpected findings in the mouse model of listeriosis. A recent publication by Lecuit et al. [194] elegantly used a transgenic mouse model to demonstrate convincingly the role of internalin in the penetration of the intestinal barrier. They constructed transgenic mice expressing human E-cadherin in their enterocytes and showed that, in these mice, L. monocytogenes mutants lacking InlA were significantly less able to cross the intestinal barrier because they were severely impaired in promoting their uptake by the enterocytes forming the intestinal barrier [192,194]. A large number of other internalins are present in L. monocytogenes as deduced from the genome sequence [38,117]; some, including InlC, InlE, InlF, InlG, and InlH [78,89,256], were already described in the pregenomic era. None of these proteins seems to be able to induce phagocytosis in mammalian cells and their roles in the infection process are largely unknown. By the construction of various combinations of in-frame deletions in most of the respective internalin genes it could recently be shown that—in contrast to InlB—InlA by itself triggers uptake into Caco-2 epithelial cells poorly and needs the help of other internalins [17]. This additional trigger function can be supplied by InlC alone or by InlC together with InlG, InlH, and InlE, or by InlB. Because no cellular receptors for internalins other than InlA and InlB are known, it is presently unclear whether these internalins trigger host cell function via the known InlB receptors or via other mammalian surface molecules. Several other L. monocytogenes surface structures have been described as involved in invasion. LpeA (Lipoprotein promoting entry) [262], a listerial lipoprotein with homology to a Streptococcus pneumoniae adherence factor, was implicated in the invasion of hepatocytes (and to a lesser extent of epithelial cells) because the respective mutant shows clearly diminished capacity of cellular invasion, but not of adhesion or intracellular growth. LpeA is the first listerial lipoprotein involved in invasion to be identified. The major extracellular protein p60 of L. monocytogenes [182,249] was initially postulated to be involved in fibroblast invasion, but its role in mediating cellular uptake is still under debate (see later discussion). The listerial surface protein ActA, a major virulence factor allowing actin-based intracellular motility [74,168] (see following), also was suggested recently to play a role in internalin-independent uptake of L. monocytogenes by epithelial cells [7]. Analysis of the invasive capacity of strains lacking or overexpressing ActA suggests that ActA may function as an invasion-mediating protein—at least when overexpressed [307]. Such an ActA-promoted attachment and invasion of CHO epithelia-like cells as well as IC-21 murine macrophages was shown to be mediated by interaction of the listerial surface protein ActA with a heparan-sulfate proteoglycan receptor [7]. It is supposed that electrostatic interactions between heparan sulfate and positively charged residues in the N-terminal part of ActA could lead to low-stringency binding to the cell surface proteoglycan

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receptors widely distributed in mammalian cells [7]. Whether the proposed low-stringency binding of L. monocytogenes to heparan sulfate proteoglycan receptors directly triggers uptake or results in adequate presentation of other bacterial factors to the host cell membrane that ultimately lead to phagocytosis remains to be clarified.

CELLULAR ADHESION The mechanisms allowing L. monocytogenes adhesion to various mammalian cells without following uptake are not well understood. InlA can clearly mediate binding to the surface of epithelial cells because mutants lacking the inlA gene bind less well to Caco-2 epithelial cells [17,262]. In contrast, InlB, which by itself triggers high-efficiency invasion of endothelial cells, is obviously not involved in cell adhesion. Mutants lacking the inlB gene adhere to human brain microvascular endothelial cells essentially as the wild-type strain [17,129]. Because even the nonpathogenic species L. innocua (which lacks the internalins of L. monocytogenes [117]), binds to endothelial cells, one must assume that common cell wall structures mediate the interaction of L. monocytogenes and L. innocua with the endothelial cells [129]. One of the listerial surface structures that may mediate internalin-independent binding to mammalian cells are the lipoteichoic acids found in listerial cells walls [91]. These polymers are known to interact with mammalian pattern recognition receptors of the scavenger- and toll-receptor families [85]. It was shown that purified lipoteichoic acid from L. monocytogenes can trigger host cell responses in macrophages [141] and such interactions may also confer cell binding. A recent paper by Abachin et al. [1] has now shown that an L. monocytogenes mutant with an altered lipoteichoic acid lacking D-alanine is severely impaired in binding to epithelial cells, hepatocytes, and even macrophages. It is believed that the mutation increases the electronegativity of the bacterial surface, which then interferes with cell binding even in the presence of internalin and InlB. Various extracellular bacterial pathogens exploit the extracellular matrix protein fibronectin as a bridging receptor for cell binding [92]. Five fibronectin binding proteins were identified in L. monocytogenes, one of which was a 55-kDa cell surface protein [114]. Additionally, the gene of one fibronectin binding protein of 25 kDa was cloned and sequenced [115]. However, the role of these listerial proteins in virulence in general and specifically in cell binding is not known. As mentioned before, the L. monocytogenes surface protein ActA can act as an invasion—at least when overexpressed. However, it is possible that in the situations tested, ActA actually acts as an adhesin allowing more efficient contact of the bacteria to the cell surface followed by interaction of the invasion-promoting molecules with the host cell [7]. Another surface protein, called p104, probably involved in listerial adhesion to Caco-2 epithelial cells was recently identified by transposon mutagenesis [243]. The mutants that lack p104 expression are reduced about tenfold in adhesion to the Caco-2 cells and antibodies against the protein also inhibit adhesion. Up to now, the only listerial protein clearly shown to mediate L. monocytogenes adhesion without promoting uptake is the surface molecule Ami [224,225]. Ami is a 102-kD autolysin with the catalytic activity in the N-terminal part of the molecule. The C-terminal cell wall anchor region is made up of repeated modules containing a GW dipeptide as also found in InlB [27]. Ami confers cell binding to epithelial cells and hepatocytes in a ∆inlAB background. It was shown by complementation that the C-terminal GW modules are responsible for mediating cell adhesion and the purified C-terminal part binds to epithelial cells in vitro [224].

UPTAKE

BY

MACROPHAGES

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Macrophages of different origin were used in in vitro studies to analyze the mechanisms of L. monocytogenes uptake by professional phagocytes, which are generally assumed to take up L. monocytogenes by conventional phagocytosis involving actin-polymerization. If present, complement factors C1q and C3 are deposited on the bacterial surface and stimulate L. monocytogenes uptake by binding the bacteria to the respective receptors [4,59,80].

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Macrophages ingest L. monocytogenes very rapidly and intracellular killing starts shortly after phagocytosis and leads to destruction of most of the ingested bacteria [64,66,259,325]. In a single macrophage, killed bacteria inside acidified phagosomes and phagolysosomes and growing bacteria that have escaped into the cytoplasm can be detected. These findings suggest competition between phagosome–lysosome fusion followed by killing of the bacteria and their escape from the acidified phagosome before phagosome–lysosome fusion occurs. The result of this competition is a population of cytoplasmic bacteria able to grow inside the macrophage. The route of uptake by the macrophages may also be important for the fate of the invading bacteria. As demonstrated by Drevets et al. [81], the mode of uptake is critical for subsequent survival because L. monocytogenes taken up in the presence of complement C3 leads to enhanced killing of the bacterium. Whether the surface protein InlA substantially contributes to the triggering of phagocytosis of L. monocytogenes by macrophages is still under debate. Using bone marrow-derived macrophages, InlA had only a slight effect on invasion because an inlAB mutant still showed more than 60% invasion when compared with the wild-type strain [146]. Uptake of L. monocytogenes by the mouse macrophage -like cell line J774A.1 was inhibited by at least 50% by the pretreatment of L. monocytogenes with anti-InlA antibodies and that recombinant InlA specifically bound to the macrophages [281]. It was suggested recently that the listerial protein p60 might enhance phagocytosis by macrophages because a Salmonella typhimurium strain that expresses and secretes p60 seems to be more invasive in phagocytic cells but not in enterocytes [146]. In line with this assumption is that pretreatment of L. monocytogenes with a polyclonal anti-p60 antiserum inhibits uptake of the bacteria by a macrophagelike cell line [146]. Another factor that could be involved in attachment and invasion of L. monocytogenes in macrophages is the listerial cell wall polymer lipoteichoic acid. L. monocytogenes binds to the macrophage scavenger receptor most likely via lipoteichoic acid [85,128]. This interaction may also trigger conventional receptor-mediated phagocytosis of L. monocytogenes. The mechanisms specifically underlying L. monocytogenes uptake by macrophages are not well understood. As expected, a functional actin cytoskeleton is necessary as deduced from inhibitor studies with cytochalasins [103,183]. In addition, microtubules also seem to be involved because the drugs nocodazole and cholchicin, which depolymerize microtubules, inhibit L. monocytogenes uptake by different macrophages [181]. The early signaling events associated with L. monocytogenes uptake by J774 macrophages were analyzed by Goldfine and coworkers (summarized in Goldfine and Wadsworth [125]). They demonstrated that very rapidly and before attachment and invasion, the addition of the bacteria to the macrophages results in three peaks of calcium mobilization within 10 min [323]. Whereas the first two peaks result from calcium influx, the third results from the mobilization of intracellular calcium stores. Linked to the calcium peaks is the mobilization of several protein kinase C isoforms that are translocated to the cell membrane [324]. Calcium signaling and protein kinase C activation depend on the listerial virulence factors LLO and PI-PLC (see later discussion) and modulate the uptake of L. monocytogenes into the macrophages. It appears that calcium mobilization and protein kinase C activation negatively influence the speed of the uptake process because mutants lacking LLO and PI-PLC are taken up more rapidly as the wild-type strain and specific inhibitors of PKC also induce the speed of uptake [323]. Another class of professional phagocytic cells important in the innate immune response against L. monocytogenes is the dendritic cells. They can be found in different tissues where they take up foreign material and present it to T-cells to stimulate adaptive responses [264]. Dendritic cells of mouse and human origin efficiently take up L. monocytogenes [135,173,174,247]. Invasion of dendritic cells seems to be independent of internalin and InlB [135,173], but requires a functional cellular cytoskeleton. Furthermore, the uptake is significantly improved in the presence of human serum and specifically enhanced by antibodies against the listerial protein p60, which act as opsonins [174]. At least some of the intracellular bacteria are released into the cytoplasm, but intracellular replication seems to be very low in dendritic cells. Because dendritic cells are able to migrate over long distances toward lymphoid tissue, they might also be important vectors during the early dissemination of the bacteria [255].

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ESCAPE FROM THE PHAGOCYTIC VACUOLE Hemolytic activity detected around colonies of L. monocytogenes growing on blood-agar plates was long supposed to represent a major virulence determinant because all clinical isolates of L. monocytogenes show this hemolytic phenotype. The hemolytic activity is due to the action of a cytolysin, called listeriolysin O (LLO). In experimental infections all virulent strains were found to be hemolytic, whereas nonhemolytic strains were avirulent. Nonhemolytic mutants obtained after transposon mutagenesis using the conjugative transposons Tn1545 or Tn916 [104,163,253] always proved to be avirulent in the mouse model. Virulence is restored in hemolytic revertants that have lost the transposon insertion or by the introduction of the cloned hly gene into a nonhemolytic L. monocytogenes transposon mutant [55]. Listeriolysin O, a secreted protein of 58 to 60 kDa, belongs to a family of thiol-activated, cholesterol-dependent, pore-forming toxins (CDTX) for which streptolysin O is the prototype [242]. All members of this family are inhibited by low concentrations of cholesterol and oxygen and activated by reducing agents like DTT. Cholesterol is the receptor for these cytolysins because only membranes containing cholesterol are attacked and this component inhibits pore formation and toxicity [242]. Upon addition to erythrocytes, toxin monomers oligomerize in the target cell membrane to form stable pores that can be visualized by electron microscopy (Figure 5.4) [246]. Listeriolysin O has been purified to homogeneity and its toxicity, determined by intraperitoneal injection into the mouse, shows LD50 of 1.7 µg per mouse. Optimal hemolytic activity is found at pH 5.5, a pH value much lower than that determined for the other CDTX [111], a property in agreement with the function of LLO in the acidified phagosome (see later discussion). The gene encoding LLO, hly, was cloned from strains of different serovars of L. monocytogenes and sequenced [71,216,220]. The deduced amino acid sequence for LLO yielded 529 amino acids including an N-terminal signal sequence of 25 amino acids. As expected, the sequence shows extended homologies with the protein sequences of other CDTX. The highest homology is observed in the C-terminal part and includes a highly conserved undecapeptide containing the unique cysteine thought to be essential for cytolytic activity. Site-directed mutagenesis revealed, however, that the cysteine is not essential for hemolytic activity. In contrast, a tryptophan residue, in close vicinity to the cysteine, appears to be required for hemolytic activity and virulence [222] as it was also shown for an alanine residue also located in that motif [152]. The three-dimensional structure of LLO is not known. However, the structure of the closely related toxin perfringolysin O (PFO) was solved. This toxin—and hence most likely also LLO—is composed of four domains that form an L-shaped molecule (Figure 5.4) [273]. The expression of domain 4 alone or of domains 1 to 3 of LLO showed that domain 4 is responsible for membrane binding and oligomerization, whereas the first three domains are necessary for full hemolytic activity [83,172]. Strikingly, when expressed simultaneously, the two secreted domains LLO-d123 and LLO-d4 reassembled into a hemolytically active form [83]. The role of LLO in virulence was determined by injection, intravenous and intraperitoneal, of wild-type and nonhemolytic mutants of L. monocytogenes into mice and by following the fate of the bacteria in liver and spleen. In contrast to the wild-type strain, the nonhemolytic mutants are eliminated from these organs within a few hours without eliciting protective immunity [55,104,163,253]. The role of LLO in the intracellular survival was determined using different mouse and human cell lines. In the human enterocyte-like cell line Caco-2 [103], mouse 3T6 fibroblasts [183], and mouse CL.7 fibroblasts [253], nonhemolytic L. monocytogenes mutants are as invasive as the isogenic wild-type strains. The nonhemolytic mutants are, however, incapable of survival and intracellular growth within these host cells and also in mouse peritoneal macrophages [183], mouse bone marrow-derived macrophages [253], and the mouse macrophage-like cell line J774 [253]. Electron microscopy of infected macrophages and epithelial cells revealed that nonhemolytic L. monocytogenes mutants found inside the cells are unable to open the phagosome and hence unable to escape into the

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A

B FIGURE 5.4 A: Pore-forming activity of LLO. Sheep erythrocyte ghost after treatment with purified LLO, showing the ring-shaped oligomeric structures of the toxin attached to the membrane (bar, 100 nm). (Reprinted with permission from Jacobs, T. et al. 1998. Mol. Microbiol. 28:1081–1089.) B: Crystal structure of PFO, a close relative of LLO. (Reprinted with permission from Rossjohn, J. et al. 1997. Cell 89:685–692.)

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cytoplasm of the host cells [103,315]. Additional evidence that LLO is essential for lysis of the phagosomal membrane was obtained by infection of macrophages with a Bacillus subtilis strain expressing LLO [18]. This engineered strain escapes from the phagosome into the cytoplasm, whereas the nonhemolytic B. subtilis parental strain stays in the phagosome as do the nonhemolytic L. monocytogenes mutants. The low pH optimum of LLO is in agreement with its function in the acidified phagosome. Bafilomycin or ammonium chloride treatment inhibits vacuolar acidification and inhibits the escape of L. monocytogenes from phagosomes of infected epithelial cells or macrophages. These findings sustain the importance of the low pH activity optimum of LLO for its role as a vacuole opener [15,51]. Recently, Portnoy and coworkers [156,157] analyzed the role of LLO (and especially the role of its low pH optimum) by constructing L. monocytogenes strains that secrete the closely related extracellular cytolysin perfringolysin instead of LLO. Such a strain escaped from the vacuole, but damaged the host cell. An elegant selection procedure was used to isolate mutants that do not damage the host cell upon perfringolysin expression in the cytoplasm. The mutated perfringolysins were reduced in activity at neutral pH, had a generally reduced hemolytic activity, or showed a shorter half-life in the cytoplasm. These results show that the low activity of LLO at neutral pH values and its short half-life in the cytoplasm are critical parameters for its suitability as a phagosome opener without concomitant cytotoxicity. The strains expressing mutated perfringolysins allowing intracellular growth without cell damage are totally avirulent, however [157]. Further study [118] now showed that a single amino acid exchange in LLO (Leu461Thr) results in an LLO variant with ten times higher hemolytic activity at neutral pH and an L. monocytogenes strain expressing this LLO variant is about 100-fold less virulent. The reduction in virulence is most likely associated with a higher toxicity of the molecule to the host cells, which are killed by membrane damage resulting from activity of LLO [118,119]. The importance of restriction of LLO activity to the phagosomal compartment is further supported by other lines of evidence. A so-called PEST sequence was recently identified in the N-terminal region of LLO [65,202]. PEST sequences target proteins to the proteasome degradation pathway [260], hence dramatically reducing their cytoplasmic half-life. LLO variants lacking the PEST sequence demonstrate normal hemolytic activity and allow vacuolar escape, but are toxic to their host cells. Their expression results in a decrease in virulence in the mouse model [65]. A recent publication by Lety et al. [201], however, showed that rather than the actual PEST sequence, some amino acids immediately downstream of PEST could be responsible for the correct function of LLO, thus questioning the results mentioned earlier. The expression of LLO in the infected host cell is tightly controlled; expression is induced when the bacteria are taken up by mammalian cells and seems to be highest in the phagosome [36], although it also continues in the cytoplasm [227]. A recent paper by Dancz et al. [61] finally showed that induction of LLO expression (with an IPTG-inducible expression system) at different time points after infection allows bacteria trapped in the cytosol to escape into the cytoplasm of macrophages and then grow intracellularly, again demonstrating the crucial role of LLO in phagosomal escape. Fusion of L. monocytogenes-containing phagosomes with endosomes has been observed in electron microscopy studies [315]. However, it is not known whether such an event is necessary for L. monocytogenes to progress through its intracellular life cycle [325]. The recent description of Rab5-regulated fusion of L. monocytogenes-containing phagosomes with endosomes and results that indicate that Rab5a controls early phagosome–endosome interactions and governs the maturation of the early phagosome leading to phagosome–lysosome fusion [6] show that phagosome maturation events take place upon ingestion of L. monocytogenes into macrophages [3]. On the other hand, live L. monocytogenes delays phagosome maturation and subsequent degradation by yet unknown mechanisms. It is believed that this allows the bacteria to prolong their survival inside the phagosome/endosome, assuring their viability as a prelude to escape into the cytoplasm [5]. Prolonged intraphagosomal survival of L. monocytogenes in macrophages was

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recently demonstrated [61]; this could be nicely explained by the data on an L. monocytogenesinduced delay in phagosome maturation. Listeriolysin O-independent escape of L. monocytogenes from primary vacuoles in human epithelial cells [253] is mediated by two listerial phospholipases and a metalloprotease [213] that also contribute to vacuole escape in other cells like bone marrow-derived macrophages [42]. Phospholipase activity of L. monocytogenes was first discovered as a zone of opacity surrounding colonies on egg yolk agar [100]. Transposon mutants of L. monocytogenes lacking phospholipase activity were identified by formation of small plaques on fibroblast cell monolayers [309] and by reduced hemolysis on blood-agar plates [164], indicating a participation of phospholipase activity in hemolysis. The gene encoding a phosphatidylinositol-specific phospholipase C (PI-PLC), called plcA [41,195,215], encodes a protein of 34 kDa that exhibits high homology to several Grampositive phospholipases and contains a typical transport signal sequence. This enzyme was purified from culture supernatant fluid of an overexpressing L. monocytogenes strain [124] and was highly specific for phosphatidylinositol with no detectable activity on phosphatidylethanolamine, phosphatidylcholine, or phosphatidylserine. It also does not cleave phosphatidylinositol-4-phosphate or phosphatidylinositol-4,5-bisphosphate, but is active, albeit with low specific activity, on glycosylated phosphatidylinositol-anchored proteins [108]. The crystal structure of PI-PLC was recently determined (Figure 5.5) [228]. The enzyme consists of a single (βα)8-barrel domain with the active site located at the C-terminal side of the β-barrel. Unlike other

FIGURE 5.5 Ribbon diagram of the structure of PI-PLC viewing toward the active site pocket where the bound myoinositol molecule (labeled Ins in yellow) is shown. α-Helices (A to G) are colored in red, β-strands (I—VIII) in blue, loops in green. β-Strands are labeled with roman numerals. The N-terminus is labeled with N′, the C terminus with C. (Reprinted with permission from Moser, J. et al. 1997. J. Mol. Biol. 273:269–282.)

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(βα)8-barrels, the barrel in PI-PLC is open. Interaction between the substrate and the active site pocket is made by specific hydrogen bonds with a number of charged amino acid side-chains. Two histidine residues (His38 and His86) not present in the active site are, however, important for the activity of the enzyme because their mutagenesis results in PI-PLC variants with activity reduced 100-fold [11]. L. monocytogenes strains expressing these variants behave like plcA-deletion mutants in cell culture assays. In addition to the highly specific PI-PLC, L. monocytogenes produces a second phospholipase C, which hydrolyzes phosphatidylcholine (lecithin), and is thus a phosphatidylcholine-specific phospholipase C (PC-PLC) or lecithinase [112], also called broad-spectrum phospholipase C. A 32-kDa protein was detected in the supernatant liquid of an L. monocytogenes culture that showed phospholipase activity on egg yolk overlays [164]. The protein was purified to homogeneity [112,123] and is a zinc-dependent phospholipase C of 29 kDa. The pH optimum of the enzyme is between pH 6 and 7 and its activity is stimulated by 0.5 M NaCl and 0.05 mM ZnSO4. In addition to phosphatidylcholine, it also hydrolyzes phosphatidylethanolamine, phosphatidylserine, and, with lower efficiency, sphingomyelin. Phosphatidylinositol is not a suitable substrate. The purified protein exhibits weak hemolytic activity but is not toxic to mice [112]. The gene plcB, encoding PC-PLC, is part of the lecithinase operon, which consists of mpl, actA, plcB, and the small orfs X and Z [319]. PC-PLC is a protein of 289 amino acids with a 25-amino-acid N-terminal transport signal and a putative propeptide of 26 amino acids. Maturation of the 32-kDa precursor of PC-PLC occurs after secretion because both forms of the protein can be found in the supernatant liquid; it is obviously accomplished by the metalloprotease of L. monocytogenes [254,258]. Use of in-frame deletions in the plcA gene enabled clear demonstration that PI-PLC is required for efficient escape of L. monocytogenes from the phagosome of mouse bone marrow-derived macrophages. However, the mutation in plcA has only a slight effect on virulence [42]. It is assumed that PI-PLC acts in concert with listeriolysin inside the acidified phagosomal vacuole of the host cell to mediate lysis of the vacuolar membrane. The broad pH optimum of PI-PLC, ranging from pH 5.5 to 7.0, is consistent with its postulated function in the acidified phagocytic vacuole of infected cells. To further assess the role of PI-PLC, the plcA gene was expressed in L. innocua, which lacks the prfA-dependent virulence gene cluster and is therefore unable to escape from the host cell vacuole. The PI-PLC expressing L. innocua strain cannot escape from the phagosome of J774 macrophages, but shows limited intracellular growth inside the vacuoles, which appear to be structurally intact [286]. The role of PC-PLC in escaping from the primary vacuole is not clear and differs from cell type to cell type. In mouse bone marrow-derived macrophages, PC-PLC has no role in lysis of the vacuole [299]. However, in the human Henle 407, HEP-2, and HeLa epithelia-like cell lines, where escape of L. monocytogenes occurs at low efficiency independently from LLO [133,213,253], PC-PLC is required for lysis of the phagocytic vacuole together with the metalloprotease. PI-PLC is not required in this system, but the efficiency of escape was reduced in a hly, plcA double mutant [213]. The metalloprotease Mpl of L. monocytogenes indirectly contributes to pathogenicity and intracellular replication of the bacteria. Transposon mutants with insertions in the mpl gene are less virulent but grow normally inside mammalian cell lines [258]. The reduced virulence was attributed to lack of proteolytic processing and hence activation of the 32-kDa PC-PLC proform [254,305]. This also seems the way in which Mpl contributes to lysis of the vacuole in Henle 407 cells, where vacuolar escape is independent of LLO but depends on PC-PLC and Mpl [258]. Located immediately downstream of the hly gene, the mpl gene [72,218] encoding Mpl is the first gene of the lecithinase operon [72,218,319]. The deduced amino acid sequence of this protease shows high homology to several zinc-dependent metalloproteases from Bacillus species and yields 510 amino acids with a typical N-terminal signal sequence and a putative internal cleavage site. Like other metalloproteases, the enzyme is activated by proteolytic maturation resulting in a mature 35-kDa protein [72,218]. A 60-kDa protein is detected with an antiserum raised against Bacillus stearothermophilus thermolysin, which probably represents the proform of the metalloprotease.

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Only small amounts of the mature 35-kDa form of the protein were detected in the supernatant liquid of an L. monocytogenes culture [72]. Recently, the protein Mpl was purified and biochemically characterized and shown to be an enzyme active at a wide range of temperatures and pH values. The protein exhibits a high thermal stability and shows a narrow substrate specificity cleaving, besides the pro-PC-PLC, also casein and actin [50]. The translocation through and release of PC-PLC from the bacterial cell wall occurs most efficiently upon a decrease in pH as regularly encountered in primary and secondary phagosomes. The release coincides with the proteolytic activation of PC-PLC by Mpl, which both colocalize at the cell wall–membrane interface [302]. Listeria monocytogenes encodes a large number of putative lipoproteins [38,117] with largely unknown functions. By deleting the gene encoding a putative lipoprotein-specific signal peptidase, Reglier-Poupet et al. [261] recently got first insights into the role of at least some members of this protein family. The deletion mutant failed to process several lipoproteins and showed reduced virulence in the mouse model. Expression of the signal peptidase is strongly induced while the bacteria reside in the phagosome and mutant bacteria are clearly impaired in phagosomal escape. The mechanism of how listerial lipoproteins contribute to lysis of the phagosomal membrane is, however, still unknown.

GROWTH IN THE HOST CELL CYTOPLASM As mentioned earlier, phagosomal escape is a prerequisite for L. monocytogenes to replicate intracellularly and is hence a critical virulence mechanism of this species. In principle, intracellular bacteria have two possibilities for intracellular multiplication: They replicate in a membrane-bound vacuole as exemplified by many pathogens, including Salmonella Typhimurium, Legionella pneumophila, Mycobacterium tuberculosis, and Coxiella burnetii or they escape into the host cell cytoplasm. This second intracellular niche is only chosen by L. monocytogenes, L. ivanovii, Shigella flexneri, and Rickettsia spp. [92]. Because the vacuole is a potentially hostile environment, bacteria that stay there have evolved many different ways to interfere with phagosome maturation that allow them to live in an intracellular compartment fulfilling their needs [138]. L. monocytogenes starts intracellular multiplication shortly after escape from the vacuole with intracellular generation times of 40 to 60 min—close to the approximately 40-min generation time observed in rich broth culture [253]. The host cell cytoplasm hence allows listerial growth with high efficiency. However, the cytosol is poorly characterized as a substrate supporting bacterial growth and the relative abundance of nutrients is unknown. Whereas various auxotrophic mutants of L. monocytogenes are able to grow intracellularly [212], expression of several metabolic genes is increased intracellularly [166], indicating that at least some metabolites may be limiting in the cytosol, but there is no general upregulation of expression of stress proteins in intracellularly growing L. monocytogenes [139]. Early studies addressing the question whether even nonpathogenic bacteria not adapted to an intracytoplasmic lifestyle can grow in the host cell cytoplasm used B. subtilis [18] or L. innocua [90] strains engineered to express listeriolysin to allow phagosomal escape after uptake. Results of these studies showed that nonpathogenic bacteria were able to multiply—at least to some extent—in the host cell cytoplasm and hence implied that this compartment may be favorable for bacterial growth. A recent study by Goetz et al. [122] addressed this question differently by directly microinjecting L. monocytogenes and other bacteria into cytoplasm of Caco-2 epithelial cells and J774 macrophages. Intracellular multiplication of the bacteria was followed microscopically because they were constructed to express the green fluorescent protein. In contrast to the studies just mentioned that used this method, only bacteria naturally capable of intracytoplasmic growth (L. monocytogenes, S. flexneri, and enteroinvasive Escherichia coli) grew in the cells; others such as B. subtilis, L. innocua, and S. Typhimurium did not [122]. Furthermore, an L. monocytogenes mutant lacking the central virulence regulator PrfA (discussed later) did not

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multiply upon microinjection, clearly pointing to the need of specific virulence determinants necessary for efficient intracytoplasmic multiplication. The obvious discrepancies in the studies concerning the ability of bacteria to use host cell cytosol for growth have been discussed in detail elsewhere [120,240,318], but further investigation is needed before a clear decision can be made. Probably some cytosolic compartments permit bacterial growth and others do not, depending on conditions of the infected cell and infecting bacterium. In search of specific bacterial factors allowing intracellular growth, a bacterial homologue of the mammalian glucose-6-phosphate translocase (Hpt) [48] and a lipoate protein ligase (LplA1) [239] were identified that are necessary for efficient intracellular proliferation of L. monocytogenes. Expression of the Hpt permease is tightly controlled by the central virulence regulator PrfA, which induces a set of virulence factors required for listerial intracellular parasitism upon entry into host cells. Loss of Hpt resulted in impaired listerial intracytosolic proliferation and attenuated virulence in mice but did not affect bacterial growth in a rich medium like BHI broth [48]. However, Hpt alone is not sufficient for cytoplasmic growth because L. innocua expressing Hpt together with LLO cannot grow intracytoplasmically upon infection of macrophages [298]. Lack of LplA1 results in bacteria that cease intracellular replication after about five rounds of replication and are clearly defective in mouse virulence assays. A major target for LplA is the E2 subunit of the pyruvate dehydrogenase enzyme (PDH) complex; in intracellularly grown lplA1 mutants, PDH is no longer lipolyated. Studies of lipoic acid metabolism have shown that little free lipoic acid exists in mammalian cytosol [241]. Thus, LplA1 may not be important in replication of L. monocytogenes when free lipoic acid is available, but it is required in the host cell wherein lipoic acid may be scavenged by host molecules [239].

INTRACELLULAR MOTILITY AND CELL-TO-CELL SPREAD Intracellular movement of L. monocytogenes inside the host cell cytoplasm as well as intercellular spread mediated by actin polymerization were initially described by Mounier et al. [229] and Tilney and Portnoy [315]. Their studies were followed by a series of analyses describing the cell biology of the process. It was shown that L. monocytogenes moves rapidly through the cytoplasm with the help of the formed actin tails with a speed of up to 1.5 µm/sec. Quite surprisingly, L. monocytogenes rotates around its long axis as it is propelled by actin polymerization [271]. The rate of actin assembly, which occurs at the barbed ends of the actin filaments near the bacterial surface, equals the rate of actin-based motility; actin polymerization provides the propulsive force for intracellular movement, which can also take place in cytoplasmic extracts from Xenopus oocytes [60,280,312,313,314]. Mutants defective in intracellular motility were obtained by transposon mutagenesis. These mutants have lost the ability to initiate actin polymerization [309] because of insertion into a gene called actA or still induce actin polymerization but are unable to rearrange actin filaments to actin tails [184]. The gene actA is located downstream of mpl in the lecithinase operon and codes for a proline-rich protein (ActA) of 639 amino acids (Figure 5.6). Its apparent molecular weight determined by SDS-PAGE is 92 kDa [74,319]. ActA is a surface protein consisting of three domains: the N-terminal domain with the transport signal sequence, the central proline-rich repeat region, and the C-terminal part, which includes a membrane anchor [74,168,319]. Mutations in the actA gene result in loss of virulence in mice [74], lack of intracellular actin polymerization around bacteria, and inability of intracellular movement [74,168]. Inside the host cells, actA mutants form microcolonies located near the nucleus [74]. Several assays were used to prove that ActA alone is sufficient to stimulate F-actin assembly and promote intracellular movement: First, the nonmotile species L. innocua was engineered to express ActA; the recombinant bacteria induce formation of actin tails and move in cytoplasmic extracts as the wild-type L. monocytogenes strain [170]. Second, actA was transfected into mammalian cells [99,250,251] where the ActA protein (including the membrane anchor) was targeted

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FIGURE 5.6 Diagram of the molecular components required for actin-based motility of L. monocytogenes. A: Interactions between host-cell proteins and ActA at the bacterial surface. Two domains of ActA are required for normal motility. The amino-terminal domain activates actin filament nucleation through Arp2/3. The central proline-rich domain binds VASP and profilin interacts with VASP, enhancing filament elongation. B: Host protein functions throughout the comet tail. In addition to the factors that act at the bacterial surface, capping protein binds to the barbed end of actin filaments to prevent elongation of older filaments, α-actinin crosslinks filaments to stabilize the tail structure, and ADF/cofilin disassembles old filaments. (Reprinted with permission from Cameron, L. A. et al. 2000. Nat. Rev. Mol. Cell Biol. 1:110–119.)

to mitochondria, which subsequently assemble F-actin on their surface. Third, polystyrene beads were coated with purified ActA that induced F-actin polymerization in in vitro systems that also support listerial movement [39]. The ActA protein is distributed asymmetrically on the surface of L. monocytogenes. After cell division, it is concentrated at the old pole [169,311] and absent or present only at low concentrations at the new bacterial pole. This asymmetric distribution of the ActA protein is required and sufficient to direct actin-based motility by coating streptococci asymmetrically with genetically engineered ActA protein [300]. In a cell-free system, these streptococci, but not uniformly coated ones, moved efficiently in cytoplasmic extracts [300]. Additionally, only polystyrene beads coated with ActA asymmetrically induce true F-actin tails and move in extracts [39]. ActA seems to be an elongated protein [49] that thus far resists crystallization efforts. It was postulated that ActA is present on the bacterial surface as a dimer [230], but these data were questioned recently by demonstration that functional ActA is a monomeric protein [207]. Elucidation of the precise mechanisms by which ActA allows actin recruitment and intracellular movement by stimulating F-actin polymerization is in the center of research interests of several laboratories and has been reviewed in detail elsewhere [14,40,53]. Expression of mutated forms of ActA in mammalian cells [99, 250] or in L. monocytogenes [49,186–188,252,297,301,306] made it possible to define regions of the ActA protein with specific functions in recruitment of cellular proteins and hence in actin polymerization and movement. Deletion of the whole N-terminal domain of ActA was followed by total abolishment of actin polymerization and intracellular movement in both systems, showing the absolute necessity of this domain in ActA function [186]. Within the N-terminal part of ActA, two smaller regions were identified that are required for filament elongation (aa 117 to 121) or for continuity of the actin tail (aa 21 to 97); their deletion led to discontinuous actin tail formation [187,188,252]. In contrast, deletion of the C-terminal domain did not inhibit actin assembly [186]. The actin tails produced for L. monocytogenes strains expressing ActA without the central proline-rich repeats were significantly shorter and movement speed was drastically reduced [301]. The protein composition of the F-actin tails was analyzed using different methods and several actin binding proteins and proteins regulating the actin dynamics were colocalized with tails, including α-actinin, tropomyosin, vinculin, talin, fimbrin, villin, ezrin/radixin, cofilin, coronin, Rac,

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capZ, profilin, VASP, Mena, and the Arp2/3 complex [44,60,63,70,113,167,311,313,328]. From these proteins only profilin, Mena, the Arp2/3 complex, and the vasodilator-stimulated phosphoprotein (VASP) are associated with the surface of moving bacteria and colocalize with ActA [44,113,313]; only VASP, Mena, and the Arp2/3 complex directly bind to ActA [44,113,252,334]. Another cellular protein, called LaXp180, directly binds to ActA-expressing intracellular L. monocytogenes. This resulted in recruitment of the cellular phosphoprotein stathmin [13,248]. However, the role of these proteins in intracellular motility, if any, is unknown. The Arp2/3 complex, named after two of its members (Actin related proteins 2 and 3), consists of seven host cell proteins and was initially characterized as a profilin-binding protein complex [205]. Arp2/3 initiates F-actin polymerization because of its nucleation activity [231], which is activated significantly by the presence of ActA [328,329]. Studies with ActA mutants clearly demonstrated that the N-terminal region of ActA is sufficient for this activation [297]. The regions of the ActA protein that directly interact with proteins from the Arp2/3 complex during activation were mapped to two regions spanning the acidic aa 41 to 46 and the basic aa 146 to 150 [26,252,334]. In this interaction, ActA is thought to mimic the activity of proteins of the WASP family, which are natural ligands and activators of Arp2/3 [26]. Arp2/3 can also bind to the side of pre-existing actin filaments and initiate a new filament at this point, creating branched structures found throughout the comet tails associated with moving L. monocytogenes [327]. In summary, current data point to a central role of the Arp2/3 complex in initiating actin-based listerial intracellular movement. However, a recent study showed that ActA can also generate new actin structures in an Arp2/3independent but VASP-dependent manner [94]. This clearly shows that a complex protein like ActA can interfere with host cell actin polymerization machinery in multiple ways. The phosphoprotein VASP binds directly to the proline-rich repeats of ActA [44] via its Ena/VASP-homology domain [238]. On the other hand, VASP is a natural ligand of profilin [263] and could stimulate actin assembly by binding to ActA and enhancing profilin concentration in the vicinity of the bacterium. In this respect it is important to note that ActA can interact simultaneously with four Ena/VASP homology domains [207] and thus recruit at least four profilins to the bacterium. Mena, which is closely related to VASP, also binds ActA and profilin directly and might function in concert with VASP to recruit profilin–actin complexes to the site of actin polymerization [113]. However, profilin is dispensable, at least in vitro, because profilin-depleted cytoplasmic extracts still supported actin assembly and bacterial movement [209]. ActA can bind G-actin with a region in its N-terminal part, but deletion of this region does not interfere with actin tail formation in infected cells [297]. It is believed that, inside cells, the VASP-mediated profilin/G-actin recruitment can bypass defects in actin binding of ActA [296]. Another function recently attributed to Ena/VASP proteins is control of temporal and spatial persistence of bacterial actin-based motility [9]. Furthermore, purified VASP binds to F-actin [189] and enhances actin-nucleating activity of wild-type ActA and the Arp2/3 complex while also reducing frequency of actin branch formation. The ability of VASP to contribute to actin filament nucleation and to regulate actin filament architecture highlights the central role of VASP in actin-based motility [279,296]. The 92-kDa ActA protein on the bacterial surface is cleaved by the listerial metalloprotease Mpl, resulting in a major 72-kDa degradation product and, depending on the Listeria strains tested, additional smaller degradation products [121,237]. These products are found on the bacterial surface or in the supernatant liquid as 65- and 30-kDa fragments [237]. ActA is also degraded inside the cytoplasm of the infected host cell [226]. However, this type of degradation seems to be mediated by the proteasome of the host cell because it can be blocked by proteasome inhibitors. Additionally, ActA becomes phosphorylated inside the host cell as shown by the presence of three distinct forms of the protein with slightly different motilities in SDS-PAGE found in infected cells [33]. A genetically engineered ActA variant, which is fully functional but lacks the C-terminal region, is no longer phosphorylated inside host cells, suggesting that phosphorylation may not be necessary for movement [186]. The roles of Mpl- or proteasome-mediated ActA degradation as well as the

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phosphorylation inside the host cell are currently not understood, but one could imagine that different variants of ActA could interact differently with host cell proteins regulating the dynamics of the F-actin cytoskeleton. Expression of ActA is controlled by PrfA (discussed later) as are the other genes of the virulence gene cluster. However, different virulence factors are required at different steps of the intracellular life cycle and in different quantities. These necessities are reflected by complex regulatory networks governing expression of the virulence genes; this is far from being completely understood. ActA expression is maximally induced when bacteria have reached host cell cytoplasm and the expression level is increased more than 200-fold in cytosolic bacteria in comparison to broth-grown cultures [291]. ActA expression in intracellularly located bacteria was analyzed by reporter gene fusions (β-galactosidase, gfp, or β-glucuronidase) to actA [36,95,217,291] or by direct measurement of actA-specific transcripts [36]. In all these studies, the principle finding was identical, showing a marked induction of ActA expression in intracytoplasmically located L. monocytogenes. L. monocytogenes can spread from cell to cell without leaving the cytoplasm by forming microvilli-like protrusions on the host cell surface that are phagocytosed by neighboring cells [229,315]. The mechanisms of microvilli formation and induction of phagocytosis by the neighboring cell are largely unknown. Listeria randomly move through the cytoplasm; bacteria are finally propelled into the host’s distended plasma membrane and long protrusions are formed (Figure 5.1). In a monolayer of cells, the protrusions enter neighboring cells; they are subsequently taken up and a secondary vacuole with a double membrane is formed. This secondary vacuole is disrupted, which requires the action of LLO and the listerial phospholipase PC-PLC. Electron micrographs of plcB mutants inside mammalian cells [319] show numerous bacteria followed by actin tails and trapped in vacuoles surrounded by double membranes, indicating that plcB mutants are unable to lyse the double membrane of the vacuole. Careful examination of the plaque formation capacity of different mutants, which is thought to be a good correlate for intercellular spread, revealed that in addition to the broad spectrum phospholipase PC-PLC, PI-PLC and the metalloprotease contribute to plaque formation, most likely by supporting lysis of the double membrane vacuole [214,299]. The listerial metalloprotease Mpl probably supports this lysis by proteolytic activation of PC-PLC, but host cell proteases may also cleave and hence activate PC-PLC in the absence of Mpl [214]. The pivotal role of LLO in escape from the secondary vacuole was shown in an elegant study using an L. monocytogenes hly mutant coated with recombinant LLO, which allowed bacteria to escape from the primary vacuole and spread into neighboring cells once. However, dilution of the recombinant LLO through bacterial replication inhibited further spread of bacteria [110]. The predominant role of LLO in lysis of the secondary vacuole and hence in cell-to-cell spread was confirmed by microinjection of hly mutants into the cytosol [122] and use of strains allowing temporal control of LLO expression [61]. Listerial factors other than ActA, LLO, Mpl, and PCPLC are not known to contribute to intercellular spread. The timeline and mechanics of cell-tocell spread were analyzed in some detail by Robbins et al. [270], who presented a model for this process. Upon membrane contact, bacteria continue to move and form the protrusion. The fitful bacterial movement stops after several minutes during which the protrusion is formed. After about 20 min, the protrusion suddenly collapses and the double membrane vacuole is formed and rapidly acidifies to activate LLO and PC-PLC. Upon lysis of the membrane, motility recovers after one to two bacterial generations.

BILE SALT HYDROLASE—A NOVEL VIRULENCE FACTOR Comparison of L. monocytogenes and L. innocua genomes [117] has revealed the presence of an L. monocytogenes-specific putative gene, termed bsh, encoding a bile salt hydrolase (BSH) [86]. Bile salts are end products of cholesterol metabolism in the liver; they are stored in the gall bladder and released into the duodenum, helping fat digestion. In addition, bile salts are known to have

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antimicrobiol activity because they are amphipathic molecules that can attack and degrade lipid membranes. Some intestinal microorganisms have hence evolved mechanisms to resist the detergent action of bile, including synthesis of porins, efflux pumps, and transport proteins [134]. Others produce bile salt hydrolases that transform and inactivate bile salts. Deletion of the bsh gene from the L. monocytogenes chromosome results in increased bile sensitivity, reduced virulence, and reduced liver colonization after infection of mice. This demonstrates that BSH is a novel L. monocytogenes virulence factor involved in the intestinal and hepatic phases of listeriosis. In addition, the bsh gene is positively regulated by the central listerial virulence regulator PrfA (see later discussion), further demonstrating that it is a true virulence factor.

ACCESSORY VIRULENCE FACTORS The listerial proteins reviewed so far are true virulence factors because their sole function is specifically connected to colonization of the vertebrate host; they are found exclusively in pathogenic Listeria species. Other listerial factors involved in virulence have been identified that also contribute to successful colonization of the host. They have functions outside the pathogenic lifestyle and are present in nonpathogenic Listeria species. Such factors are sometimes called accessory virulence factors. P60,

PRODUCT

OF THE IAP

GENE

Protein p60 is a major protein secreted by all L. monocytogenes isolates [34,182]. It is also found on the cell surface of the bacteria [278]. Because it possesses murein hydrolase activity that appears to be involved in a late step of cell division, p60 is an important enzyme for cell metabolism of L. monocytogenes cells [333]. The gene for this protein, initially called iap (invasion associated protein), codes for an extremely basic protein of 484 amino acids, with a 27-amino-acid signal sequence and an extended repeat domain consisting of 19 threonine-asparagine units separated by a proline–serine–lysine motif. A single cysteine found in the C-terminal part of p60 is probably essential for its enzymatic activity [171,333] and amino acids in the N-terminal region of the protein define its intracellular stability [295]. p60 is a member of a protein family in L. monocytogenes with three members: p45, which is a peptidoglycan lytic protein [283] encoded by the spl gene; the putative protein encoded by lmo394 [117]; and p60. At least one type of rough mutant of L. monocytogenes characterized by expression of reduced amounts of p60 shows significantly reduced uptake by 3T6 fibroblast cells [182]. These mutants form long cell chains that possess double septa between the individual cells. Treatment of L. monocytogenes rough mutants with partially purified p60 protein disaggregates the cell chains to normally sized single bacteria, which again become invasive for fibroblasts. Ultrasonication leads to physical disruption of the cell chains, producing similar single cells that are noninvasive. The reduced invasiveness of the p60 mutants is only observed with certain mammalian host cells. Cell chains of p60 mutants adhere normally to Caco-2 epithelial cells and are perfectly invasive for these cells upon disruption of bacterial cell chains by ultrasonication without addition of p60 [34]. Other rough mutants of L. monocytogenes that show normal or even increased levels of p60 expression have been isolated [277]. These mutants are adherent and invasive like WT bacteria despite formation of long filaments. The genetic basics of these phenotypes are unknown. p60 was long regarded as an essential protein [333]. However, viable mutants with in-frame deletions in the iap gene recently demonstrated that p60 is not an essential protein for L. monocytogenes [199,249]. Detailed characterization of one of the deletion mutants showed that the mutant forms cell chains but is nearly as invasive as the WT strain. However, the mutant grows in microcolonies inside host cells and does not form F-actin tails; it only induces actin clouds around the bacteria. A defect in polar ActA distribution caused by impaired cell division is the reason for lack of intracellular motility in this p60-lacking strain. According to these findings, p60, renamed

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CwhA for cell wall hydrolase A, seems not to be linked directly to cell invasion; rather, it indirectly modifies bacterial behavior via its impact on cell division [249]. Another L. monocytogenes peptidoglycan hydrolase, called MurA or p66, was recently identified [43]. Deletion of the gene encoding MurA, which shows homology with p60 in its C-terminal domain, also results in the formation of long cell chains. It is currently believed that p60 and p66 may act in concert to control proper cell separation during the last step of bacterial division [43]. Two recent papers by Portnoy and coworkers [199,200] also shed light on genetic and biochemical bases of reduced p60 expression and the rough phenotype. They described an additional secA gene in L. monocytogenes, called secA2, located immediately upstream of the iap gene and involved in the smooth–rough transition. They found that rough mutants with reduced p60 expression have a deletion in the secA2 gene or express truncated SecA2 proteins. Furthermore, deletion of the secA2 gene resulted in reduced p60 expression and conversion to the rough phenotype. Rough mutants displaying normal p60 levels are not affected in their secA2 gene. A number of additional proteins, the secretion of which is secA2 dependent, have been identified [199]; it has been shown that the hydrolytic activity of p60 is crucial for L. monocytogenes virulence through generation of glucosaminylmuramyl dipeptide, which modifies host inflammatory responses.

SUPEROXIDE DISMUTASE

AND

CATALASE

Possible roles of catalase and superoxide dismutase in the virulence of L. monocytogenes have recently been reviewed [137,320]. Both enzymes act in concert to detoxify potentially harmful superoxide radicals. Generated by the oxidative burst in a phagocytic cell, these radicals are converted into hydrogen peroxide by action of superoxide dismutase, which is then cleaved by catalase into water and molecular oxygen. Bacterial catalases and superoxide dismutases have long been suspected to be important virulence factors of intracellular bacteria, but no correlation of superoxide dismutase expression with virulence was found in L. monocytogenes [326]. The gene for superoxide dismutase from L. monocytogenes [29], called lmsod, reveals an ORF coding for a protein of 202 amino acids with high homology to manganese-containing superoxide dismutases from other organisms. Catalase mutants obtained by transposon mutagenesis show wild-type virulence in infected mice [190]. Whereas catalase-negative L. monocytogenes mutants are killed by mouse resident macrophages already at low serum concentrations, killing wild-type bacteria requires high serum concentrations, suggesting that resistance to fully activated macrophages is partially mediated by catalase activity [316]. Isolation of catalase-negative L. monocytogenes strains from listeriosis patients supports the notion that catalase does not seem to be necessary for intracellular growth of L. monocytogenes [35,88]. Also in line with these data is the finding that all species of Listeria produce the same type of SOD, which is constitutively expressed and not regulated by environmental factors [317], as true virulence factors are.

STRESS RESPONSE MEDIATORS Bacterial survival under stress conditions requires an adaptive response mediated by a set of conserved proteins that are upregulated upon exposure to many different stress conditions and when bacterial growth is restricted. However, in contrast to other facultative intracellular bacteria, L. monocytogenes does not induce expression of general stress proteins during intracellular proliferation in macrophages [139]. Nevertheless, stressful conditions are likely to be encountered by L. monocytogenes during transient residence in the phagosome upon uptake by macrophages. In that stage, expression of the hly gene encoding LLO is induced as it is under other stress conditions like heat treatment [303–305]. A group of virulence-associated stress mediators involved in phagosomal escape and intracellular multiplication were identified in L. monocytogenes. The first was ClpC, an ATPase belonging

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to the heat-shock protein 1000 family [282]. The gene encoding the ClpC ATPase, called clpC, was identified by Tn917 mutagenesis and a selection for mutants depending on iron [274]. The clpC mutants are highly susceptible to stress, including iron limitation, elevated temperatures, and high osmolarity. Virulence of these mutants is severely impaired in the mouse with restricted capacity to grow in bone marrow-derived macrophages [275,276]. Electron microscopy of infected macrophages showed that clpC mutants remained inside the phagocytic vacuole, indicating that ClpC is involved in phagosomal lysis or helps the bacteria to survive in the harsh conditions of this environment to allow production of sufficient amounts of LLO and PI-PLC to destroy the vacuolar membrane [275]. Expression of ClpC is not directly controlled by PrfA and no PrfAboxes (discussed later) are found in the promoter region of the clpC gene. In contrast, overexpression of the PrfA regulon leads to a loss of ClpC expression, showing that expressional cross-talk between PrfA and ClpC is most likely mediated by a negative regulator activated by PrfA [269]. Recent evidence also points to a role for ClpC in expression of internalin, InlB and ActA, because ClpC is required for efficient uptake into several cell lines [235]. ClpE is closely related to ClpC and is also involved in virulence of L. monocytogenes [234]. L. monocytogenes mutants with deletions in the clpC and clpE genes are totally avirulent in mice; it is believed that both proteins have redundant functions in stress tolerance because the absence of one is compensated by the transcriptional upregulation of the other [234]. ClpP is a stress-induced L. monocytogenes serine protease of 22 kDa belonging to a highly conserved protein family. ClpP is required for growth under stress conditions and for survival in macrophages and infected animals [107]. ClpP seems to be involved in stress-induced upregulation of LLO expression because clpP-mutants secrete only small amounts of functional LLO [106]. Expression of ClpC, ClpE, and ClpP is negatively controlled by ctsR, the first gene of the operon containing ClpC [233]. L. monocytogenes CtsR is homologous to the B. subtilis CtsR repressor of stress response genes. Consistent with the function of CtsR as a repressor is the fact that a ctsR deletion mutant showed enhanced survival under stress conditions and normal virulence, whereas overproduction of CtsR resulted in a significantly attenuated virulence in the mouse model of infection [233].

IRON UPTAKE SYSTEMS Iron is a key element for all bacteria, serving as a cofactor in many proteins involved in electron transport processes. Inside the host organism, however, iron is not freely available because it is tightly bound to transferrin or ferritin. Pathogenic bacteria therefore had to evolve specific mechanisms to capture iron from host tissues and these uptake mechanisms play an important role in virulence. In L. monocytogenes, different iron-uptake mechanisms have been described [32]: The first uses the direct transport of ferric citrate [2] and the second involves an extracellular ferriciron reductase that uses iron-loaded catecholamines as a substrate [12,57,67]; the third system may use a surface-located transferrin-binding protein [140]. Unlike many other pathogenic bacteria, L. monocytogenes does not secrete siderophores to capture extracellular iron [58]. The genetic basis of different iron uptake systems has not been studied and there is no experimental evidence whether these systems directly contribute to pathogenesis of L. monocytogenes infection.

PRFA AND REGULATION OF VIRULENCE GENE EXPRESSION IN L. MONOCYTOGENES THE POSITIVE REGULATORY FACTOR A (PRFA) The first indications for coordinate regulation of virulence genes in L. monocytogenes by a trans acting factor were obtained from analysis of spontaneously occurring nonhemolytic mutants of L. monocytogenes, which carry deletions in a region upstream of the hly gene [126,197]. Cloning and

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sequencing of the locus affected by the deletion led to identification of the prfA (positive regulatory factor A) gene. Its product, PrfA, a cytoplasmic protein of 27 kDa [198,217], regulates all virulence genes of the virulence gene cluster. The prfA deletion mutants can be complemented in trans by introduction of the cloned prfA gene to yield a wild-type phenotype again [198]. Site-specific mutations or transposon insertions in the prfA promoter or the prfA coding region block transcription of the entire gene cluster, i.e., plcA, hly, mpl, actA, and plcB [47,217]. This indicates that the prfA gene encodes a transcriptional activator required for expression of the L. monocytogenes virulence gene cluster. Additional evidence for this presumptive role of PrfA was provided by transcriptional activation of the cloned hly gene by PrfA in B. subtilis [98]. Also present in the closely related species L. ivanovii and L. seeligeri are prfA-like genes with high sequence similarity to prfA from L. monocytogenes [178,185]. In L. seeligeri the prfA gene is silenced [178] and activity of the protein is not known; however, PrfA from L. ivanovii also controls sets of virulence genes similar to those of L. monocytogenes [185]. PrfA is a member of the Crp/Fnr family of transcriptional activators, which have been identified so far mainly in Gramnegative bacteria. Like all members of this family, PrfA contains a conserved helix–turn–helix motif in its C-terminal part. In addition, adjacent to this motif, PrfA carries a sequence with features of a leucine zipper and a second helix–turn–helix motif at its N-terminus [30,185,288] (Figure 5.7). In hyperhemolytic strains of L. monocytogenes, an altered PrfA, termed PrfA*, has been identified and characterized and it was shown that all these PrfA variants carry a Gly145Ser

FIGURE 5.7 Structure of PrfA (A) and its target DNA sequences in PrfA-regulated promoters (B). In PrfA, amino acid coordinates correspond to the peptide sequence deduced from the prfA gene (position 1 corresponds to the Met residue of the first triplet), whereas in Crp they correspond to the actual amino acid position in the primary structure of the native protein, which lacks the residue encoded by the first triplet. Therefore, the amino acid numbering of Crp is shifted one position with respect to that of PrfA and hence position 144 of Crp, where the Crp* mutation Ala144Thr lies, aligns exactly with position 145 of PrfA, where the Gly145Ser substitution leading to the PrfA* mutant phenotype is located. In Crp, A to F designate the α-helical stretches of the protein and AR1 is activating region 1 (two other activating regions [AR2 and AR3] are embedded within the β-roll structure). In PrfA, structures specific for this protein are in light gray. (B) N indicates any nucleotide. (Reprinted with permission from Vazquez-Boland, J. A. et al. 2001. Clin. Microbiol. Rev. 14:584–640.)

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substitution [23,267]. Furthermore, all genes of the virulence gene cluster are highly expressed in these strains and do not respond to conditions under which expression of virulence genes is induced in wild-type strains [267]. Interestingly, Crp mutants are known that carry a mutation at similar positions and lead to a transcriptionally active conformation without the need of the cofactor cAMP (Figure 5.7). This suggests that wild-type PrfA also needs a conformational change for activity and that the Gly145Ser substitution results in a constitutively active form of PrfA [321]. Three other mutations in PrfA were recently reported to increase PrfA function: a Gly155Ser mutation that appears to be similar in nature to Gly145Ser and a Glu77Lys substitution located in the β-roll structure in the N-terminal part of the protein [292]. A Ser183Ala substitution located in the second HTH motif that also resulted in increased PrfA activity was described by Sheehan et al. [288]. On the other hand, it was shown that inactive PrfA can be present in large quantities in L. monocytogenes [266]. In addition, PrfA mutants were selected with single point mutations rendering the protein partially or totally inactive [144]. These mutants were unable to bind to DNA and harbored mutations in the leucin zipper or the HTH motif or they still bound to the target DNA but were unable to form a stable complex with RNA polymerase and were located in the β-roll structure. Other studies suggested that PrfA-mediated activation of gene expression requires presence of a coactivator protein [22, 69] the nature of which is still unknown.

PRFA-DEPENDENT PROMOTERS, TRANSCRIPTS, AND MECHANISM OF TEMPERATURE-DEPENDENT VIRULENCE GENE EXPRESSION A 14-bp palindromic sequence first identified in the promoter region of the hly gene [221] was found to be present in promoters of all PrfA-dependent genes, located about 40 bp upstream of the transcriptional start site. However, the 14-bp palindrome, called PrfA-box, is not perfectly conserved in all promoters and the differences could contribute to differential regulation of the adjacent genes by PrfA (Figure 5.7) [30,176,289,331]. Meanwhile, it was shown that PrfA directly binds to these palindromic sequences [22,24,69,288], thereby activating the virulence genes. Purified PrfA alone is able to bind specifically to the target sequence as shown by gel motility shift assays. However, addition of PrfA-free cell extracts results in formation of additional PrfA-containing DNA-binding complexes that also contain RNA polymerase [24,69]. Whether PrfA first binds to the PrfA-box and enhances binding activity for RNA polymerase or whether the PrfA/RNA polymerase complex may already form before interaction with DNA is still unknown. The observation that PrfA can exist in an inactive form and the striking similarity of the cAMP-independent mutant Crp* and the constitutively active PrfA* strongly suggest involvement of a cofactor in regulation of PrfA activity. Despite extensive investigations, such a cofactor remains unknown [178]. The transcriptional organization of most PrfA-regulated genes—especially the virulence gene cluster—is complex. Three transcripts of the prfA gene have been identified: a long (2.1 kb) transcript cotranscribed with the plcA gene and autoregulated by PrfA, and two shorter ones (0.8 and 0.9 kb) transcribed from three distinct promoters located in front of the prfA gene [97]. The listeriolysin gene, hly, is the only gene in the virulence gene cluster transcribed in a monocistronic mRNA from two PrfA-dependent promoters, P1 and P2, located in the intragenic region between hly and plcA [221]. A third hly promoter, P3, downstream from P1 and P2, was recently identified and shown to be PrfA independent; it results in low-level transcription of the hly gene [73]. The three genes of the lecithinase operon are transcribed from at least two PrfA-regulated promoters: One, located in front of the mpl gene, yields a 5.4- to 5.7-kb transcript comprising mpl, actA, and plcB, as well as an additional mRNA of 1.8 kb comprising mpl alone [24]. A second promoter, located directly in front of the actA gene, leads to a 3.6-kb bicistronic transcript comprising actA and plcB [24]. The inlAB operon is transcribed from multiple PrfA-dependent and -independent start sites upstream of inlA. In different studies, three or four start sites were mapped in the promoter region upstream of inlA that harbors a degenerated PrfA-box with two mismatches

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(Figure 5.7) [79,203]. In addition, an inlB monocistron is detectable and a putative transcription terminator is located between inlA and inlB. Overall, transcriptional control of inlAB expression is complex and not fully understood. Full expression of PrfA-dependent virulence genes requires synthesis of monocistronic prfA transcripts and the bicistronic plcA-prfA transcript [96,42]. In an initial step of the infectious process, it is believed that transcription of prfA via the prfA promoters results in synthesis of a limited amount of PrfA sufficient to activate the high-affinity PrfA-dependent hly and plcA promoters. This, in turn, would result in synthesis of the plcA-prfA transcript, which leads to enhanced PrfA synthesis. The higher cellular level of PrfA activates the mpl and actA promoters, which seem to have a lower affinity for PrfA because of base mismatches in their palindromic PrfA boxes [53]. As in many other instances [149,175], PrfA-dependent virulence gene expression in L. monocytogenes is thermoregulated and the shift from 30 to 37°C results in a dramatic increase in expression of virulence genes [196,217]. Detailed recent analysis of this phenomenon culminated in identification of an RNA thermosensor controlling temperature-dependent gene expression [155]. Low expression of virulence genes at temperatures below 30°C coincides with absence of PrfA protein. However, quite surprisingly, the prfA gene is still transcribed under these conditions from its own promoter, resulting in a monocistronic prfA transcript [196,266]. At 37°C, prfA is transcribed from the prfA promoter and the PrfA-dependent plcA promoter, resulting in monocistronic and bicistronic plcA-prfA messengers [196,217,266]. Johannson et al. [155] could now demonstrate that, at 30°C, the monocistronic prfA messenger is not translated because the upstream untranslated mRNA (UTR) preceding prfA forms a secondary structure that masks the ribosome-binding region. This secondary structure is thermosensitive and hence unstable at higher temperatures (37°C), which then allow efficient translation resulting in an approximately fivefold increase in PrfA levels. As mentioned earlier, in the presence of PrfA, the bicistronic plcA-prfA messenger is transcribed from the PrfA-dependent promoter upstream of plcA, further increasing the amount of PrfA in the bacterial cell and finally allowing virulence gene expression from low-affinity promoters. L. monocytogenes has hence evolved a sophisticated twostep mechanism to control PrfA-dependent virulence gene expression (reviewed in Johansson and Cossart [154] and Newman and Weiner [236]). Before completion of the genomic sequence of L. monocytogenes [117], knowledge of the PrfA regulon was very limited. At that time, the only known genes that did not belong to the virulence gene cluster but were also regulated by PrfA were inlAB, [79] and inlC [75,89]. The availability to sequence the complete genome of L. monocytogenes allowed in silico screening of the sequence for genes preceded by putative PrfA-boxes. This screen identified four additional previously unknown genes harboring PrfA-boxes. One of the genes, later called hpt, encodes for a putative hexose permease, expression of which was shown to depend strictly on PrfA [48]. The other three genes preceded by PrfA-boxes are genes of unknown function. A recent systematic approach to elucidate the complete PrfA regulon used a whole genome macroarray based on the complete genome sequence of L. monocytogenes [223]. With this macroarray, expression profiles of the wild-type strain and a prfA-deletion mutant were compared upon growing the bacteria in standard BHI medium or in media known to induce or to repress virulence gene expression. With this approach, three groups of differently regulated genes were identified. In addition to the 10 already known PrfA-regulated genes, Group I comprises 2 new genes, positively regulated and preceded by a PrfA box. Group II comprises eight negatively regulated genes: One is preceded by a PrfA box and the others form an operon. Group III comprises 53 genes of which only 2 are preceded by a PrfA box and that are activated or repressed under different conditions; most of the genes in this group are transcribed from sigma B-dependent promoters. Taken together, the results suggest that PrfA positively regulates a core set of 12 genes preceded by a PrfA box that is probably expressed from a sigma A-dependent promoter and negatively regulates 8 genes. A second set of PrfA-regulated genes lacks PrfA boxes and is expressed from sigma B-dependent promoters. The data presented reveal that PrfA can act as an activator or a repressor and suggest

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that PrfA may directly or indirectly activate sets of genes in association with different sigma factors [223]. The important role of sigma B in expression of L. monocytogenes virulence genes was also confirmed by recent studies [165,308] showing that sigma B regulates not only stress response genes but also several well-known virulence genes like those encoding the internalins and the bile salt hydrolase.

ENVIRONMENTAL SIGNALS AFFECTING VIRULENCE GENE EXPRESSION Pathogenic bacteria that can also live in the free environment are forced to sense their surroundings in order to regulate expression of genes needed for living inside or outside their host. Facultative intracellular pathogens additionally should be able to know whether they are inside or outside their individual mammalian host cell. An increasing number of signals have been shown to affect virulence gene expression in L. monocytogenes (reviewed in Brehm et al. [30] and Kreft and Vazquez-Boland [178]). The signals can be classified into physicochemical signals (temperature, iron, glucose, cellobiose, salt, pH, activated charcoal) or stress conditions (heat shock, oxidative stress, nutritional stress, growth inside host cells). The mechanisms of altered gene expression under such conditions are unknown or only poorly understood. However, in all systems analyzed, PrfA plays a role in regulation of environmentally modulated gene expression. At temperatures below 30°C, the PrfA-dependent genes are not transcribed because of a lack of translation of the monocistronic prfA transcript (see earlier discussion) [155]. A shift in temperature to 37°C results in onset of prfA expression followed by transcription of the virulence cluster genes [79,196]. Treatment of culture medium with activated charcoal probably depletes a signal molecule from the medium; this would result in increased transcription of prfA and the PrfA-dependent genes [268]. Carbohydrates modulate virulence gene expression in a complex and poorly understood manner. Glucose directly influences prfA gene expression and thereby interferes with PrfA regulation. Addition of larger amounts of glucose to the medium results in acidification, which reduces LLO expression by unknown mechanisms [62,97]. The disaccharide cellobiose results in inhibition of hly and plcA expression without reduction in monocistronic prfA mRNA levels and with only slightly decreased amounts of PrfA protein. This indicates that the inhibitory mechanism exerted by cellobiose leads to a change in PrfA activity [16,166,245]. Two genomic loci have been identified [31,148] that contribute to cellobiose-mediated virulence gene repression; one of these, the bvrABC system, most likely encodes for a sensor system for extracellular cellobiose [31]. The mechanisms of stress-mediated altered gene expression are even less understood. Heat-shock conditions increase hly, plcA, and actA expression. P60 expression is inhibited by heat shock and also by oxidative stress (H2O2) [303–305]. Shift of L. monocytogenes from a rich medium into a minimal essential medium (MEM) induces expression of the virulence cluster genes as well as other surface-associated proteins [268]. Phagocytosis and intracellular localization are supposed to be natural stress factors. It has been shown that expression of numerous proteins is selectively induced during phagocytosis of L. monocytogenes by macrophages [139]. An insertional mutagenesis study [166] and two IVET studies [84,101] tried to identify genes preferentially expressed inside mammalian cells using the hly gene as a reporter. The first study resulted in identification of genes involved in nucleotide biosynthesis, an arginine transporter, and plcA [166]; the IVET studies only identified some of the known virulence genes and some unknown genes whose function is unknown in the intracellular life cycle. Experiments directly measuring bacterial mRNA levels inside host cells by RT-PCR or gfp-reporter gene assays revealed that the genes hly, actA, and inlC are heavily expressed inside the mammalian cell [24,36,89,95,291]. It was also shown that PrfA is upregulated during interaction of L. monocytogenes with host cells [265]. The complexity of regulation of gene expression inside the host cell is, despite all progress, far from understood.

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Signal transduction mechanisms allowing bacteria to modulate gene expression in response to diverse stimuli often involve two-component systems (TCSs) composed of a sensor kinase and a response regulator [132]. The sensor kinase is often localized in the cytoplasmic membrane to sense outside signals and contains a highly conserved cytoplasmic kinase domain which, upon activation, phosphorylates the cognate response regulator. The phosphorylated response regulator then binds to specific DNA sequences and activates transcription of its target genes. TCSs have not been studied intensively in L. monocytogenes; lisR/K [52], cheY/A [93], agrA/C [10], and cesRK [161] were characterized in some detail and shown to contribute to virulence of L. monocytogenes. Kallipolitis et al. [160] identified a total of seven putative TCSs in a PCR-based approach including the already known lisR/K. Availability of the complete genomic sequence of L. monocytogenes strain EGD-e allowed the in silico identification of 16 putative two-component systems in addition to a large array of other putative regulatory systems belonging to different families. The largest of these are GntR-like regulators and BglG-like antiterminators, many of which are associated with PTS. Surprisingly, only 1 of 16 putative TCSs identified had no counterpart in the closely related but totally apathogenic species L. innocua. This points to a minor role of TCSs for virulence gene regulation in L. monocytogenes [117]. To study the role of the listerial TCS for in vitro and in vivo survival and growth of L. monocytogenes more systematically, in-frame deletion mutants in 15 of 16 TCSs were constructed by introducing large deletions in the individual response regulator genes, removing most of the open reading frames [330]. The mutants are currently characterized by testing them in various in vitro growth assays and several cell culture assays and by assessing their virulence potential in experimentally infected mice. At least one of the previously undescribed response regulators (degU) (lmo2515) also contributes to virulence because the L. monocytogenes degU mutant is severely impaired in colonizing experimentally infected mice [330].

LESSONS LEARNED FROM GENOME SEQUENCE OF L. MONOCYTOGENES Availability of complete genomic sequences of L. monocytogenes and its close nonpathogenic relative L. innocua [117] opens new possibilities for investigation of listerial virulence. Sequencing of the 2,944,528 base pairs of the circular chromosome of L. monocytogenes revealed the presence of 2,853 protein-coding genes, from which about 35% are without a predicted function. Comparison of this sequence with that of L. innocua and the closely related species B. subtilis revealed some special features of L. monocytogenes [37,117]. The first surprise was the presence of many putative surface proteins belonging to six families (19 internalins, 21 other LPXTG proteins, 9 GW module-containing proteins, 11 hydrophobic-tail proteins, 4 p60-like proteins, and up to 68 putative lipoproteins). The high number of related members in each family seems to be—at least in part—because of extensive gene duplications. Of particular interest are surface proteins characterized by the common LPXTG motif in their C-terminus necessary for covalent linkage of the proteins to the cell wall peptidoglycan. L. monocytogenes harbors a total of 41 genes encoding for LPXTG proteins (more than any other bacterial species analyzed). These can be further grouped into the family of internalins and internalin-like proteins (characterized by the leucine-rich repeat motif) and the LPXTG proteins lacking this leucine-rich repeat. The function of the vast majority of these genes is unknown, but one may speculate that internalins and internalin-like proteins confer host-species and cell-type specificity during infection. They may also mediate attachment of bacteria to other surfaces during their life outside the mammalian host [38,117]. Another surprising feature of L. monocytogenes revealed by analysis of the genome was the high number of transport proteins (331 in total), of which 88 are devoted to carbohydrate transport by phosphoenolpyruvate-dependent phosphotransferase systems (PTS). Hence, L. monocytogenes

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has nearly three times as many PTS systems as B. subtilis, which probably gives L. monocytogenes the ability to take up, and therefore grow on, a large number of carbohydrates. As mentioned earlier, one specific hexose-phosphate transporter is necessary for intracellular multiplication of L. monocytogenes and further studies will most likely reveal the importance of other uptake systems for adaptation to different environmental conditions. As anticipated, because of the wide range of different conditions faced by L. monocytogenes during extracellular and intracellular growth, a high number of transcriptional regulators (209 in total) have also been identified. This number is second only to that of Pseudomonas aeruginosa, another ubiquitous opportunistic pathogen [117].

EVOLUTIONARY ASPECTS Based on in vitro data, all Listeria species are regarded as normally noncompetent [25]. It was therefore totally unexpected to find genes in L. monocytogenes and L. innocua coding for putative DNA uptake systems homologous to B. subtilis competence genes [25,117]. The uptake apparatus may no longer be functional, or its regulation or signals that induce competence may differ from those of B. subtilis and correct conditions to induce competence have not yet been found during laboratory culture. Nevertheless, the possibility of gene transfer by transformation could well explain genomic differences between sequences of the two Listeria species characterized on a genomic level to date. These differences are mainly found in blocks dispersed around the chromosome, resulting in a mosaic genome structure. Furthermore, the collinearity identified for L. monocytogenes and L. innocua chromosomes (see Figure 1.1) also extends to numerous regions of the B. subtilis chromosome. The hundreds of insertions found in the three chromosomes are best explained by multiple independent transformation events followed by DNA integration at various sites in the chromosomes. The origin of the known virulence genes in Listeria is still unclear. The large family of internalins seems to have evolved in Listeria after initial combination of the LPXTG membrane anchor motif with a leucine-rich repeat motif (both of unknown origin) to form a proto-internalin. This then (probably) duplicated several times and evolved further by recombinations and by point mutations. The virulence gene cluster was obviously acquired a long time ago because traces of the transfer event are barely detectable, or it evolved in Listeria. Of the individual genes present in the virulence gene cluster, homologues of the listeriolysin gene [242], the metalloprotease gene [72,218], and the two phospholipase genes [195, 215, 319] are present in many related species but never found in a cluster as in L. monocytogenes. The origin of the actA gene is unknown because no bacterial protein with significant homology has yet been isolated. It has been speculated, however, that the actA gene may be of eukaryotic origin because parts of the protein show some homology to eukaryotic cytoskeletal proteins [45]. At present, fully functional virulence clusters are found in L. monocytogenes and in the animal pathogen L. ivanovii; a similar cluster with additional genes is present in the nonpathogenic species L. seeligeri (Figure 5.2) [45,127]. Interestingly, one of the ORFs with unknown function at the right border of the cluster shows some weak homology to genes of Listeria phages [45,204]. This might point to phage transduction events involved in early evolution of this gene cluster. Analysis of the sequences flanking the virulence gene cluster in different Listeria species indicates that it is ancestral to the genus Listeria because it is present in all isolates exactly at the same chromosomal position [45,46,179]. Most likely, the virulence gene cluster was lost by two independent events from the nonpathogenic species L. innocua and L. welshimeri as indicated by the presence of short DNA sequences believed to belong originally to the virulence gene cluster [179]. The situation in L. grayi is still unknown, but current sequencing efforts [116] will soon resolve the situation in this more distantly related species. Availability in the near future of genome sequences of L. ivanovii, L. welshimeri, L. grayi, and L. seeligeri—in addition to those of L. monocytogenes and L. innocua (thus, the complete genome sequences of the whole Listeria genus) [46,116]—and their comparison will certainly greatly enlarge understanding of the evolution of the genus Listeria.

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OPEN QUESTIONS The last years have seen an enormous increase in understanding of the molecular basis of infectious diseases. Knowledge of the genes determining virulence of L. monocytogenes and the role played by virulence gene products in the infectious process is rapidly expanding. However, many problems concerning the virulence of L. monocytogenes remain unsolved. For instance, L. ivanovii, a species largely apathogenic for humans [287], resembles L. monocytogenes in its intracellular life cycle [162]. Genes homologous to most of the known virulence genes of L. monocytogenes are also detectable in this species [127,136,177] and the complete PrfAregulated gene cluster identified in L. monocytogenes is also present in L. ivanovii [127]. However, L. ivanovii is virulent for animals and avirulent for humans; an experimental infection in mice yielded a different outcome than that of L. monocytogenes [147]. What is the molecular explanation for this obvious difference in the pathogenic potential of these two Listeria species? Is it the result of a different mechanism in regulation of known virulence genes inside the infected cells or differences in specific activity of the known virulence gene products? What is the role of the many small internalins present in L. ivanovii but absent in L. monocytogenes? The availability of the genomic sequence of L. ivanovii in the near future [116] will probably help to answer at least some of these questions. Expression of L. monocytogenes virulence genes inside infected mammalian host cells and tissues is another important but unsolved problem. The expression pattern of known L. monocytogenes virulence determinants is complex under in vitro growth conditions and regulated by PrfAdependent and PrfA-independent mechanisms. Very little is known of how PrfA and other putative regulatory factors control these genes while the bacteria reside inside host cells and tissues. Application of available high-throughput methods like macro- and microarrays to measure gene expression on the mRNA level [223] or two-dimensional gel electrophoresis on the protein level [257] of many genes in parallel under different conditions will certainly dramatically increase understanding of gene expression regulation of L. monocytogenes. These data, together with others, will greatly add to understanding of how this fascinating human pathogen thrives under very different conditions inside and outside the human host.

ACKNOWLEDGMENTS We thank T. Williams and J. Kreft for carefully and critically reading this manuscript. We apologize to all those who contributed to our current knowledge on the biology of L. monocytogenes but were not mentioned in this chapter. Work from this group was supported by the Deutsche Forschungsgemeinschaft through the grants SFB 479-B1 (WG) and SFB 479-B5 (MK), the European Union through the grants BMH4-CT96-0659, BIO4-CT98-0036, and QLG2-CT1999-00932 (WG), and the Fonds der Chemischen Industrie (WG).

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of Listeria 6 Characteristics monocytogenes Important to Food Processors Beatrice H. Lado and Ahmed E. Yousef CONTENTS Introduction ....................................................................................................................................158 Temperature....................................................................................................................................159 Growth Temperature.............................................................................................................159 Range and Optimum ................................................................................................159 Growth Kinetics........................................................................................................160 Cold Tolerance..........................................................................................................161 Stress Adaptation at Elevated Sublethal Temperatures............................................163 Variations in Virulence with Temperature ...............................................................163 Freezing ................................................................................................................................163 Lethal Temperature...............................................................................................................164 Mechanisms of Thermal Inactivation.......................................................................164 Kinetics of Thermal Inactivation..............................................................................165 Extrinsic Factors in Resistance to Heat ...................................................................167 Intrinsic Factors in Resistance to Heat ....................................................................167 Surrogate Microorganisms for Thermal Studies..................................................................168 Acidity ............................................................................................................................................169 Survival at Low pH ..............................................................................................................169 Inactivation at Low pH.............................................................................................169 Factors Influencing Acid Tolerance .....................................................................................170 Mechanisms of Acid Damage and Tolerance ......................................................................170 Consequences of Enhanced Acid Tolerance ........................................................................171 Water Activity ................................................................................................................................171 Water Activity and Growth or Survival ...............................................................................171 Osmotolerance Factors .........................................................................................................172 Antimicrobial Components in Food ..............................................................................................174 Salt ........................................................................................................................................174 Survival at Extreme Salt Concentrations .................................................................174 Physiology at High Salt Concentrations ..................................................................174 Organic Acids and Their Salts .............................................................................................175 Lactate.......................................................................................................................176 Sodium Diacetate......................................................................................................176 Sodium Propionate ...................................................................................................177 Potassium Sorbate ....................................................................................................177

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Sodium Benzoate......................................................................................................178 Parabens and Other Benzoic Acid Derivatives ........................................................178 Fatty Acids and Related Compounds...................................................................................178 Free Fatty Acids .......................................................................................................178 Fatty Acid Monoesters .............................................................................................179 Sodium Nitrite ......................................................................................................................180 Antioxidants..........................................................................................................................180 Smoke ...................................................................................................................................181 Spices, Herbs, and Plant Extracts ........................................................................................181 Lysozyme..............................................................................................................................182 Hydrogen Peroxide...............................................................................................................183 Lactoperoxidase System.......................................................................................................183 Lactoferrin ............................................................................................................................184 Biocontrol.......................................................................................................................................185 Live Fermentate....................................................................................................................185 Bacteriocins ..........................................................................................................................185 Modified Atmosphere.....................................................................................................................186 Alternative Processing Technologies .............................................................................................187 Irradiation .............................................................................................................................187 Ionizing Radiations...................................................................................................187 Ultraviolet Radiation and High-Intensity Pulsed Light...........................................188 High-Pressure Processing.....................................................................................................189 Pulsed Electric Field Processing..........................................................................................189 Attachment and Biofilm Formation...............................................................................................190 Sanitizers ........................................................................................................................................192 Chlorine and Chlorinated Compounds.................................................................................193 Quaternary Ammonium Compounds ...................................................................................194 Acid Sanitizers......................................................................................................................194 Ozone....................................................................................................................................194 Miscellaneous Sanitizing Agents .........................................................................................195 Active Packaging............................................................................................................................196 Multiple Antimicrobial Treatments................................................................................................197 References ......................................................................................................................................198

INTRODUCTION The goal of food processing is to produce a safe, wholesome product that has a suitable shelf life and is acceptable to the consumer. Food manufacturers rely on a variety of processing and preservation methods to reach this goal. These methods inactivate or inhibit growth of spoilage and pathogenic microorganisms, suppress undesirable chemical and biochemical changes and hence ensure food’s safety, and maintain its desirable physical and sensory properties. Methods currently used in food preservation involve physical, chemical, or biological factors. Physical preservation factors include heating, cooling, freezing, radiation, high-pressure processing, and packaging. Chemical treatments include addition of antimicrobial agents (e.g., benzoate, propionate, and sorbate), acidifying agents (e.g., acetic and lactic acids) or curing agents (e.g., sodium chloride and sodium nitrite). Preservation by biological means (biopreservation) includes fermentations that control spoilage and pathogenic microorganisms through competition for substrate, gradual lowering of pH, and release of antimicrobial metabolites. Success of a preservation technology depends on meeting the processing goal described earlier. Heat is the most reliable and commonly used preservation factor, but thermal processing alters

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quality of food and decreases availability of nutrients. Alternative technologies, such as radiation and high-pressure processing, may maintain the critical balance between food’s safety and its quality. Similarly, treatment combinations are used in food processing and the value of using multiple preservation factors is best expressed as the hurdle concept. Growth, inhibition, or inactivation of Listeria monocytogenes in response to food-processing and preservation techniques will be detailed in this chapter. To eliminate any ambiguities, some basic concepts will be defined, and every effort will be made to use these terms uniformly in this chapter. The expression “log” or “log count” refers to the microbial count in “log10 CFU/mL or g.” When measurable increase in count is encountered, it is described as such, unless multiplication of the microorganism is reported; in this instance, the increase will be described as “growth.” Growth of microorganisms is enhanced when the lag phase decreases, generation time decreases (i.e., maximum specific growth rate increases), or the gain in count attained after a given growth period increases. Conversely, “growth inhibition” or simply “inhibition” is the condition when opposite change in the growth parameters just described occurs. Growth inhibitors may also be described as bacteriostatic and, for inhibition of Listeria, as listeriostatic agents; such agents do not usually cause measurable inactivation. “Inactivation” refers to a decrease in cell population; it will be expressed as a decrease in log count. An agent that causes microbial inactivation is described as bactericidal, but may also be reported as listericidal when it is active against Listeria. The D-value is the time of exposure to a lethal factor (e.g., heat or radiation) required to inactivate 90% (i.e., one log) of the population of a given microorganism at a given dose of the deleterious factor. D-value is a measure of resistance of the microorganism to the deleterious (lethal) factor; the larger the D-value is, the greater is the resistance. When the count does not change appreciably, the status of the microorganism is best described as survival. The word “survival” also refers to ability of the microorganism to maintain its viability during the treatment. Some generalizations and conclusions in this chapter should be viewed with caution. These are based on published research using bacterial strains available to researchers at the time of the study. Recent reports show that strains of L. monocytogenes vary considerably in resistance to processing and new processing-resistant strains are occasionally discovered [190,310]. If highly resistant pathogenic strains are discovered in the future, the conclusions in this chapter will need to be modified accordingly.

TEMPERATURE The temperature to which food is exposed may have growth-conducive, preserving, or lethal effects on microorganisms in the product. At ~0 to 45°C, L. monocytogenes grows to various extents when present in a suitable medium. Temperatures below 0°C freeze the culture or food and preserve or moderately inactivate the pathogen. Temperatures greater than 50°C are lethal to the pathogen. These three ranges of temperature will be addressed separately.

GROWTH TEMPERATURE Range and Optimum The temperature range that permits growth of L. monocytogenes is of particular interest to food processors because this pathogen is a psychrotrophic bacterium. Listeria monocytogenes was reported to grow at temperatures between –1.5 and 45°C [148,261]. In contrast, L. innocua (19 strains), L. murrayi (1 strain), and L. grayi (1 strain) failed to grow at temperatures below 1.7 (± 0.4), 2.8, and 3.0°C, respectively [161]. Difficulty in determining experimentally the minimum temperature for growth of L. monocytogenes led Tienungoon et al. [316] to use mathematical modeling to estimate this value. According to these investigators, the estimated minimum growth temperature, under optimum pH and aw, was –1.6 and 0.41°C for L. monocytogenes Scott A and L5, respectively. Limits of growth at refrigeration temperature depended strongly on medium pH [316].

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Microorganisms grown at optimum incubation conditions exhibit a short lag phase, short generation time during the exponential growth phase, and high cell count or density at the stationary phase. Interestingly, incubation conditions producing the shortest generation time do not always result in the shortest lag phase or the largest cell density. Therefore, reported “optimum” growth temperatures are only estimated determinations. Optimum temperature for growth of L. monocytogenes, as frequently reported in publications, occurs between 30 and 37°C. Growth Kinetics This section addresses the growth rate of L. monocytogenes, related parameters, and influence of temperature on parameters. Specific growth rate (µ), a commonly used kinetic parameter, reaches a maximum at the midexponential phase of growth. Specific growth rate and generation time (or doubling time) are inversely related as follows: Generation time = 0.693/µ Bacteria at the lag phase show no detectable growth; however, duration of this phase is a useful indicator of subsequent growth. Researchers from the U.S. Department of Agriculture, Eastern Regional Research Center (USDAERRC) modeled growth of L. monocytogenes in broth culture at different incubation conditions [88]. According to these models, lag phase and generation time were smallest (1.7 and 0.3 h, respectively) when the bacterium was incubated aerobically at 37°C, and the medium has 0.997 water activity (aw) and pH 7.0 initially. Decreasing incubation temperature progressively increased these two growth parameters. Robinson et al. [277] used a different model to estimate growth parameters of L. monocytogenes at different incubation temperatures. These authors observed a shorter lag phase at 15°C than at 20 or 25°C. Zaika and Fanelli [351] reported minor variation in generation time of L. monocytogenes when the bacterium was grown in brain–heart infusion at temperatures ranging from ~30 to 42°C; the corresponding value was ~0.6 h. Determination of L. monocytogenes growth kinetics in food produced different results. The generation time of L. monocytogenes decreases substantially as temperature increases from –1.5 to 30°C. The pathogen grew at –1.5°C in vacuum-packaged sliced roast beef with a calculated generation time of 100 h [148]. Generation times of 62 to 131 h in chicken broth and pasteurized milk were observed during extended incubation at –0.1 to –0.4°C [330]. Generation time was 28.5 to 46 h, 1.8 h, and 0.7 h when the pathogen was incubated at 4, 21, or 35°C in dairy products, respectively [280]. Therefore, food storage at refrigeration temperature retards but does not prevent growth of Listeria contaminants. In the absence of antimicrobials, holding food at 20 to 43°C supports rapid growth of the pathogen. A relationship between incubation temperature and generation time of Listeria would be useful in risk assessment studies, but large variations have been observed at a given temperature. Analysis of some published data (e.g., [280]) shows a linear relationship between the square root of the maximum specific growth rate ( µmax ) and incubation temperature (Figure 6.1). Other studies produced a nonlinear relationship between these two variables, with ( µmax ) showing a plateau after the optimum growth temperature is reached [277,351]. Medium composition contributes to the large variations in generation time at a given temperature. High salt (4.5 to 7.5%) or high EDTA (0.1 to 0.3 mM) concentration, for instance, may mask the effect of temperature on growth by increasing generation time, therefore contributing to deviation from linearity [351]. The listeriostatic activity of 7.5% NaCl or 0.3 mM EDTA was higher at 37 to 42°C than at 19°C [351]. Strains of L. monocytogenes vary considerably in growth characteristics. Lag phase durations for 39 strains varied from 70 to 270 h at 4°C and from 36.5 to 70 h at 10°C in trypticase soy broth–yeast extract [15]. Scott A, a strain extensively used in Listeria-related research, had the longest (209 h) and the second longest (62.8 h) average lag phase at 4 and 10°C, respectively.

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1

Square root of max

0.8

0.6

0.4

0.2

0

0

10

20

30

40

Incubation Temperature (°C) FIGURE 6.1 Linear relationship between incubation temperature and the square root of the maximum specific growth rate ( µmax ) of L. monocytogenes in various foods. Note that µmax values were calculated from minimum generation times (h) as follows: µmax = 0.693/generation time. (From Hudson, J. et al. 1994. J. Food Prot. 57:204–208; Iturriaga, M. H. et al. 2002. J. Food Prot. 65:1745–1749; and Rosenow, E. M. and E. H. Marth. 1987. J. Food Prot. 50:452–457.)

However, correlation between growth parameters and strains’ serotype was not established [15]. Variability among strains shows the importance of selecting a target strain for use in challenge studies. Selecting a fast growing and process-resistant target strain would be particularly valuable for food processing optimization research. Such a strain could be used to illustrate the worst-case scenario for lethality of a process, as well as survivability or growth during subsequent storage. Cold Tolerance The ability of L. monocytogenes to grow at refrigeration temperature is problematic to food processors. Mechanistic studies on bacterial cell membrane and osmoprotectants help explain how this pathogen maintains its physiological functions in a cold environment. Membrane phospholipids must remain in a liquid–crystalline state to maintain membrane fluidity and therefore growth at low temperature. The fatty acid composition determines whether membrane phospholipids are in the liquid–crystalline state. Membranes of L. monocytogenes contain >95% branched-chain fatty acids [12]. When grown at 37°C, major fatty acids are anteiso-C15:0 (41 to 52%), anteiso-C17:0 (24 to 51%), and iso-C15:0 (2 to 18%) (Figure 6.2). When grown at 5°C, the anteiso-C15:0 form becomes a strongly predominant group, representing 65 to 85% of total membrane fatty acids [12]. This reduction in the proportion of long aliphatic chains (C17:0) and the increase in asymmetric branching reduce van der Waals

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O

CH3 C

OH

H3C Iso-C 15:0

O C OH H3C

CH 3 Anteiso-C 15:0

O C OH H3C

CH 3 Anteiso-C 17:0

FIGURE 6.2 Structure of the major membrane fatty acids in L. monocytogenes. (From Annous, B. A. et al. 1997. Appl. Environ. Microbiol. 63:3887–3894.)

bonds among membrane constituents. Tight packing of membrane phospholipids at low temperature is therefore reduced; this helps maintain the pathogen’s membrane fluidity. Food-grade agents interfering with the biosynthesis of anteiso-C15:0 would be useful in controlling growth of L. monocytogenes in refrigerated foods. Highest anteiso-C15:0 fatty acid proportions in the membrane were found when the pathogen was grown in the presence of glycine betaine [12]. Growth of L. monocytogenes at low temperature is stimulated by presence of glycine betaine and carnitine [10,226]. When L. monocytogenes was grown at 4°C, addition of 130 µM glycine betaine nearly doubled the specific growth rate [181]. Listeria does not synthesize glycine betaine and carnitine, but imports these compounds from the environment. Plants are rich in betaine, and meat is rich in choline, a precursor of betaine [26]. Processed meat contains approximately 340 to 480 nmol/g glycine betaine [297]. Abundant levels of carnitine are found in foods of animal origin; processed meats (sausages and ham) and skim milk contain 230 to 950 and 120 to 140 nmol/g free carnitine, respectively [11,297]. The ATP-dependent glycine betaine porter II (Gbu) and, to a lesser extent, the glycine betaine-Na+ symporter (BetL) allow accumulation of glycine betaine into the cytoplasm at refrigeration temperature, even when the osmolyte concentration is very low in the growth medium [226]. Uptake is higher in the late exponential phase than in the stationary phase [11], but this uptake does not appear to be under σB control [180]. Carnitine was transported into the pathogen cytoplasm via an ATP-dependent transporter (OpuC) [120]. Prior treatment of a microorganism affects its subsequent physiology and growth kinetics. Temperature downshift, from 25 to 4°C, increased σB transcription [17]; expression of this alternative sigma factor is known to contribute to resistance of L. monocytogenes to heat, carbon starvation, acid, and osmotic and oxidative stresses [17,109]. Another study showed that growth of L. monocytogenes at 10°C, compared to 37°C, upregulated genes involved in cold-adaptive response (flaA and flp), regulatory adaptive response (rpoN, lhkA, yycJ, bglG, adaB, and psr), general

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microbial stress responses (groEL, clpP, clpB,flp, and trxB), amino acid metabolism (hisJ, trpG, cysS, and aroA), cell surface alteration (fbp, psr, flaA), and degradative metabolism (eutB, celD, and mleA) [201]. An oligopeptide permease (OppA) and a high-affinity potassium uptake system (Kdp) were required for growth at low temperature [29,37]. Stress Adaptation at Elevated Sublethal Temperatures Physiological changes caused by exposure to elevated, yet sublethal, temperature has been extensively studied for its consequences on stress resistance, or heat-shock response. Heat-shock response of L. monocytogenes has been triggered at temperatures ranging from 43 to 52°C, i.e., ~8 to 17°C above the pathogen’s optimal growth temperature [105,124,296]. Temperature upshift, from 25 to 48°C, increased transcription of the σB general stress regulon [17]. Expression of major molecular chaperones DnaK, DnaJ, GrpE, GroEL, and GroES also increased [124,136]. These molecular chaperones are referred to as heat-shock proteins because they fold newly synthesized proteins, repair misfolded proteins, and prevent protein aggregation upon heat shock [124]. In general, heatadapted L. monocytogenes cells are more resistant to stress than cells grown at optimal temperature. Holding food at temperatures in the heat-shock range should be avoided because the pathogen may gain resistance to subsequent processing treatments. Variations in Virulence with Temperature Virulence of Listeria increases when the pathogen is grown at refrigeration rather than optimum temperature. Durst [84] reported that 7 of 36 weakly virulent L. monocytogenes strains became markedly virulent to mice by intraperitoneal injection after maintaining the cultures on agar slants for 6 months at 4°C. Similarly, Wood and Woodbine [340] found a strain of L. monocytogenes that was more virulent to chick embryos when grown at 4°C, instead of 37°C. Cold storage, therefore, may enhance virulence of some L. monocytogenes strains. In contrast, activity of listeriolysin O appeared lost after several weeks of storage at 4°C [47]. The decrease in activity of listeriolysin O was more pronounced at pH 7.0 than at pH 5.5. However, pathogenicity was recovered in ≤ 24 h by incubating these refrigerated cells at 37°C [47]. Listeriolysin and other PrfA-regulated virulence genes are thermoregulated and expressed only in Listeria cultures grown above 30°C [192]. Heat shock and other environmental stresses affect the virulence of L. monocytogenes. When L. monocytogenes was heat shocked at 48°C for 2 h, listeriolysin O was almost totally lost; however, subsequent growth of the heat-shocked cells at 37°C resulted in production of listeriolysin 40 times greater than that present immediately after heat shock [174]. Restoration of virulence after heat shocking reinforces the importance of eliminating L. monocytogenes from minimally processed, ready-to-eat food.

FREEZING Although L. monocytogenes does not grow below –1.5°C, this pathogen can readily survive at much lower temperatures. The Listeria population decreased < 1 log over 3 months’ storage at –18 to –20°C in inoculated samples of fish, shrimp, ground beef, ground turkey, frankfurters, corn, and ice-cream mix [140,250]. Populations of the pathogen in tomato soup (pH 4.7) decreased ~3 logs of L. monocytogenes under similar inoculation and frozen storage conditions [250]. The high inactivation rate for Listeria in tomato soup was attributed to freeze–thaw injury [250]. Freezing–thawing ruptured the cell wall, altered plasma membrane integrity, and caused leakage of cytoplasmic contents [92]. Survival and injury of L. monocytogenes during storage at frozen temperature vary with the temperature, freezing rate, and freezing menstruum [91,92,94]. A low freezing temperature and rapid freezing rate were most favorable to bacterial survival [94]. No evidence of cell death was observed when L. monocytogenes was frozen and stored at –198°C in liquid nitrogen. Freezing and storage at –18°C inactivated 1 to 2 logs and injured >50% of the pathogen population.

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Similar ranges of inactivation were observed in five foods (spinach, cheese, fish, chicken, and beef) during initial quick freezing to –50°C in 57 min and subsequent storage at –18°C for up to 300 days [128]. The quick freezing and subsequent storage only inactivated 0.1 to 1.6 and 0.0 to 1.0 logs, respectively. Injury of the surviving population ranged from a nondetectable level to 90%. Multiple freeze–thaw cycles are more detrimental to survival of Listeria than is a single cycle [94]. Such treatment is also far more damaging to the pathogen when frozen at –18°C than at –198°C. Four freeze–thaw cycles in phosphate buffer inactivated > 2 logs L. monocytogenes and caused no detectable injury when the freezing temperature was –18°C. In phosphate buffer lethality was only 34% and injury was 15% when the pathogen was subjected to four freeze–thaw cycles and the freezing temperature was –198°C [94]. Freezing and subsequent storage cause limited inactivation of L. monocytogenes, so contamination of frozen food should be prevented through good manufacturing practices and, whenever possible, by subjecting the food or ingredients to listericidal processes (e.g., pasteurization) before freezing. Some food ingredients may protect Listeria against freeze–thaw injury. Addition of 2 to 4% glycerol or 2% milk to phosphate buffer markedly decreased the extent of cell death and injury during freezing at –18°C [91]. Survival of Listeria over 5 months of frozen storage is higher in the presence of 2% glycerol than in 2% milk [91]. Casein, lactose, and fat were identified as the main milk fractions protecting Listeria against freeze injury. Cryoprotection by lactose and milk fat was observed during the first month of storage at –18°C. Cryoprotection by glycerol and casein exceeded 5 months under similar storage conditions and was, therefore, longer lasting than that of lactose or milk fat [91]. Simulated milk ultrafiltrate, compared to phosphate buffer, caused almost no change in death rate, but decreased cell injury during the first 24 h of frozen storage at –18°C [91]. Adaptation of L. monocytogenes to sublethal levels of environmental stress from acid, ethanol, sodium chloride, heat, or starvation increases survival of the pathogen during freezing, frozen storage, and freeze–thaw cycles [204]. Although freezing and frozen storage may cause a limited decrease in viability of L. monocytogenes, such treatments can cause injury and thus sensitize L. monocytogenes cells to listeriostatic or listericidal agents. After frozen storage, viability of the pathogen decreased in the presence of acid [23,250], lysozyme, or lipase [93]. Cross-contamination of meat during freezing by immersion in nitrogen was reduced, although not fully prevented, by 2% lactic acid wash before freezing [23].

LETHAL TEMPERATURE Thermal processing, such as pasteurization, is the most widely used method to preserve food. These processes target microorganisms of concern in a given food, thus rendering the product safe for human consumption. Listeria monocytogenes is ubiquitous in the environment, and it has been increasingly associated with foodborne diseases and food recalls [312]. The pathogen appears relatively resistant to processing compared to other nonsporing bacteria; processors consider L. monocytogenes to be the primary pathogen of concern in minimally processed and ready-to-eat products. Researchers, therefore, have been actively pursuing new intervention strategies and evaluating efficacy of conventional preservation processes, particularly those involving heat. Mechanisms of Thermal Inactivation Elevated temperature causes multiple irreversible cellular damage in Listeria that results in cell death. Heating L. monocytogenes at temperatures above 56°C causes ribosomal damage, protein unfolding and denaturation, and, consequently, enzyme inactivation [8,49]. Upon heating, ribosomes lose Mg2+, which results in dissociation of the 30S and 50S ribosomal subunits [233,342]. The 30S is more heat labile than the 50S subunit, which is in turn more heat labile than the entire 70S ribosome [79]. Ribosomal damage, particularly denaturation of the 30S ribosomal subunit, has been

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associated with thermal inactivation of vegetative bacteria and is believed to be the main cause of bacterial death [233]. Superoxide dismutase and catalase activities in L. monocytogenes decreased at temperatures higher than 45 to 50°C and 55 to 60°C, respectively. Higher processing temperatures may completely inactivate these enzymes [72], sensitizing the pathogen to aerobic storage conditions [177,254]. However, no apparent correlation between these enzymes and the pathogen’s heat resistance was found [72]. Heating probably releases Mg2+ from teichoic acids in the cell wall of Gram-positive bacteria [8]. Mild heat (56°C), however, did not damage the membrane permeability of Listeria [49]. Recovery of thermally injured Listeria requires synthesis of mRNA and repair proteins [49]. Kinetics of Thermal Inactivation Measurement Measuring thermal inactivation kinetics involves heating an inoculated medium uniformly at the desired temperature and monitoring survivors during the course of the treatment. Small thickness of samples maximizes the rate of heat transfer, minimizes temperature come-up and cooling down times, and ensures treatment homogeneity [18,99]. Researchers used capillary tubes, sealed bags, sealed tubes, or open tubes to measure Listeria thermal inactivation kinetics [18,78,99], but variations among these methods led to conflicting results [78]. For fluid products, capillary tubes are generally recommended to establish reliable survivor plots [18,99]. Data are analyzed and inactivation rates or thermal death times are calculated. Before implementation in food processing, it is recommended that the kinetic results be confirmed in pilot-scale pasteurizers, using inoculated food; operating parameters, pasteurizer design, and treated matrix may affect the efficacy of thermal processes. D-Value and z-Value The relationship between thermal treatment time and the log count of survivors is commonly referred to as a “survivor plot.” If this relationship is linear, thermal resistance parameters can be readily calculated. Time required to inactivate one log of the microbial population at a given temperature (i.e., D-value) is a popular expression of its thermal resistance. If the survivor plot is nonlinear, the D-value cannot be determined accurately. Implications of the nonlinearity of inactivation data have been addressed in a recent publication [142]. Pooled data from multiple sources (411 data points) show a log-linear relationship between treatment temperature and D-values of L. monocytogenes (Figure 6.3). Thermal inactivation rates at any given temperature varied considerably among studies and when the pathogen was heated in different media. Using raw and reconstituted nonfat dry milk, for example, produced D63°C and D71.7°C values of ~0.33 and 0.015 min, respectively [32,99]. In contrast, the pathogen appeared more resistant to heat when present in ice-cream mix; D-values at 68.3, 73.9, and 79.4°C were 3.9, 0.53, and 0.043 min, respectively [33]. Although Figure 6.3 shows large variability in thermal resistance of L. monocytogenes in different studies, these data were pooled and used to estimate the temperature change necessary to vary the D-value of L. monocytogenes by one log (i.e., z-value); this calculation resulted in a z-value of 7.6°C. Recently, a large number of published inactivation studies were reviewed and D- and z-values of L. monocytogenes were compared for various laboratory media, and dairy, meat, egg, seafood, and vegetable products [79]. The authors reported average and median z-values of 7.1 and 6.4°C, respectively (minimum: 4.3°C; maximum: 29.3°C). Heat treatment of low-acid refrigerated food should be sufficient to decrease the target pathogen ≥ 5 logs. Manufacturers of fruit juice and other acid foods also adopt this principle. Presence of L. monocytogenes in ready-to-eat food is not permitted in the United States. This zero-tolerance policy, in addition to the pathogen’s ubiquity and resistance to adverse conditions and mild processing, makes L. monocytogenes a suitable target for thermal processing.

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100

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Temperature (°C)

FIGURE 6.3 Decrease of L. monocytogenes D-values with heating temperature of inoculated meat products, dairy products, seafood, fruits, juices, and vegetables. (From Chhabra, A. T. et al. 1999. J. Food Prot. 62:1143–1149; Doyle, M. E. et al. 2001. J. Food Prot. 64:410–429; Juneja, V. K. and B. S. Eblen. 1999. J. Food Prot. 62:986–993; Mazzotta, A. S. 2001. J. Food Prot. 64:315–320; and Murphy, R. Y. et al. 2002. J. Food Prot. 65:53–60.)

The following are examples of thermal treatments that successfully inactivated L. monocytogenes in various foods. Cooking ham and brined salmon to internal temperatures of 65 and 82.8°C, respectively, eradicates Listeria from these foods [79]. Pasteurization of apple cider at 71.1°C for 11 sec inactivates > 5 logs L. monocytogenes. When processed at 68.1°C for 14 sec, low numbers of injured Listeria were recovered from artificially contaminated apple cider with relatively high pH (4.1) and low sugar content (11°Brix); however, survivors died when the processed cider was stored at 4°C for 24 h [216]. Minimum temperature-time pasteurization processes required by the U.S. Food and Drug Administration (FDA) for dairy products are sufficient to eliminate ≥ 105 CFU/g L. monocytogenes [13,51,99,341]. Processing food at a different temperature should achieve equivalent microbial inactivation [114]. The hardiness of L. monocytogenes to mild thermal processes has frequently been observed. At temperatures < 60°C, Listeria spp. had a substantially higher D-value than Salmonella spp. in meat products, especially in chicken [239]. Equal or slightly lower D-values for Listeria were observed at 70°C, indicating that the z-value for Listeria was probably lower than that for Salmonella in these products. In fruit juices processed at 56 to 60°C, Listeria was more heat resistant than Salmonella, but

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less heat resistant than Escherichia coli O157:H7 [220]. The z-value of the latter pathogen, however, was smaller than that of Listeria [220]. Populations of L. monocytogenes decreased only 1.6 and 0.38 logs during heat treating egg white (56.7°C for 3.5 min) and salted/sweetened egg yolk (63.3°C for 3.5 min), respectively [230]. Because low populations of the pathogen are typically found in contaminated egg products, current pasteurization protocols for whole egg and egg yolk may be sufficient to eliminate the organism, even though these thermal processes do not provide a wide safety margin [230]. Extrinsic Factors in Resistance to Heat Food Composition Some food components may protect L. monocytogenes against heat. Listeria monocytogenes was more resistant in presterilized or repasteurized milk than in raw milk, but the reason for this increased resistance has not been identified [34]. Resistance of L. monocytogenes to mild heat increases with the food’s pH [160], fat content [210], salt concentration [160], freedom from antimicrobials [215], highfructose corn syrup solids concentration [146], and presence of stabilizers such as guar gum and carrageenan [266]. However, acid, salt, and phosphate in beef gravy caused only a minor increase, if any, in D-values at temperatures higher than 62.5°C [160]. High salt concentration increased the denaturation temperature of Listeria’s 30S ribosomal subunit, which contributes to heat tolerance of the pathogen [302]. The fat fraction from sheep milk protected L. monocytogenes against heat [210]. Increased viscosity in the presence of gum decreased the rate of heat transfer in the food; this may have been sufficient to induce heat adaptation [266]. Variations in heat resistance because of food composition may be associated with availability of nutrients that support growth of Listeria. Starvation of Listeria can trigger a stress-adaptive response and thereby increase the pathogen’s tolerance to heat [205]. Listeria monocytogenes Engulfment Most in vitro inactivation studies processed freely suspended cells of L. monocytogenes. Animals suffering from listeric mastitis produce milk in which L. monocytogenes is usually entrapped within phagocytic leukocytes. Bunning et al. [52] reported average D71.7°C values of 5.0 and 3.1 sec for intracellular (zintracellular = 8.0°C) and freely suspended (zsuspension = 7.3°C) L. monocytogenes, respectively. The higher thermal resistance of intracellular compared to freely suspended Listeria was also observed by Doyle et al. [80], except when contaminated milk was stored ≥ 4 days at refrigeration temperature. During cold storage of milk, heat resistance of Listeria decreased in parallel to leukocyte breakdown; therefore, the researchers speculated that leukocytes protected the engulfed pathogen against thermal inactivation [80]. Homogenization of raw milk disrupts phagocytes; this frees the pathogen, which loses thermal protection during the subsequent thermal process. As infected cows develop fever, L. monocytogenes grown at these elevated temperatures may become adapted to heat [80]. Consistent with this hypothesis, heat shocking (48°C, 15 min) Listeria in milk increased the D71.7°C value from 3.0 to 4.6 sec [51]. The latter value is comparable to that measured for intracellular L. monocytogenes [52]. Intrinsic Factors in Resistance to Heat Strain Listeria monocytogenes strains vary in resistance to heat [99]. Strains from serovar 4b tend to be slightly more heat resistant than those from serovar 1/2a [46]. This trend, however, is not always confirmed. One of the most commonly studied strains, Scott A (serovar 4b), has low heat resistance, compared to V7 (serovar 1a) [99]. Sporadic survival of hardy strains may be encountered if process parameters are based on inactivation studies using heat-sensitive strains, such as Scott A. Process validation, therefore, should aim at destruction of a target Listeria strain selected for its high resistance to heat. Some studies have assessed thermal resistance using mixtures of

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strains (“cocktails”). Presence of at least one problematic (target) strain and strains from various serovars is recommended in these cocktails to minimize the risk of underestimating the pathogen’s thermal resistance. Physiological State As frequently observed in bacteria, Listeria is more heat resistant in the stationary than in the exponential phase of growth. When L. monocytogenes is exposed to sublethal stress, it may develop an adaptive response to subsequent thermal treatments. Environmental and processing stresses that may cause this phenomenon include sublethal heat, acids, oxidants, starvation, and high osmolarity [158,205]. Prewarming, slow heating, or cooking food; hot water washing; underprocessing; and holding food in warm trays (as may happen in food service establishments) are examples of sublethal heat shock. Holding L. monocytogenes at sublethal temperatures (e.g., 45 to 48°C, for 15 min to 1 h) induces adaptive thermotolerance [124,206]. The D52 to 71.1°C-values of heat-adapted cells were 1.5 to 4 times greater than that observed in cells incubated at optimal growth temperature (30 to 38°C) before the thermal process [51,175,199,296]. A nearly twofold increase in D62°C was observed when the pathogen was heated in ground pork at 1.3°C/min compared to 8°C/min [175]. The z-value of heat-adapted L. monocytogenes, however, remained similar to that of unadapted cells [199]. The average D56°C of L. monocytogenes suspended in a phosphate buffer (pH 7.0) was 4.1, 8.8, and 2.9 min, when cells had been previously exposed to pH 4.5, 4 to 8% (v/v) ethanol, and 500 ppm hydrogen peroxide, respectively. For comparison, the corresponding nonadapted L. monocytogenes strain had a D56°C of 1.0 min [205]. Starvation at 30°C for up to 163 h in phosphate buffer increased the D56°C value of the remaining viable cells to 13.6 min [205]. The pathogen’s thermotolerance at 60°C increased 1.3- to 8-fold in the presence of 0.5 to 1.5 mol/L NaCl, compared to cells exposed to 0.09 mol/L NaCl [211]. Adaptive thermotolerance is a transient, nonheritable property [157,158]. Acquired thermotolerance of L. monocytogenes lasted at least 24 h at 4°C in a sausage mix [105], <1 h at 35°C (optimum temperature for growth) in tryptic soy—0.6% yeast extract broth (TSB-YE) [50], and ≥4 h at 42°C in TSB-YE (heat-shock temperature) [50]. Thermotolerance in liquid media was lost in <5 min after osmotic downshock [158]. Potential heat adaptation, therefore, should be taken into account when establishing thermal-processing parameters.

SURROGATE MICROORGANISMS

FOR

THERMAL STUDIES

Because of safety concerns, researchers look for nonpathogenic indicator microorganisms that may replace a pathogen such as L. monocytogenes in thermal inactivation trials. A suitable indicator microorganism, called a surrogate, should be at least as resistant to the thermal process as the most heat-resistant L. monocytogenes strain. Listeria innocua, which is nonpathogenic to humans, is frequently used as a surrogate for L. monocytogenes because of the genetic relatedness of the two species. Listeria innocua has growth rates and resistance to common food preservation factors similar to or higher than those of L. monocytogenes [81,112,260]. Listeria innocua M1 is a potential surrogate for thermal process validation. This strain is resistant to rifampin and streptomycin, which aids in its enumeration in food [102]. When determining survivor plots, Fairchild and Foegeding [102] showed the importance of enumerating uninjured and injured cells to prevent underestimation of Listeria counts after processing. In spite of the merits of using L. innocua as a surrogate, it is judicious to select Listeria surrogates from different bacterial genera. Screening protocols used in food processing facilities commonly target Listeria spp. rather than L. monocytogenes specifically. Because using L. innocua in lieu of L. monocytogenes may lead to accidental release of the bacterium in the processing environment, detection of this surrogate by screening tests may raise unwarranted concerns. Generally recognized as safe (GRAS) bacteria, such as those used in food fermentations, are ideal surrogates for pathogens.

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ACIDITY Like many other bacteria, L. monocytogenes grows optimally at a pH close to neutrality. Growth of L. monocytogenes has been reported at pH values ranging from 4.0 to 9.6 [261,263]. In the absence of other growth-limiting factors, highest final populations were reached at pH 6.0 to 8.0 [42,261,284]. Lag phase and generation time increase considerably as pH decreases to below 6.5 [88,284]. Listeria will grow in some low-acid foods, including fermented products depending on water activity, SOH content, and other intrinsic factors. Camembert [283], brick [286], and white pickled cheese [1] have a pH ≥ 5.9 and support growth of L. monocytogenes.

SURVIVAL

AT

LOW PH

Low pH is one of the most critical safety and quality determinants of fermented foods. Although growth of L. monocytogenes at pH < 4.0 has not yet been documented, this organism appears to be fairly acid tolerant under such conditions, with the pathogen surviving 1 to 4 days in orange juice (pH 3.6) stored at 4°C [252] and > 200 days and >365 days in cheddar cheese (pH 5.1) stored at 13 and 6°C, respectively [282]. The effect of pH on cell viability, however, depends strongly on other environmental factors and on the physiological state of the microorganism. Inactivation at Low pH Absence of growth and decrease in cell viability may be observed at pH ≤ 5.5, when other environmental conditions (e.g., temperature) are not optimal for survival of L. monocytogenes. The Pathogen Modeling Program from the USDA estimated the decrease in count in nutrient broth at pH 3.2 to 4.4 [88]. Within this pH range, the predicted D-value decreased with pH in a log-linear manner (Figure 6.4). This decrease in D-value was also more marked as the temperature increased from 4 to 35°C.

1000

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3 x D-value (hours)

10000

Temperature (°C) FIGURE 6.4 Exposure time, under lethal acidic conditions, needed to decrease L. monocytogenes populations 3 logs (i.e., 3 × D-values) at different storage temperatures, as determined by the USDA Pathogen Modeling Program. (From Eastern Regional Research Center. 2003. USDA. Pathogen Modeling Program [version 7.0]. http://ars.usda.gov/services/docs.htm?docid=6786.)

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During prolonged storage in orange serum at pH ≤ 4.8, counts of L. monocytogenes were first constant (~40- and 20-day lag periods at pH 4.8 and 4.0, respectively), then decreased at a rate of approximately 1 log/5 to 8 days [252]. When pH of the orange serum decreased, the lag period decreased and the subsequent inactivation rate increased [252]. At 5°C, the pathogen population decreased < 2 logs after 49 days and >4 logs after <10 days in cabbage juice acidified to pH 4.8 and 4.4, respectively [68]. The specific listeriostatic and listericidal activity of organic acids is reviewed later in this chapter. Listeria counts decreased during ripening of parmesan [349] and stretching and storage of mozzarella cheese [39,328]. Inactivation was likely related to the combined antilisterial effect of the cheeses’ low pH (pH ≤ 5.2) and elevated processing temperature.

FACTORS INFLUENCING ACID TOLERANCE Refrigeration inhibits growth, but favors survival of L. monocytogenes in acid food. This bacterium, for example, survived well in hard salami (pH ~4.4) during refrigerated storage [156]. When milk was inoculated with Listeria and made into yogurt, the pathogen survived in the product for up to 27 days at 4°C [62]. The pathogen persisted in cottage cheese (pH 5.0) more than 35 days at 5°C [265]. This also suggests that refrigerated acid products should be considered as possible vehicles of infection in epidemiological investigations of human listeriosis. Temperature influences the growth behavior of L. monocytogenes under acidic condition. When L. monocytogenes is incubated at 5 to 35°C in growth-permitting acidic media (i.e., pH 4.5 to 6.5); greatest inhibition occurs at lowest pH and temperature [88]. Listeria monocytogenes populations (104 CFU/mL-inoculum) increased to 109 CFU/mL in cabbage juice (pH 5.2) incubated at 30°C for 3 days [68]. In similarly processed juice, the population remained unchanged during 22 days of storage at 5°C [68]. Rapid growth at 30°C, however, was followed by equally rapid bacterial death, and the pathogen was no longer detectable after 15 days’ storage. Although the pathogen’s viability in acidic food is expected to decrease as storage temperature increases from refrigeration to ambient, such storage is likely to lessen overall quality of perishable low-acid foods and is therefore inadvisable. Sensitivity of L. monocytogenes to high acidity (pH <4.5) increases with temperature (Figure 6.4), especially when the medium is not conducive to growth [88]. Acid resistance varies substantially with physiological state of the pathogen. Exposure to some environmental stresses, for example, may elicit an adaptive response that increases tolerance of L. monocytogenes to acid. Counts of L. monocytogenes in a culture medium at pH 3.5 were 3.1 to 6.4 times higher when the population was pre-exposed to mild acidity (pH 4.5 to 5.5), a stepwise pH decrease to 4.5, or 5% ethanol, compared to the unadapted control [206]. Prolonged survival of acidadapted cells compared to unadapted cells was confirmed in cottage cheese (pH 4.7), cheddar cheese (pH 5.2), yogurt (pH 3.9), salad dressing (pH 3.0), and orange juice (pH 3.8) stored at refrigeration temperature [123]. Acid-adapted survivors were detected in these foods when stored at refrigeration temperatures for ≥ 15 days, 70 days, 2 days, 7 h, and 7 h, respectively [123]. Lactic and acetic acid are more effective than hydrochloric acid in inducing acid adaptation in L. monocytogenes [246]. Acid-tolerant spontaneous mutants also have been isolated after prolonged incubation at pH 3.5 [246].

MECHANISMS

OF

ACID DAMAGE

AND

TOLERANCE

Viability of bacteria depends on the cell’s ability to maintain an intracellular pH close to neutrality, regardless of environmental pH. The cell can cope with mild acidity by eliminating excess protons from the cytoplasm. Below a threshold pH, cells are unable to expel protons fast enough and a decrease in intracellular pH becomes inevitable. Cytoplasm acidification reduces the proton motive force of membranes, thus depriving the cell of essential energy for its metabolism. Excess H+ alters enzymatic activity and may denature proteins [304,344]. Therefore, depending on food composition, processing, and storage conditions, low pH can be listeriostatic or listericidal.

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Constitutive and induced mechanisms increase the pathogen’s acid tolerance. The glutamate decarboxylase system of L. monocytogenes contributes to the pathogen’s survival in acid foods that contain ≥ 0.22 mM free glutamate (e.g., salad dressing, mayonnaise, fruit juices, yogurt) [70]. It is believed that internalized glutamate acts as a proton sink and therefore prevents cytoplasm acidification. A shift in gene expression characterizes induction of acid tolerance. Cells in the stationary phase are naturally more acid tolerant than those in the exponential phase, regardless of the nature of the acidifying agent [246]. Transition to the stationary phase and a mildly acidic environment increases expression of σB factor. This activates a general stress regulon, which increases survival to various growth-limiting factors, such as high acidity or elevated temperature [109]. Increased expression of the following proteins was observed in acid-adapted L. monocytogenes: molecular chaperones, ATP synthase, a thioredoxin reductase homologue of B. subtilis, and a ferric uptake regulator [264]. Molecular chaperones refold damaged bacterial proteins and therefore contribute to acid resistance.

CONSEQUENCES

OF

ENHANCED ACID TOLERANCE

Acid adaptation cross-protects L. monocytogenes against a variety of deleterious environments; these include lethal doses of hydrogen peroxide, heat, NaCl, and ethanol [205,206]. Therefore, prolonged storage of mildly acidic food (pH ≥ 4.5) may harden L. monocytogenes to processes designed to inactivate the pathogen. Strains with constitutive acid resistance are more likely to persist in plant environments than are other strains. To cause foodborne listeriosis, L. monocytogenes must cross the intestinal epithelial cells, and survive defense by phagocytes. Acid adaptation increased adhesion, entry, and growth in an intestinal cell line, Caco-2 cells [69]. Entry efficiency in phagocytes was similar in acid-adapted and nonadapted cells. However, acid-adapted cells and those of an acid-tolerant mutant multiplied approximately four times faster than nonadapted wild-type cells [69]. Mice infected intraperitoneally died faster when injected with an acid-tolerant mutant than with the corresponding wildtype L. monocytogenes strain [246]. Virulent L. monocytogenes excrete listeriolysin O, which has a maximum activity at pH 4.0 to 5.0 [224]. Growth rate is not proportional to virulence of the pathogen. Some weak organic acids enhance pathogenicity of the bacterium, while others reduce it. Citrate, acetate, and lactate increased secretion of listeriolysin O, whereas sorbate inhibited secretion of this hemolysin [182,183].

WATER ACTIVITY Availability of moisture for microbial growth is best expressed as water activity (aw), which is defined as the ratio of the water vapor pressure of a food substrate to the vapor pressure of pure water at the same temperature [88]. High osmolarity, i.e., low water activity, decreases turgor pressure in a bacterial cell. Bacterial turgor pressure provides the force that expands the cell and therefore contributes to cell wall growth and cell division. Reduction in turgor pressure inhibits bacterial growth [6]. Most fresh foods have a water activity > 0.98 [88]. Drying and addition of salt (e.g., during sausage making) or sugar (e.g., in fruit preserves) are traditional methods to lower water activity of food and therefore enhance its shelf life. Listeria monocytogenes survives or even grows in foods with relatively low water activity [242]. Tolerance of L. monocytogenes to low aw should be considered when developing food applications that use minimal processing or involve dehydration or brining.

WATER ACTIVITY

AND

GROWTH

OR

SURVIVAL

Like most bacteria, L. monocytogenes grows optimally at aw ≥ 0.97 [261]. However, when compared to other common infectious foodborne pathogens, this bacterium has a rather unique ability to multiply at aw values as low as 0.90. The lowest water activity values allowing growth to ≥ 6.5 log

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A

10

15

20

25

30

35

0.997

5

35

0.997

1.0

Temperature (°C)

0.970

Wa

te

c ra

0.930

10.0

0.950

100.0

0.990

Lag phase (h)

1000.0

tivi

ty

100.0

0.1 5

10

15

20

25

30

Temperature (°C)

0.970

Wa

a ter

0.930

1.0

0.950

10.0

0.990

Generation time (h)

B

ctiv

ity

FIGURE 6.5 Changes in lag phase (A) and generation time (B) of L. monocytogenes at different water activities and incubation temperatures (pH 7.0), as determined by the USDA Pathogen Modeling Program. (From Eastern Regional Research Center. 2003. USDA. Pathogen Modeling Program [version 7.0]. http://ars.usda.gov/services/docs.htm?docid=6786.)

CFU/mL in presence of glycerol, sucrose, NaCl, and propylene glycol were 0.90, 0.92, 0.92, and 0.97, respectively [232,242]. These water activities correspond to approximately 30% glycerol, 39.4% sucrose, 11.5% NaCl, and 16.7% (w/w) propylene glycol. Lag phase and generation time increase as water activity of the medium decreases (Figure 6.5). Although L. monocytogenes does not appear to grow at aw < 0.90, the bacterium survives in these environments, particularly under refrigeration, for long periods. Listeria monocytogenes survived in fermented hard salami (aw: 0.79 to 0.86) at 4°C for at least 84 days [156]. When commercial cheese brines were inoculated with L. monocytogenes (2 × 104 to 2 × 105 CFU/mL) and stored at 4°C, the pathogen was detectable for up to 259 days [191]. Low aw (< 0.90) is listeriostatic, but rapid growth may resume as aw increases. Cheese brines, therefore, should be considered as potential sources for cross-contamination [191].

OSMOTOLERANCE FACTORS The combined effect of aw and incubation temperature on growth of L. monocytogenes is profound; the greatest inhibition is observed when both aw and temperature are minimum (Figure 6.5). Survival of L. monocytogenes at reduced moisture levels depends on the dominant solute in the medium.

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Survival in media of equal aw, but containing different solutes, was in the following increasing order: propylene glycol, NaCl, sucrose, and glycerol [232,242]. The capacity of L. monocytogenes to grow on processed meats was related to accumulation of high levels of glycine betaine, carnitine, and glycine- and proline-containing peptides (200 to 1,000 nmol/mg cell protein) [6,297]. These osmoprotectants are essential to maintain intracellular turgor pressure [6]. Glycine betaine is preferentially used upon cell exposure to severe osmotic or cold stress [6]. Glycine betaine transporters (BetL and Gbu) and carnitine uptake-transporter (OpuC) pump osmoprotectants into the cytoplasm at high environmental osmolarity [10]. The BetL symporter instantaneously accumulates glycine betaine upon osmotic upshock, at a rate of 2 nmol/min/mg cellular protein. The Gbu transporter is essential to long-term osmotolerance or to survival under high (≥ 6%) salinity; this transporter activity started a few minutes after upshock, increased progressively, and accumulated 8 nmol glycine betaine/min/mg cell protein after 4-h exposure to 4%NaCl [226]. Dimethyl glycine, trigonelline, triethylglycine, and γ-butyrobetaine are glycine betaine analogs and potent inhibitors of glycine betaine and/or carnitine uptake (Figure 6.6) [226]. Osmotic upshift induces expression of the σB factor in L. monocytogenes [17]. As observed in heator acid-adapted cells, induction of the σB regulon increases resistance of osmotically adapted cells to mild stresses, such as acid, heat, and freeze injury [109,160,206]. The extent of cross-protection is minimized when combining mild hurdle factors adequately. A combination of stresses was probably responsible for the rapid demise of Listeria in parmesan cheese [349]; this cheese curd had a relatively low pH, was cooked at 51°C for ~45 min, and had low moisture content (30.1 to 31.4%). Glycine betaine

Carnitine

CH3 | O CH3 — N + — CH2 — C O| CH3

•Triethylglycine •Trigonelline

CH3 OH O | | CH3 — N + — CH2 — CH — CH2 — C O| CH3

•Dimethylglyc ine •Triethylglycine

•Dimethylglycine

• -butyrobetaine

Na+

BetL

Gbu

Gbu

OpuC OpuC

CELL MEMBRANE

Na+

ATP

CH3 | O CH3 — N + — CH2 — C O| CH3

CYTOPLASM

CH3

O NH + — CH2 — C

CH3

O-

Dimethylglycine

O CH3 —

Trigonelline

ATP

ADP + Pi

ATP

ADP + Pi

CH3 OH O | | CH3 — N + — CH2 — CH — CH2 — C O| CH3

CH3CH2 CH3CH2 — N + — CH2— C CH3CH2

O O-

Triethylglycine

—C N+

ADP + Pi

O-

CH3 O CH3 — N + — CH2 — CH2 — CH2 — C OCH3 -Butyrobetaine

FIGURE 6.6 Glycine betaine and carnitine transport across the cell membrane and uptake impairment by glycine betaine analogs. Figure insert shows the structure of glycine betaine analogs that were found to inhibit uptake of the two osmoprotectants. (From Mendum, M. L. and L. T. Smith. 2002. Appl. Environ. Microbiol. 68:5647–5655.)

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ANTIMICROBIAL COMPONENTS IN FOOD Antimicrobial ingredients are frequently added to control foodborne microbiota, with some (e.g., salt and spices) contributing to the food flavor profile. Antilisterial activity of salt, organic acids, fatty acids, nitrite, antioxidants, smoke, plant extracts, and related compounds has been thoroughly investigated over the past two decades.

SALT Salt (i.e., sodium chloride) is an important food ingredient. High salt concentrations reduce the growth of L. monocytogenes by decreasing the food’s water activity [261] and electrochemical potential across the cell membrane [253]. Influence of water activity on microbial growth, survival, and death of L. monocytogenes was described earlier, with NaCl-specific interactions reported in this section. Survival at Extreme Salt Concentrations Listeria monocytogenes tolerates extremely high salt concentrations. The pathogen survived in commercial cheese brine (23.8% NaCl, pH 4.9) stored at 4°C for 259 days [191]. Consequently, use of concentrated brine solutions in food applications, such as in cheese manufacturing and salmon processing, should not be considered a reliable means to eliminate L. monocytogenes. Survival of Listeria in the presence of >12% NaCl decreases with increasing salt concentration or incubation temperature. Presence of 16% NaCl was listeriostatic for at least 33 days at ≤4°C. Presence of 26% NaCl decreased Listeria populations 2 and 3.5 logs when the pathogen was incubated at 0 and 4°C, respectively, for a similar storage period [147]. Incubation of L. monocytogenes in the presence of 14% NaCl for 36 days at 10 and 25°C decreased populations ~2 and ≥ 6 logs, respectively [299]. Physiology at High Salt Concentration Listeria formed colonies with a rough surface and irregular borders on agar media containing ≥6% NaCl [152]. Cell morphology changed from short rod to filamentous and deformed shapes [22,351], with a strongly hydrophilic surface [22]. Morphological changes were less pronounced when incubating the cells at refrigeration temperature [38]. Interestingly, elongated cells were also observed when L. monocytogenes was grown in media adjusted to pH 5.0 to 6.0 with citric acid adjusted to pH > 9.0 with NaOH containing ≥ 1.75 mM H2O2 containing ≥ 0.3 mM EDTA This suggests that filamentous morphology contributes to adaptation to adverse conditions [152,351]. Changes in cell morphology, in response to medium salinity, altered the adhesion properties of the pathogen [22]. Whether these changes facilitate persistence of the pathogen in foodprocessing environments or on food and equipment surfaces has not been reported. Viability of cells requires maintenance of an electrochemical potential across the membrane as well as a low cytoplasmic Na+ concentration regardless of the extracellular NaCl concentration. Regulation of potassium uptake (kdp), Na+ efflux (gbu and ykpA), and multidrug efflux (mdrL) were overexpressed under high salinities, presumably to expel excess Na+ ions from cytoplasm [37,126]. An increase in anteiso-C15:0 and decrease in anteiso-C17:0 fatty acid levels in the Listeria membrane have been observed upon exposure to high salinities [61]. A similar membrane fatty acid profile has been observed when the pathogen is stored at refrigeration temperature. Reasons for this change in membrane fatty acid profile have not been determined and may be linked to increased osmoprotectant (e.g., glycine betaine) concentration in the cell cytoplasm. Activation of the osmotic stress response was discussed in the previous section.

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Up to 6% sodium chloride protected L. monocytogenes against thermal inactivation in beef gravy (pH 4.0) [160]. This thermoprotective effect was particularly evident at treatment temperatures ≤ 60°C. The highest D55°C-value was observed in gravy containing 1.5 to 4.5% NaCl. When heating the gravy at 55°C, increasing the salt concentration to 4.5% decreased the rate of thermal inactivation of L. monocytogenes. Furthermore, thermotolerance of the pathogen increased slightly with NaCl concentration when sodium pyrophosphate was included in the gravy formulation. As explained earlier, high salt concentration increases the temperature of denaturation of the pathogen’s ribosomes and hence increases its thermotolerance [302].

ORGANIC ACIDS

AND

THEIR SALTS

Organic acids and their salts (Table 6.1) are frequently incorporated into foods as acidulants (e.g., acetic acid) or preservatives (e.g., sodium diacetate). Acid sprays and acid dips are used to inactivate

TABLE 6.1 Organic Acids Commonly Used as Acidulants or Antimicrobial Agents in Food Name

Molecular Weight (g/mol)

pKa

Acetic acid

60.05

4.74

Sodium diacetate

142.09

Citric acid

192.1

3.13 4.76 6.40

Benzoic acid

122.12

4.19

Lactic acid

90.08

3.79

Parabens: e.g., methylparaben

152.14

8.47

137.13

4.65 4.80

Propionic acid

74.08

4.87

Sorbic acid

112.12

4.76

Para-aminobenzoic acid

Source: Budavari, S. 1989. The Merck index. Rahway, NJ: Merck & Co., Inc.

Structure

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L. monocytogenes on food surfaces. According to the USDA Pathogen Modeling Program [88], bactericidal activity of acid increases with temperature. When listeriostatic doses of organic acids are used, storage at refrigeration temperature is essential to prevent further growth of the pathogen [151]. Under listericidal conditions, however, refrigeration diminishes acid lethality (Figure 6.5). The growth rate of L. monocytogenes in the presence of organic acids varies markedly with type and concentration of acid and medium pH. Acetic and lactic acids (50 mM) inhibited growth of the pathogen at 37°C when the medium pH was 4.7, but not when it was 6.0 [344]. Growth of Listeria on turkey frankfurters was retarded when frankfurters were dipped into 15 to 25% sodium diacetate or sodium benzoate and to a lesser extent when they were dipped into 15 to 25% potassium sorbate or sodium propionate [151]. At equal pH and equimolar total acid, growth inhibition of L. monocytogenes was in the following order: acetic > lactic > citric [344]. However, at equal pH and equimolar undissociated acid concentration, listeriostatic activity was in the following order: citric > lactic > acetic. These organic acids were more potent bacteriostatic agents than was HCl [344]. Although relatively high concentrations of citric acid (>0.5 M ) have listeriostatic activity, smaller concentrations promoted growth of L. monocytogenes; these data suggest that citric acid contributes to the pathogen’s metabolism [41,344]. When multiple organic acids are applied simultaneously, these acids may act additively or synergistically against targeted microorganisms. A combination of 1.5 to 2.5% sodium lactate and 0.15% sodium diacetate successfully prevented growth of L. monocytogenes in cured meat products (wieners, ham, light bologna, and Cotto salami) at 4°C during 18 weeks [292]. It is widely accepted that antimicrobial activity of weak organic acids, such as acetic acid, is associated with the acid’s undissociated form. The undissociated acid freely permeates the cytoplasmic membrane and dissociates inside the cytoplasm, thus accumulating protons and anions within the cell. This decreases the proton motive force and interferes with microbial metabolism [304,344]. Concentration of the undissociated form of a weak organic acid at a given pH can be calculated according to the Henderson–Hasselbalch equation: pH = pKa + log ([dissociated form]/[undissociated form]) Concentration of the undissociated (uncharged) organic acid form increases as the pH decreases. Contrary to the weak-acid theory, some researchers believe that hydrophobicity of sorbic acid is the cause of its antimicrobial properties [304]. According to these researchers, weak organic acids are membrane-active agents that act to disrupt cytoplasmic membrane function, producing cell death. Lactate Salts of lactic acid (Table 6.1) are used at 1 to 4% as additives in baked goods, meat, and poultry products. When added to food, lactates do not change pH of the products [59]. Sodium or potassium lactate (4%) is listeriostatic at refrigeration temperature [59]. Sodium, potassium, and calcium lactates were equally effective in inhibiting growth of L. monocytogenes in cooked strained beef stored at 20°C [59]. Calcium lactate, however, was most antilisterial in pork-liver sausage [335]. Application of NaCl (2 to 3%), nitrate (125 ppm), or low temperature enhanced the listeriostatic effect of lactate against L. monocytogenes in meat and smoked salmon [259,335]. A combination of lactate (4%) and nisin (400 IU/mL) is listericidal at pH 5.5 and 4°C, and the inactivation rate increases with addition of polyphosphate (0.5%) [48]. Lactate–nisin and lactate–nisin–polyphosphate combinations caused ~2.3- and 4.2-log reductions in 28 and 20 days, respectively; nisin alone only resulted in an initial 1.1-log decrease and did not prevent subsequent growth of L. monocytogenes [48]. Addition of 0.3% sorbate did not improve antilisterial activity of lactate (4%) [48]. Sodium Diacetate Sodium diacetate (Table 6.1) is a GRAS additive [115]. The additive is used as an acidulant, flavoring compound, and antimicrobial agent in foods. When sodium diacetate was added to brain

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heat infusion (BHI) broth at the 18- to 35-mM level (mixture pH of 6.3 to 5.25), a small inoculum of L. monocytogenes (103 CFU/mL) was inhibited in a concentration-dependant manner [293]. A low incubation temperature (5°C, compared to 35°C) enhanced inhibitory action of the diacetate. Minimum inhibitory concentrations of diacetate in the broth were 35, 32, and 28 mM at 35, 20, and 5°C, respectively. Based on equal levels of undissociated acetic acid at different pH values, sodium diacetate was more effective and had lower minimum inhibitory concentrations at 35°C than did acetic acid; minimum inhibitory concentrations were 5, 20, 30, 40, and >100 mM for sodium diacetate and 5, 20, >50, >100, and >150 mM for acetic acid, at pH 4.7, 5.0, 5.5, 6.0, and 6.5, respectively. Dipping turkey frankfurters into 15 to 25% sodium diacetate (pH 4.6) at 22°C for 1 min decreased L. monocytogenes populations 1.7 to 2.0 logs [151]. Sodium acetate on the surface of these frankfurters inhibited Listeria growth ≥14 days when the treated product was stored at 4 to 13°C. The pathogen population increased 2.5 logs over the same time period when frankfurters were stored at 22°C [151]. Sodium diacetate also reportedly enhanced listericidal activity of gamma irradiation [298] and 5,000 AU/mL of pediocin [289]. Sodium Propionate Propionic acid (Table 6.1) and its salts are useful antimycotic agents, and their potential role as antilisterial preservatives has been investigated. Broth media containing >2,000 ppm sodium propionate inhibited growth of L. monocytogenes at pH 5 [96]. Generation times for L. monocytogenes in tryptose broth at pH 5.6 and without sodium propionate decreased from 68 to 49 min as the incubation temperature increased from 4 to 35°C. When 3,000 ppm sodium propionate was added to the medium, generation times decreased from 3.0 days to 4.5 h as the incubation temperature increased from 4 to 35°C [96]. Sodium propionate (3,000 ppm) at pH 5.0 was listeriostatic at 4°C and listericidal at 35°C in tryptose broth incubated for 67 days. Combinations of propionate and acetic acid in growth media produced strong antilisterial action [98]. Lowering the incubation temperature from 35 to 13°C not only diminished the rate of growth of L. monocytogenes, but also decreased maximum populations of the bacterium in the presence of propionate and other organic acids [98]. Sodium propionate (3,000 ppm) was less effective than sorbic acid in eliminating L. monocytogenes from cold-pack cheese at pH 5.2 to 5.5 [285]. Growth of the pathogen on the surface of frankfurters dipped into 15 to 25% sodium propionate (pH ~9.0) was inhibited at 4°C for ≥14 days [151]. Refrigerated storage is essential for long-term listeriostatic activity of sodium propionate at pH >5.5. When contaminated frankfurters were stored at 13 and 22°C, growth was observed after ~7 and 3 days, respectively [151]. Potassium Sorbate Sorbic acid (Table 6.1) is primarily active against yeasts and molds, but this antimicrobial agent also inhibits a wide range of bacteria, particularly aerobic, catalase-positive organisms. Hence, several investigators have assessed the ability of potassium sorbate and sorbic acid to inhibit L. monocytogenes in laboratory media and various foods. Sorbate had a minimum inhibitory concentration of 400 to 600 and >5,000 mg/L at pH 5.0 and 6.0, respectively, when L. monocytogenes was cultured in BHI broth at 35°C [234]. Antilisterial activity of sorbate was enhanced by other organic acids, with acetic and tartaric acids being most effective [97]. The lower the storage temperature and pH of the medium were, the greater was the effectiveness of sorbate against L. monocytogenes. Strong listericidal effects were observed when 0.3% sorbate was used in combination with 125 ppm sodium nitrite, 0.5% polyphosphate, or 400 IU/mL nisin [48]. Potassium sorbate is widely used to extend the shelf life of many foods, including butter, cheese, meat, cereals, and bakery items. Listeria did not grow on turkey frankfurters (pH ~ 9.0) dipped for 1 min in 15 to 25% potassium sorbate when the frankfurters were subsequently stored at 4°C for

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≥14 days [151], with any decrease in viability being minimal (<0.6 log CFU/mL). Even though potassium sorbate retarded the pathogen, growth was observed on frankfurters stored at ≥13°C with the growth rate increasing with storage temperature. Sodium Benzoate Benzoic acid (Table 6.1) is a potent antimicrobial agent in acid foods; optimum activity is in the pH range of 2.5 to 4.0. Listeriostatic activity of this antimicrobial agent at pH value near neutrality is more pronounced at refrigeration than ambient temperature. Growth of the pathogen on the surface of frankfurters dipped into 15 to 25% sodium benzoate (pH ~7.8) was inhibited at 4°C for ≥14 days [151]. When contaminated frankfurters were stored at 13 and 22°C, slow growth was observed after ~10 and 3 days, respectively. Listeria cells sublethally injured in the presence of benzoate have impaired mRNA synthesis [40]; therefore, processes requiring mRNA synthesis for survival of the pathogen (e.g., high acidity) would likely enhance listericidal action of benzoate synergistically. Parabens and Other Benzoic Acid Derivatives Parabens are esters of p-hydroxybenzoic acids (Table 6.1). Of these esters, methyl, propyl, and heptyl parabens are approved in several countries for direct addition to food. Parabens exhibit antimicrobial activity in low-acid (pH ≥4.6) and acid (pH <4.6) foods. Antilisterial activity of parabens increases with decreasing pH and temperature [257]. Parabens (1,000 ppm) were listeriostatic in milk [257], but their action was questionable in processed meat products [28]. Synergistic listericidal activity was observed when methylparaben was used in the presence of anise, basil, or fennel oil [122]. Para-aminobenzoic acid had greater inhibitory activity against L. monocytogenes than propionic, formic, acetic, citric, and lactic acids, in order of decreasing activity [276]. Highest activity of p-aminobenzoic acid against this pathogen was observed as pH decreased. The high potency of p-aminobenzoic acid may result from dual damage to the pathogen; this antimicrobial agent interferes with peptidoglycan synthesis and alters the cell’s metabolism. The minimum inhibitory concentration of p-aminobenzoic acid against L. monocytogenes in BHI (pH ~6.7) at 37°C after 24 h was 9 to 12 mmol/L [276].

FATTY ACIDS

AND

RELATED COMPOUNDS

Antilisterial activity of free fatty acids, particularly those of medium chain length, has been demonstrated. Some fatty acid esters, such as monoacylglycerols (monoglycerides) and esters of sucrose, inhibit a wide spectrum of microorganisms, including L. monocytogenes, in addition to their primary function as food emulsifiers. Free Fatty Acids Lauric (C12:0), linoleic (C18:2), and linolenic (C18:3) acids (Table 6.2) exhibited listeriostatic to listericidal activity and reduced the invasiveness of Listeria [262,332]. Presence of these fatty acids at ≥ 200 µg/mL was listericidal in a storage-conditioning solution (1% NaCl) for mozzarella cheese [262]. The potassium salt of conjugated linoleic acid (100 to 300 µg/mL) was listeriostatic in whole milk [332]. Duration of the L. monocytogenes lag phase increased with an increase in fatty acid concentration [332]. Listeriostatic activity of lauric, linoleic, and linolenic acids varied with pH, presence of other organic acids, and antioxidants. These fatty acids were more active against Listeria at pH 5.0 than at pH 6.0 or 7.0 [262,332]. Listeriostatic activity of K-conjugated linoleic acid (200 µg/mL) in whole milk was enhanced by addition of 2,000 µg/mL citrate, 200 µg/mL butylated hydroxyanisol, or 200 µg/mL ascorbate [332]. Other fatty acids have variable impact on the pathogen’s growth or viability. Counts of L. monocytogenes decreased below the detection level when incubated at 37°C in a storage-conditioning

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TABLE 6.2 Minimal Listeriostatic Concentration of Lauric, Linoleic and Linolenic Acids in Brain Heart Infusion Broth Incubated at 37°C for 24 h and These Fatty Acids’ Effect on Invasion Efficiency of Caco-2 Cells by Listeria monocytogenes Fatty Acid

Lauric acid Linoleic acid Linolenic acid

Minimum Inhibitory Concentration (µg/mL) pH 5.0

pH 6.0

10 50 20

20 200 100

Invasion Efficiency (%)a

0.1 1.26 0.04

a

In absence of fatty acids (control), 23% of inoculated L. monocytogenes were internalized in Caco-2 cells. Sources: Petrone, G. et al. 1998. Lett. Appl. Microbiol. 27:362–368, and Wang, L. L. and E. A. Johnson. 1992. Appl. Environ. Microbiol. 58:624–629.

solution of mozzarella (pH 5.0) supplemented with 200 µg/mL butyric (C4:0), caproic (C6:0), caprylic (C8:0), capric (C10:0), or myristic (C14:0) acid for 24 h. Survival (in the presence of capric or myristic acid) or growth (in the presence of butyric, caproic, or caprylic acid) of the pathogen was observed when the pH of the solution was 7.0 [262]. Medium supplementation with 200 µg/mL palmitic (C16:0), stearic (C18:0), or oleic (C18:1) acid did not inhibit growth of the pathogen [262,332]. Fatty Acid Monoesters Monoacylglycerols (i.e., monoglycerides) are active against L. monocytogenes. Minimum concentrations inactivating ~103 to 104 L. monocytogenes CFU/mL in BHI broth (pH 6.0) after 24 h at 37°C were 25, 75, and 50 µg/mL for monolaurin, monocaprin, and monomyristin, respectively [334]. At concentrations ≥ 200 µg/mL, monocaprin was more effective than monolaurin in inactivating L. monocytogenes in milk, possibly because of the higher water solubility of monocaprin [334]. Listeria monocytogenes was not inhibited by 300 µg/mL monopalmitin, monostearin, monoolein, or monolinolein [334]. Monoacylglycerols were listericidal in beef frank slurries and seafood salad stored at 4°C [333]. These compounds were listeriostatic in turkey frank slurries, cooked shrimp, imitation crabmeat, and Camembert cheese stored at 4°C [333]. Monolaurin showed stronger antilisterial activity in 2% chocolate milk than in 2% milk [334]. The listericidal activity of monolaurin has been observed in media with low pH and low fat content stored at refrigeration temperature [332,333]. Although 100 µg/mL and ≥ 200 µg/mL monolaurin in skim milk at 4°C were listeriostatic and listericidal, respectively, 200 µg/mL monolaurin showed no antilisterial activity in whole milk [332]. Listericidal activity is enhanced when combining monolaurin with organic acids, chelating agents, high salinity, antioxidants, or other monoacylglycerols [272,333]. Propyl gallate (200 µg/g) and lactic acid (0.2%) enhanced the bacteriostatic activity of monolaurin and monocaprin against L. monocytogenes in seafood and Camembert cheese, respectively, stored at 4°C [333]. Monomyristin (200 µg/mL) and monocaprin (200 µg/mL) synergistically inactivated L. monocytogenes in skim milk at 4°C [334]. Coconut oil contains high levels of monolaurin and monomyristin. Synergistic interaction between these monoacylglycerols may have accounted for higher listericidal activity of coconut oil compared to that of monolaurin [334]. Monolaurin has been proposed for decontamination of food contact surfaces [325]. Poor water solubility and the soapy flavor resulting from a high concentration of monolaurin limit its use in food. Monoacylglycerols and derivatives of monolaurin with greater solubility compared to monolaurin were investigated. Listeria was inhibited by triglycerol-1,2-laureate (380 µg/mL) in the presence of 380 µg/mL EDTA [272]. Sucrose monolaurate (400 µg/mL) was

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listericidal in tryptose phosphate broth at 30°C [235]. The listeriostatic activity of 200 µg/mL sucrose monolaurate decreased as temperature increased from 4 to 15°C [235]. Presence of EDTA (50 to 100 µg/mL) enhanced synergistically the listericidal activity of sucrose monolaurate [235]. Sucrose monolaurate alone, or in combination with EDTA, synergistically increased the thermal inactivation rate for L. monocytogenes [235]. Sucrose fatty acid esters (e.g., 0.01% sucrose palmitate) also reportedly acted synergistically with nisin (250 IU/mL) against L. monocytogenes [315].

SODIUM NITRITE Sodium nitrite is frequently used to preserve meat and fish and occasionally as a preservative in certain cheeses. This curing agent slightly inhibits growth of L. monocytogenes [88]. Lag phase and generation time increased as the nitrite concentration increased from 0 to 150 ppm in nutrient broth. Inhibition increases when the addition of nitrite is combined with low temperature, pH, or oxygen level, or when the concentration of sodium chloride in the medium increases [43]. Nitrite antilisterial activity has mainly been reported at pH ≤ 5.5. At pH 6.3, combining 103 ppm sodium nitrite and 3.5% sodium chloride in meat did not control growth of L. monocytogenes at 32°C [132]. Nitrite (30 ppm) did not increase the listeriostatic activity of sodium diacetate in turkey slurries (pH 6.2) [289]. Growth of L. monocytogenes was suppressed for ≥12 weeks in hard salami sausage (aw = 0.79 to 0.86) that contained sodium nitrite (156 ppm before meat fermentation) and NaCl (5.0 to 7.8%), had a pH ~4.4, and was stored at 4°C [156]. However, the contribution of low water activity and low pH was probably more important in preventing growth than was the presence of sodium nitrite. Addition of EDTA (0.9 g/L) or nisin (0.45 g/L) enhanced the listeriostatic activity of sodium nitrite (0.18g/L) in BHI (pH 6.0) [131]. Viability of L. monocytogenes at 4°C decreased up to 3.7 logs in 12 days in BHI (pH 5.5) supplemented with nitrite (125 ppm) and one or several of the following compounds: sorbate (0.3%), lactate (4%), nisin (400 IU/mL), and polyphosphate (0.5%) [48]. The mechanism of nitrite action against L. monocytogenes in processed food is unclear. Filtersterilized nitrite at 50 µg/mL and pH 5.0 prevented visible growth of Listeria within 48 h at ≤ 20°C, whereas 200 µg/mL autoclaved nitrite at the same conditions did not retard Listeria growth [223]. Listeriostatic activity of nitrite, however, may be linked to reactive species derived from nitrite rather than nitrite itself [55]. These compounds may be formed during food processing. Production of low levels of nitrous acid may contribute to growth inhibition at pH < 6.0. Under aerobic conditions, peroxynitrite is formed upon reaction of nitric oxide with superoxide or nitroxyl ion with oxygen. Iron–sulfur–nitrosyl complexes are formed during heating. Minimum inhibitory concentration of Roussin’s black salt (NH4+[Fe4S3(NO)7]–), an iron–sulfur–nitrosyl complex, against L. monocytogenes was 3 µmol/L. Nitric oxide and nitrosothiols break DNA strands, impair ribonucleotide reductase activity, and damage the cell membrane.

ANTIOXIDANTS Antioxidants such as butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ), and propyl gallate comprise an important category of food additives [116]. Primarily used to prevent oxidation of fat, some of these antioxidants also possess antimicrobial activity. TBHQ is the most inhibitory to L. monocytogenes, followed by BHA, propyl gallate, and BHT. Minimum inhibitory concentrations of these antioxidants were ~64, 128, 256, and > 512 ppm, respectively, when they were used in tryptose phosphate agar and the inoculated medium was incubated at 35°C for 18 h [257]. Listeria monocytogenes exhibited an increasingly longer lag phase and generation time as well as a lower maximum population in tryptose broth incubated at 35°C that contained BHA at 100 to 200 ppm, added as a DMSO solution [346]. Concentrations of BHA ≥300 ppm were listericidal [346]. Increasing the concentration of TBHQ from 10 to 30 ppm in the medium increased the duration of the lag phase from 6.5 to 34 h at 35°C, but did not appreciably affect generation time or maximum population. Presence of TBHQ (150 µg/mL) in milk was listericidal

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and listeriostatic after 24 h at 35°C when this medium was inoculated with 1 × 101 CFU/mL and 1 × 103 CFU/mL, respectively [257]. Propyl gallate inhibited growth of L. monocytogenes in cabbage juice at room temperature, but ellagic acid, the hydrolytic product of propyl gallate, failed to inhibit the bacterium [63]. Although these findings appear promising for the food industry, FDA regulations stipulate that BHA and similar additives can only be used as antioxidants and cannot be added indiscriminately to foods for other purposes. Phenolic antioxidants are believed to damage bacterial cell membranes [2,321]. Carvacrol, an antioxidant naturally found in oregano and thyme, is structurally related to BHA. Carvacrol (1 mM) depletes bacterial membrane potential and alters membrane permeability to cations [322]. Exposure to BHT sensitized bacteria to osmotic shock [321]. Development of BHA resistance was observed in the presence of glycerol and was attributed to the increase in lipid content of the cell wall and membrane of the pathogen [2].

SMOKE Smoking has traditionally been used to preserve meat and fish and enhance their sensory quality. Addition of 0.2 and 0.6% (v/v) of a commercial liquid smoke to wiener exudates inactivated L. monocytogenes with D-values of 36 and 4.5 h, respectively; Listeria in the smoke-free exudate grew from an initial population of 105 CFU/mL to ~108 CFU/mL after 3 days at 25°C [103]. Hot smoking (core temperature at 65°C for 20 min) also inactivates this pathogen [154]. Smoking time was proportional to the reduction in Listeria counts [287]. Cold smoking of fish was recommended at a temperature < 21°C [279]. The combination of 20 ppm phenols and 4% NaCl was listericidal at 4 to 12°C in nutrient broth [225]. Nisin (4,000 to 6,000 IU/mL) and sodium lactate (60%) can be injected before smoking to further reduce the viability of Listeria in processed fish [245]. Despite numerous studies showing listericidal activity of smoke compounds, L. monocytogenes may be detected in smoked fish [159]. Therefore, observing good hygiene and good manufacturing practices during processing and refrigerated storage is essential to minimize risks of contamination with this pathogen. Liquid smoke flavorings owe their antimicrobial activity to the presence of phenolic compounds and acetic acid, both of which are bacteriostatic at relatively low concentrations [305]. Isoeugenol, for example, is a phenolic compound that retarded growth of Listeria in exudates of smoked wieners [103]. Listeriostatic activity of 100 ppm isoeugenol was higher at pH 5.8 than at pH 7.0 [103]. This compound seemed to interact with the bacterial membrane [331].

SPICES, HERBS,

AND

PLANT EXTRACTS

Although primarily used as flavoring and seasoning agents, many spices contain chemicals or essential oils that inactivate or inhibit growth of pathogenic and spoilage organisms. Consequently, surveys were made to identify spices with activity against L. monocytogenes, especially those suitable for use in minimally processed foods. Cilantro (6%), rosemary (1%), sage (1%), oregano (0.1 to 0.7%), clove (0.5%), thyme (0.1%), cinnamon (0.1%), pimento (0.1%), and pelargonium (0.05%) showed listericidal activity [14,129,141,200,251,317]. Sensitivity to spices varied among strains of Listeria [141]. Listeriostatic and listericidal activity of plant oils have been observed in food. Essential oil of oregano reduced Listeria counts in packed meat, regardless of the composition of the atmosphere within the packet [320]. Cilantro oil (6%) was bacteriostatic against Listeria in vacuum-packed ham [129]. Pelargonium oil prevented growth of Listeria in quiche filling at 25°C for ≥24 h [200]. Adding thyme’s essential oil to minced pork at a level of 0.08% decreased the population of L. monocytogenes ~1 log during 8 days’ storage at 4 and 8°C. The organism grew in the untreated controls from 1 × 107 to 6 × 107 and 2 × 108 CFU/g, respectively [14].

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TABLE 6.3 Concentration of Compounds from Plant Oils That Inactivated 50% of L. monocytogenes Population in Phosphate-Buffered Saline Solution after Incubation at 37°C for 1 h (BA50%) Active Compound Benzaldehyde Carvacrol Carvone S Cinnamaldehyde Citral Estragole Eugenol Geraniol Menthol Perillaldehyde Salicylaldehyde Thymol

Oil Source Bitter almond Thyme, oregano Dill seed Cinnamon Lemon grass Tarragon Clove Rose Peppermint Mandarin peel Almond Thyme, oregano

BA50% 0.36–0.46 0.083–0.086 0.17–0.35 0.008–0.019 0.099–0.200 0.35–0.36 0.061–0.081 0.28–0.51 0.48–0.57 0.30–0.35 0.43–0.45 0.077

Source: Friedman, M. et al. 2001. J. Food Prot. 65:1545–1560.

Antioxidant extract from rosemary (0.3%) or encapsulated rosemary oil (5%) substantially retarded growth of L. monocytogenes in pork liver sausage [251]. Listeria populations in these sausages increased from 103 CFU/g to ~2 × 105 CFU/g during 50 days’ storage at 5°C in the presence of the antioxidant extract from rosemary (0.3%) or encapsulated rosemary oil (5%). In the absence of rosemary extract, maximum counts of 109 CFU/mL were reached after 25 days at 5°C [251]. Fat from food and an emulsifier reduced the antimicrobial activity of plant oil [138]. Partitioning plant oil (hydrophobic) in the fat component of food may have decreased availability of the antimicrobial agent for bacterial cells [129]. Compounds from plant oil that express listericidal (Table 6.3) or listeriostatic activity have been identified. Carvacrol, cinnamaldehyde, eugenol, geraniol, and thymol, for instance, were listericidal within 30 to 60 min of exposure [121]. Pinene was responsible for the listeriostatic activity of rosemary oil, and 0.1 µl α-pinene/100 mL was sufficient to suppress growth of Listeria in BHI at 35°C for >24 h [251]. These active compounds (e.g., thymol) damage the cell membrane and may interfere with peptidoglycan synthesis [271]. Because of the potential link between consumption of chocolate milk and listeriosis, Pearson and Marth [258] studied the effect of caffeine and theobromine, two methylxanthine compounds in cocoa, against L. monocytogenes (103 CFU/mL, initially) in skim milk at 30°C. Limited antilisterial activity was observed with 2.5% theobromine; however, the authors found that 0.5% caffeine exhibited antilisterial activity in skim milk through extending the lag phase ~3 to 6 h, increasing the generation time 1 h, and decreasing the final maximum population ~1.4 log. The combination of 2.5% theobromine and 0.5% caffeine slightly increased antilisterial activity when compared with 0.5% caffeine alone.

LYSOZYME Lysozyme is an antimicrobial enzyme naturally present in foods of animal origin, including hen’s eggs and milk. The enzyme is active against many Gram-positive bacteria [267] and has been proposed for use in cheese [113]. Lysozyme is particularly attractive as a food preservative because this enzyme is active between 4 and 95°C and over the pH range generally encountered in food

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[155]. The mechanism of inactivation of Listeria by lysozyme is unclear, but appears to be nonlytic; cell leakage but no peptidoglycan hydrolysis was observed in lysozyme-treated L. innocua [218]. Inhibition of L. monocytogenes by lysozyme in tryptic soy broth increased as temperature and pH decreased [155]. The presence of lysozyme in food, on food surfaces, or on packaging surfaces contributes to control of L. monocytogenes [31,130,169]. Listeriostatic or listericidal activity has been reported in vegetables, fresh meat, processed meat products (e.g., ham), fish fillets, mayonnaise, milk, and cheese [101,149,150,169]. Lysozyme activity in food, however, is diminished in the presence of minerals and meat [150,169]. Addition of EDTA (1 to 5 mM) or lipase (12.5 IU/mL) enhances lysozyme (0.02 to 0.1 mg/g) activity against the pathogen [150,198]. Lysozyme sensitized the pathogen to mild heat [267]. Combinations including 0.05% potassium sorbate, 5 mM sodium acetate, 0.95% ethanol, or 10 mM ascorbic acid did not substantially increase antilisterial activity of lysozyme [149].

HYDROGEN PEROXIDE The United States permits the use of hydrogen peroxide solution (< 35% H2O2) to sterilize multilayer packaging materials in aseptic processing systems. According to FDA regulations, residual hydrogen peroxide must be < 0.5 ppm [117] immediately after packaging. Hydrogen peroxide is used as a preservative, particularly with raw milk, in several parts of the world. Use of this agent as a direct food additive in the United States is very limited. The FDA permits adding 0.05% (w/w) hydrogen peroxide to raw milk for making cheese [115]. Addition of 0.02% hydrogen peroxide to cheese brine inactivates L. monocytogenes, when the brine is held at 12°C; lower levels were sufficient as the storage temperature decreased [191]. Hydrogen peroxide (0.05%) was relatively ineffective in decreasing the Listeria count in raw milk or milk artificially contaminated with equal numbers of S. aureus and S. faecalis [77]. It was listericidal when combined with mild heat [327], lactic acid [327], or peroxyacetic acid [179]. The population of L. monocytogenes decreased >5 logs when inoculated produce was sprayed or dipped in a solution containing a mixture of hydrogen peroxide and lactic acid, at 1.5% each [327]. Alternatively, Listeria may be controlled when lactic acid bacteria that produce hydrogen peroxide are immobilized on the surface of produce [138]. Hydrogen peroxide causes oxidative damage in bacterial DNA, RNA, protein, and lipids [271]. Catalase from L. monocytogenes detoxifies low levels of hydrogen peroxide. A high concentration of this antimicrobial agent, however, overwhelms a cell’s catalase and hence overcomes the natural resistance of this pathogen. Resistance of L. monocytogenes to 0.1% hydrogen peroxide was observed after the bacterium was adapted to pH 4.5 to 5.0, 500 ppm H2O2, 5% ethanol, 7% NaCl, or 45°C heat for 1 h [206]. Adaptation of L. monocytogenes to H2O2 increased the D56°C value of this pathogen 2.9-fold [205].

LACTOPEROXIDASE SYSTEM The lactoperoxidase system is a naturally occurring antimicrobial mechanism in milk. Activation of this system has been proposed to extend the shelf life of raw milk in countries with inadequate refrigeration. A functional lactoperoxidase system in raw milk may inactivate or inhibit growth of Listeria. The lactoperoxidase system extended the lag phase of L. monocytogenes in the presence of 35 mg/L thiocyanate, 0.2 g/L glucose, and 1 mg/L glucose oxidase [30,295]. Listericidal activity of the lactoperoxidase system was reported in ultrahigh temperature-treated milk supplemented with potassium thiocyanate (84 mg/L), glucose (10 g/L), and glucose oxidase (2 mg/L) [87]. The lactoperoxidase system enhanced destruction of Listeria during thermal processing [166], high-pressure processing [125], and nisin treatment [30]. However, processors should consider

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the biphasic inactivation patterns of L. monocytogenes when the lactoperoxidase system is activated [166]. Lactoperoxidase catalyzes oxidation of thiocyanate (SCN–) by hydrogen peroxide to hypothiocyanate (OSCN–) and hypothiocyanous acid (HOSCN) [187] as follows: 2SCN − + H 2O 2 + 2H + Lactoperoxidase →(SCN)2 + 2H 2O (SCN)2 + H 2O → HOSCN + H + + SCN − HOSCN
OSCN − + H +

( pKa = 5.3)

The hypothiocyanate and hypothiocyanous acid oxidize sulfhydryl residues (R-SH) in membrane proteins of bacteria, generating sulphenic acid derivatives (R-SOH). When the lactoperoxidase system is activated, leakage of potassium ions, amino acids, and peptides was first observed. Uptake of glucose, amino acids, purines, and pyrimidines was subsequently inhibited. Consequently, bacterial replication and protein synthesis decreased [187]. Efficacy of the lactoperoxidase system depends on sufficient quantities of lactoperoxidase and reactants in milk. Lactoperoxidase represents 1% of whey proteins [274], which is adequate for a functional lactoperoxidase system. Bovine milk naturally contains 1 to 7 ppm thiocyanate. Supplementation of milk with thiocyanate (>35 mg/L, final concentration) is necessary for satisfactory listeriostatic activity of the lactoperoxidase system [87]. Hydrogen peroxide must also be added exogenously or generated by exogenous enzymes such as glucose oxidase [75]. This enzyme oxidizes glucose to gluconic acid and hydrogen peroxide. Production of gluconic acid decreases the pH; this may increase the bactericidal effect of the lactoperoxidase system beyond what would have been observed in similar model systems with pH values near neutrality [75]. Listeria monocytogenes was inhibited and inactivated in milk containing ≥0.75% gluconic acid during extended incubation at 13 and 35°C, respectively [95]. The lactoperoxidase system in TSBYE was more effective at 5 to 10°C than at 20 to 30°C; corresponding increases in lag time were 32 to 98 h and 2.8 to 8.9 h [295].

LACTOFERRIN Lactoferrin is an iron-binding glycoprotein found in mammalian milk. At 23 to 46 mg/mL, lactoferrin was slightly listeriostatic in milk stored at 35°C for 18 h [255]. Its listericidal action is localized in the N-terminus domain of the protein and not at its iron-binding site [20,76]. Dialysis reduces the degree of lactoferrin saturation with iron, and the resulting product is known as apo-lactoferrin. At 15 and 30 mg/mL, apo-lactoferrin was listeriostatic and listericidal in milk stored at 35°C for 18 h, respectively [255]. Lactoferricin B is a small antimicrobial peptide (25 amino-acid residues) resulting from hydrolysis of the N-terminal region of bovine lactoferrin by gastric pepsin [76]. At 1 to 3 µg/mL and ≥15 µg/mL, respectively, lactoferricin B was listeriostatic and listericidal in peptone glucose yeast extract broth at 30°C [19]. Storage of milk at refrigeration temperature was recommended for optimal lactoferrin hydrolysate activity [237]. Activity of lactoferrin and related compounds strongly depends on composition of the medium. Lactoferricin maintained its antilisterial activity over a pH range of 5.5 to 7.5. The listericidal activity of lactoferrin or lactoferricin decreased as cation concentrations in the medium increased [19]. Listeria monocytogenes grew in milk supplemented with ferric ammonium citrate (0.125 M) and apo-lactoferrin (30 mg/mL) after incubation at 35°C for 18 h. In this study, Listeria counts decreased 1.5 logs in the absence of ferric ammonium citrate [255]. A combination of 15 mg/mL apo-lactoferrin and 150 µg/mL lysozyme retarded growth of L. monocytogenes in milk at 36°C [256].

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BIOCONTROL The terms “biopreservation” and “biocontrol” refer to use of microorganisms or their metabolites to inhibit or inactivate undesirable microorganisms in food. Biocontrol may be attributed to the starter microorganisms, their metabolites, and fermentation end products. Biocontrol of L. monocytogenes in food is achieved by adding a bacteriocin-producing microorganism, bacteriocin-containing fermentate, bacteriocin crude extract, or purified bacteriocin [238]. Bacteria that produce antilisterial bacteriocins include strains of Lactococcus lactis, Lactobacillus bavaricus, Lb. reuteri, Lb. acidophilus, Lb. curvatus, Lb. saké, Lb. plantarum, Leuconostoc carnosum, Leuconostoc mesenteroides, Carnobacterium piscicola, Pediococcus acidilactici, Propionibacterium thoenii, and Enterococcus spp. [186,209,238,347]. Numerous studies listed potential applications of identified bacteriocins in food. This section gives an overview of the most potent bacteriocins against Listeria and regulatory issues regarding applications of bacteriocins in food. To list all bacteriocins with known efficacy against Listeria is beyond the scope of this review. Bacteria that have been used traditionally in food fermentation (i.e., most lactic acid bacteria) include bacteriocin-producing strains. Starter cultures, including these bacteriocin-producing strains, may be used to ferment food or food ingredients. Legal issues arise, however, when purified bacteriocins are applied to foods. Approval of U.S. regulatory agencies, mainly the FDA, is required before commercial use of purified bacteriocins [110]. A company can self-affirm whether a bacteriocin of interest is GRAS; however, the company is required to justify application of the bacteriocin if requested by the FDA [110]. Among purified bacteriocins, FDA has approved only nisin, for use in pasteurized cheese spreads [115].

LIVE FERMENTATE Live fermentate is a preparation with high microbial biomass that is generally the product of fermentation. The microorganism of interest is used as starter culture, but the exact composition of the live fermentate is unknown because it contains microbial metabolites and the unused portion of substrates. Live fermentate used for antimicrobial purposes may be incorporated in the food, distributed over the food surface, or immobilized on the packaging surface in contact with the food [138,238,303]. Lactic acid bacteria are most suitable for biocontrol purposes because these bacteria are used in many traditional fermented foods and generally recognized as safe. These bacteria compete with other microorganisms for nutrients or produce antimicrobial compounds, such as weak acids, hydrogen peroxide, and bacteriocins [303]. Biocontrol of L. monocytogenes in refrigerated storage requires the use of suitable antagonistic psychrotrophic bacteria. Nisin-producing lactococci are poor biocontrol microorganisms because they do not grow well at refrigeration temperature or in meat products. Pediocin-producing pediococci were not suitable meat biopreservatives because they were effective against L. monocytogenes only at abuse temperatures [347]. The psychrotrophs C. piscicola L103 [290], Leuconostoc carnosum 4010 [45], and several strains of Lb. bavaricus [339] were proposed as alternative bacteriocin producers to prevent growth of Listeria at refrigeration temperature. Growth of the pathogen may also be retarded in ready-to-eat, fresh-cut vegetables inoculated with Lb. delbrueckii [138].

BACTERIOCINS Bacteriocins are polypeptides synthesized by bacteria; these agents are generally active against strains related to the producer [238]. Bacteriocins are suitable as processing aids, complementing other preservation methods. Heat, freezing–thawing, acid [238], high hydrostatic pressure [163],

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and pulsed electric field [163] were more listericidal in the presence of bacteriocins than they were in their absence. Nisin and pediocin are the most investigated bacteriocins against L. monocytogenes. Nisin is produced by a limited number of Lc. lactis subsp. lactis strains [67]. This bacteriocin is useful in preventing outgrowth of Clostridium spp., including C. botulinum [64]. Pediocins are produced by strains of Pediococcus spp. [74,197]. Dipping meat [104,352] or salmon [306] in a nisin solution (1.3 × 103 to 5.0 × 103 IU/mL) inhibited growth of L. monocytogenes during refrigerated storage. Listeria populations decreased ~1.5 logs on packaged cheese stored at 4°C for 84 days when nisin (61.4 mg/cm2) was immobilized on the package [288]. Nisin activity, however, varies with environmental conditions. Listeriostatic activity of nisin increased with decreasing temperature [306] and pH [139]. Addition of 2% sodium chloride enhanced the listericidal activity of nisin in laboratory media, particularly at levels < 10 µg nisin/mL [139]. Stearic acid (≥10%) decreased the antimicrobial efficacy of nisin (1.0 × 105 IU/mL) [67] and carbon dioxide sensitized the pathogen to nisin [241]. After 10 days, L. monocytogenes decreased ~4 logs in the presence of 2.5 µg/mL nisin in phosphate buffer (pH 6.2) with 100% carbon dioxide atmosphere at 4°C [241]. Maximum counts were reached after 35 days’ incubation; in the absence of nisin or when aerobic incubation was used, maximal counts were reached after 10 to 15 days [241]. Listeria monocytogenes strains vary in their sensitivity to nisin [21]. Listeriostatic activity of pediocin has been demonstrated in numerous foods, including meat and dairy products [74,134,197]. Conflicting results were reported on pediocin activity in milk and liquid eggs, likely because compounds in the crude extract offset pediocin activity [197]. Antilisterial activity of this bacteriocin in food slurries was improved when pediocin was encapsulated in phosphatidylcholine-based liposomes or when 0.1% Tween 80 was added to the product [74]. Unlike nisin, pediocin is not in the FDA’s list of food-grade GRAS substances. Food supplementation with purified pediocin, therefore, requires regulatory approval in the United States. Pediocin bound to heat-killed producer cells [134] or in dehydrated fermented whey permeate [197] was recommended for food applications. Bacteriocins such as nisin and pediocin disrupt the bacterial membrane, thereby depleting the proton motive force of the membrane [134]. Acid adaptation or starvation increased resistance of L. monocytogenes to nisin and pediocin [204]. Resistant Listeria subpopulations were not detected when nisin was immobilized on edible cellulose-based packaging material [288]. Relatively stable populations of bacteriocin-resistant mutants have been isolated from bacterial suspensions exposed to a bacteriocin for extended periods [202]. Application of bacteriocin cocktails and rotation of bacteriocins could ensure their efficacy against Listeria over extended processing times. Incorporation of bacteriocins in food, however, should not preclude use of proper sanitary practices.

MODIFIED ATMOSPHERE “Modified atmosphere” refers to an altered gaseous environment that aims to extend shelf life of a food. Proper packaging maintains the food under this modified atmosphere. A defined mixture of nitrogen (N2), carbon dioxide (CO2), or oxygen (O2) commonly replaces air in modifiedatmosphere packaging [65]. A vacuum replaces air in vacuum-packaged food. Sous vide-processed foods are vacuum packaged, then cooked, chilled, and finally stored at refrigeration temperature. Modified-atmosphere packaging is frequently used to maintain the quality of fresh meat, vegetables, and fruits at refrigeration temperatures [65]. Listeria monocytogenes grows well under aerobic and anaerobic conditions and at refrigeration temperatures. These properties make L. monocytogenes a potential threat to the safety of foods packaged under vacuum or modified atmospheres [65]. Growth of L. monocytogenes is not inhibited in food that has been packaged under vacuum [148] or superatmospheric oxygen (>5 kPa O2) [4]. Presence of ≥80% carbon dioxide or 100% nitrogen gas increased the lag phase of L. monocytogenes in Stilton cheese stored at 4°C by 2 to 3 weeks [338]. The presence of 10% oxygen in a nitrogen-rich (e.g., 80% nitrogen) environment did not satisfactorily inhibit growth of the pathogen [338]. An increase in the amount of carbon dioxide

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increases the length of the lag phase and the generation time [106,338]. The presence of > 80% carbon dioxide is recommended to retard growth of Listeria [106,184,338]. Growth inhibition by carbon dioxide (≥ 50%) was more pronounced at 4°C than at 10°C, and at pH 5.5 than at pH 6.5 [106]. Incorporation of organic acids or bacteriocins increases the safety of modified-atmosphere packaged foods. Inhibition of L. monocytogenes was increased by incorporating 0.5% sodium acetate, 2% sodium lactate, or 0.26% potassium sorbate into vacuum-packaged bologna stored at 4°C [336]. The presence of nisin in vacuum or modified-atmosphere packaging (gaseous mixture not described by the authors) was slightly listericidal during storage of cheese and cooked ham at 4°C [288].

ALTERNATIVE PROCESSING TECHNOLOGIES Heat is most commonly used for food preservation, but alternative methods are gradually being implemented in food processing. Processors hope these alternative technologies will preserve nutritional quality and fresh taste and texture while decreasing the risk of disease transmission. Some alternative technologies may be applied to packaged food to overcome accidental postprocess contamination of the product with L. monocytogenes. Physical alternative processes include radiation, high hydrostatic pressure, pulsed electric field, and high-intensity pulsed light [176,189,281]. These alternative technologies are gradually gaining acceptance from regulatory agencies as well as from consumers. For example, the United States has approved irradiation for control of foodborne pathogens in fresh or frozen meat, dehydrated spices, seeds for sprouting, and whole eggs [118]. Alternative technologies are perceived as nonthermal, although some heat may be generated during their application [82,190]. However, processing conditions are generally selected so that heat is minimal and quickly dissipated [189]. Control of pathogens is therefore accomplished mainly by nonthermal means.

IRRADIATION Microwaves, radio frequencies, high-intensity pulsed light, ultraviolet, electron beam, and gamma are the forms of radiation of interest to food manufacturers. These radiations differ in wavelength and penetrating power [27]. Gamma radiation and electron beams are the most effective and applicable forms for decontamination of food [66,309]. Ultraviolet radiation has poor penetrating power and is therefore limited to decontamination of surfaces, water, and juices [118]. Pulsed light treatment controls microorganisms on food surfaces only [118]. Microwave penetrates food, but treatment uniformity is questionable: Listeria spp. survived on chickens cooked in microwave ovens [107]. Because microwave lethality is caused by heating, it will not be addressed in this section. Ionizing Radiations Food irradiation, i.e., processing food with ionizing radiation, generally refers to treatment with gamma radiation or electron beam. Cobalt-60 or cesium-137 emits gamma rays for food treatment. Accelerators (<10 MeV) generate electron beams [118]. Contrary to gamma irradiation, electron beam technology does not result in nuclear waste, and the treatment can be switched on and off. Irradiation with x-rays for elimination of foodborne pathogens is not commonly reported in the literature, even though the FDA allows its use [118]. Irradiation technology allows in-package decontamination of refrigerated and frozen foods and thus results in safer, fresh-like, nonthermally treated, ready-to-eat food. Packaging products before processing is preferable to prevent postprocess contamination with pathogens such as L. monocytogenes. A radiation dose is frequently limited to 2 to 3 kGy to prevent development of off-odors, off-colors, and aftertaste in food [66,165]. Inactivation of L. monocytogenes by gamma radiation varies with the treated product (Table 6.4). Successive irradiation treatments were more listericidal than an equivalent single treatment at 20°C,

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TABLE 6.4 Gamma Irradiation Dose Necessary to Inactivate 90% of an L. monocytogenes Populationa in Selected Food Products Food Ground beef Raw turkey nuggets Cooked turkey nuggets Sandwich Ice cream Broccoli Corn Lima beans Peas

D-Value (kGy)

Temperature (°C)

0.51–1.00 0.56 0.69 0.71–0.81 0.38 0.51 0.61 0.77 0.92

21 4 4 –40 –72 –5 –5 –20 –20

Ref. 100 313 313 66 165 240 240 240 240

a

Radiation D-value.

but not at refrigeration (4°C) or freezing (–80°C) temperatures [9]. Listeria innocua NADC 2841 in ground pork was substantially more resistant to electron beam irradiation than were three L. monocytogenes strains [309]. This L. innocua strain is therefore a potential surrogate for process validation studies [309]. Extrinsic and intrinsic resistance factors caused variations in the inactivation rate of L. monocytogenes by irradiation. Resistance of L. monocytogenes decreases with treatment temperature [240]. Some food ingredients scavenge cell-damaging free radicals produced during irradiation and therefore contribute to the resistance of pathogens to radiation treatment. Resistance of L. monocytogenes to irradiation varied among strains [100] and decreased with initial counts [9]. The reduced recovery of the pathogen on selective compared to nonselective media shows that substantial numbers of Listeria were sublethally injured during exposure to irradiation [100,309]. Irradiation breaks DNA strands and this disrupts replication. Radiationinduced radicals may alter other cell components, such as the bacterial membrane [309]. Sublethally irradiated cells are therefore sensitized to strong selective agents that may be present in agar media [100,309]. Ultraviolet Radiation and High-Intensity Pulsed Light The FDA permits food processing with ultraviolet radiation at 253.7 nm [118]. A population of L. monocytogenes decreased ≥ 5 logs when treated with ultraviolet irradiation (100 µW/min/cm2) for 2 min [348]. Survivor plots, however, indicated the presence of small Listeria subpopulations that survived extended exposure to ultraviolet radiation [348]. Causes of tailing in this survivor plot have not been identified. A population of L. monocytogenes attached to stainless steel decreased ~4 logs when exposed to 750 µW/min/cm2 ultraviolet radiation at 25°C [176]. This radiation dose was not sufficient to eliminate the pathogen from the surface of chicken. Titanium dioxide (1 mg/mL) generates free radicals when exposed to ultraviolet light (< 385 nm) and therefore synergistically enhances ultraviolet activity against foodborne pathogens, including L. monocytogenes [170]. Pulsed light processing is based on emission of radiation of broad spectral range (200 to 1,100 nm) by xenon flash lamps [118]. In the United States, total treatment permitted in food must be ≤ 12.0 J/cm2 [118]. Pulsed light (1 J/cm2) inactivated ~6 logs L. monocytogenes on the surface of nutrient agar [83]. The population of L. monocytogenes decreased > 3 logs when exposed to white light (200 to 900 nm, with peak emission at 500 to 650 nm) for 100 pulses (100 nsec/pulse) at a frequency

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TABLE 6.5 High-Pressure Processing Treatments Decreasing L. monocytogenes 4 Logs in Selected Foods Food

Pressure (MPa)

Temperature (°C)

Time (min)

Ref.

250 300 300 414 414 300

30 20 5 2 25 25

5.0 5.0 15 5.1 8.7 6.7

5 269 127 7 7 207

Fruit juices Jams Ovine milk (6% fat) Fresh pork loin Fresh pork loin Frankfurters

of 1 Hz [281]. In this study, no inactivation was observed when cells were treated with white light that was low in ultraviolet rays (350 to 900 nm), which suggests that pulsed light inactivation results from the bactericidal action of ultraviolet radiation [281].

HIGH-PRESSURE PROCESSING Treating food with high hydrostatic pressure may improve a product’s safety without damaging its quality. Pressures ≥ 300 MPa are commonly used and food pressure processors able to deliver up to 900 MPa are currently available. The pressure is transferred instantaneously and homogeneously throughout the treated product, thus making this technique suitable for surface and in-depth decontamination of food in flexible packages [350]. Adiabatic heating is generated as pressure increases [270]. The treatment temperature refers to the average temperature during the pressure-holding time. Listeria is particularly susceptible to inactivation at pressures ≥250 MPa (Table 6.5). Inactivation increases with pressure [207]. D-values are difficult to estimate because survivor plots have a sigmoid shape with considerable tailing during prolonged processing time [222,310]. With respect to pressure resistance, heterogeneity among cells within a bacterial population may explain the tailing [324]. The efficacy of high pressure against L. monocytogenes varies with treatment conditions, medium composition, and different strains of the pathogen. High-pressure inactivation of Listeria follows a second-order relationship with processing temperature [7,127]. Inactivation of Listeria in milk was minimal at around 20°C, intermediate at ~5°C, and highest at 50°C [127]. Food with high levels of proteins or glucose favored survival of the pathogen during the pressure treatment [294]. The level of fat in food did not consistently affect survival of Listeria [127,294]. Food formulations containing bacteriocins [162], the lactoperoxidase system [125] or carvacrol [167] synergistically enhanced the action of high-pressure processing against Listeria. Sensitivity to high pressure varied among Listeria strains [125,310]. Interestingly, strains resistant to high pressure [310] also expressed superior resistance to pulsed electric field and heat [188,190]. Pressure ≥ 200 MPa causes irreversible protein denaturation, rupture of cytoplasmic membrane, and leakage of cell contents [189]. This range of pressure also induces autolysis of Listeria [218]. Expression of cold-shock proteins is induced in survivors of high-pressure processing [218]. During storage, expression of cold-shock proteins also enhances the cell’s resistance to high pressure [337].

PULSED ELECTRIC FIELD PROCESSING Pulsed electric field (PEF) treatment involves exposing food to pulses ≥20 kV/cm for a total treatment time of microseconds to milliseconds [82]. The continuous PEF process is well adapted to decontamination of liquids. The PurePulse system received a no-objection letter from the FDA and can be used for acid and low-acid foods. Other systems are currently being developed for industrial-scale processing of acid foods.

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TABLE 6.6 Pulsed Electric Field Processing Parameters for Decreasing Listeria spp. 2 Logs in Selected Foods Food Whole milk 50% Acid whey (pH 4.2) Liquid whole egg

Electric Field (kV/cm)

Treatment Time (µs)

Ref.

35 25 40

300 ≤48 64

273 190 53

Pulsed electric field rapidly inactivates Listeria in fluid products (Table 6.6). Numerous processing parameters influence the bactericidal efficacy of the process. Inactivation of Listeria spp. with PEF increases with the electric field intensity, pulse length, pulse frequency, gap between electrodes, number of product passages between electrodes, and process temperature [82]. Process efficacy decreases as a product’s flow rate increases [82]. The efficacy also varies with food composition. Inactivation of Listeria spp. increases with decreasing pH and conductivity [82]. The population of L. monocytogenes Scott A decreased ~0.2 and 3.4 logs in sweet whey (pH 6.8; 0.46 S/m) and sweet whey acidified with lactic acid (pH 4.2; 0.46 S/m), respectively, when processed with a PEF at 25 kV/cm and 23°C for 48 µsec [188]. Cell injury was detected when treated cells were grown on acidified agar [343]. Inactivation of L. monocytogenes by PEF varies among strains. Listeria monocytogenes OSY8578, a meat isolate with superior resistance to the process compared to other strains, is a potential target strain for PEF process optimization [190]. Lactobacillus plantarum ATCC 8014 has greater resistance to the process than does L. monocytogenes OSY-8578 in acid whey; therefore, it is a potential surrogate microorganism for PEF process validation in this medium [188]. The electric field permeabilizes the bacterial membrane, resulting in leakage of intracellular contents [54]. Expression of molecular chaperones (DnaJ, GroEL, and GroES) decreases ~5 to 20 min after the PEF process [188]. Cell permeabilization [342] and chaperone decrease [188] are more pronounced in process-sensitive than process-resistant strains. When the input energy and maximum treatment temperature exceeded ~80 J/mL and 60°C, respectively, damage of Listeria cells resulted from the combined effect of heat and electric field [82]. Nonthermal treatment with mild electric pulses strongly sensitized L. monocytogenes to mild heat [188]. The population of L. monocytogenes decreased 3.3 to 6.1 logs when treated with heat (55°C for 10 min) 10 min after PEF treatment (27.5 kV/cm for 144 µsec). Treatments with PEF or heat killed ~0.5 log only. Heat sensitization subsequent to PEF treatment paralleled the decrease in expression of molecular chaperones [188].

ATTACHMENT AND BIOFILM FORMATION Listeria monocytogenes attaches to numerous surfaces, including stainless steel, glass, wood, porcelain, iron, plastic, polyester, propylene, rubber, waxed cardboard, and paper [85,185, 212,236,301]. Consequently, equipment surfaces, conveyor belts, floor sealants, and drains are potential reservoirs for Listeria spp. in food-processing plants and may lead to secondary food contamination. Floor drains should therefore not be located next to filling and packaging equipment, and using high-pressure hoses to clean these drains should be avoided to prevent formation of an L. monocytogenes aerosol [300]. The ability of Listeria to attach to objects with different surface properties suggests that packaging material is a potential source of contamination of food with this pathogen. Different materials, however, were not equivalent in hosting the pathogen. Attachment was stronger on polyvinyl chloride and polyurethane than on stainless steel [231]. Stainless steel was more supportive of surface colonization by Listeria at refrigeration to ambient temperatures than was polytetrafluoroethylene [58].

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1. Planktonic cells

2. Cell deposition on surface • Hydrophilic interactions • Flagella

3. Cell adhesion to surface • Hydrophilic interactions • ±Fibrils • Synthesis of exopolymers

4. Surface colonization • Cells monolayer

5. Biofilm formation • Layers with variable cell density • Homogeneous cell distribution horizontally

6. Biofilm development • Biofilm growth • Presence of capillary water channels (3-D structure)

FIGURE 6.7 Formation of L. monocytogenes biofilms under static conditions, on a flat nonporous surface. Deviations from this representation may be observed among strains and with the properties of attachment surface. (From Chae, M. S. and H. Schraft. 2000. Int. J. Food Microbiol. 62:103–111; Chavant, P. et al. 2002. Appl. Environ. Microbiol. 68:728–737; Kalmokoff, M. L. et al. 2001. J. Appl. Microbiol. 91:725–734; Mafu, A. A. et al. 1990. J. Food Prot. 53:742–746; Midelet, G. and B. Carpentier. 2002. Appl. Environ. Microbiol. 68:4015–4024; and Vatanyoopaisarn, S. et al. 2000. Appl. Environ. Microbiol. 66:860–863.)

Attachment and biofilm formation of L. monocytogenes on solid surfaces progress in the following sequence: cell deposition on the surface, adhesion to the surface, surface colonization, biofilm formation, and biofilm development (Figure 6.7) [56,58,164,212,231,326]. Adhesion of Listeria to surfaces has been attributed to hydrophilic interactions, presence of flagella, fibrils, and synthesis of exopolysaccharides. Listeria monocytogenes is a hydrophilic microorganism with surface free energy ~66 mJ/m2 [213]. Therefore, cell attachment and surface colonization are rapid (≤ 2 h at 20 and 37°C) on hydrophilic substrata [58]. Because of its flagella, Listeria movement overcomes electrostatic repulsion forces of the surface thereby facilitating attachment of the pathogen [58,326]. Strongest negative electrophoretic mobility of L. monocytogenes (3.95 µm/s) was observed at pH 6.0 and 20°C [229]. Listeria produces flagella at 20°C but not at 37°C; these are rich in negatively charged groups (COO–) [229]. The net charge of the pathogen was neutral at pH 2.0 to 3.5 [213,229]. Listeria flagella also act as adhesive structures during early stages of cell attachment [326]. Highly adherent strains synthesize fibrils [164]. Fibril formation was observed at ≤ 21°C and pH 6.0 to 9.0, after 9-h incubation on an iron surface [300].

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Exopolysaccharide fiber secretion, after a 1-h incubation at 20°C, seemed to aid attachment of the pathogen to polypropylene and glass surfaces [212]. Attachment to stainless steel, polyvinyl chloride, or polyurethane at 25°C was greater for Listeria, compared to Staphylococcus sciuri, Pseudomonas putida, and Comamonas sp. [231]. Attachment of L. monocytogenes to surfaces varies in the presence of other bacteria. The adherence of L. monocytogenes was enhanced in the presence of Flavobacterium spp. [35], but decreased when the pathogen was cocultured with S. sciuri [195] or with a nisin-producing strain of Lc. lactis [196]. The three-dimensional structure of L. monocytogenes biofilms changes with time, and it is influenced by incubation conditions (i.e., static vs. constant agitation). After 2 to 3 days’ incubation at 37°C, dense bacterial mats (i.e., biofilm) were formed on the colonized surface [56,58]. The bottom layer of the biofilm had a higher cell density than the upper layer, after 72-h incubation at 37°C on a glass slide [56]. Cell distribution horizontally appeared homogeneous [56]. Water channels are clearly observed with scanning electron microscopy as the biofilm develops [58,217]. Water channels are important to circulate nutrients and dissolved gases inside the biofilm, as well as to eliminate metabolic waste products [58]. The structure and physiology of adherent cells are different from those of planktonic (i.e., suspended) cells. Listeria monocytogenes in the stationary phase in a biofilm changed from a rod to a coccoid shape as the population aged [319]. The pathogen grew more slowly when immobilized than in a planktonic mode. Strains of L. monocytogenes varied in their ability to adhere and form biofilms, even when exhibiting a growth pattern similar to that of planktonic cell populations [56,164]. The ability to mount a stringent response, via induction of the relA gene, and physiological adaptation to nutrient deprivation were essential for growth of adhered Listeria [311]. Microscopic analysis shows an increase in cell density within the Listeria biofilm over time, while enumeration on agar indicates a constant colony count [319]. This difference in count by these two enumeration methods may have been caused by an increase in noncultivable or dead L. monocytogenes as the biofilm aged [319]. During biofilm development, synthesis of the following proteins was upregulated: pyruvate dehydrogenase, 6-phosphofructokinase, the 30S ribosomal proteins YvyD and rpsB, superoxide dismutase, a sensing protein CysK, a DNA repair protein RecO, and a cell division initiation protein DivIVA. These proteins are associated with carbohydrate metabolism, stress response, quorum sensing regulation, DNA repair, and cell multiplication [319]. Synthesis of flagellin, a flagellar protein, decreased during biofilm growth [319]. This decrease was expected because flagella help during early stages of cell attachment [326], but they may impair formation of a structured biofilm. Listeria in a biofilm is harder to remove and inactivate than when it is present as freely suspended (planktonic) cells. Planktonic Listeria is less resistant to inimical treatments than is adherent Listeria. Resistance to antimicrobial agents increased with the initial inoculum size [196], age of the biofilm [247], and when the environmental temperature decreased [249]. Food soils protected biofilms against removal by cleaning [143]. Proteins enhanced attachment to silica surfaces in the following decreasing order: bovine serum albumen < β-casein and α-lactoglobulin < β-lactoglobulin [3]. Active packaging is proposed as a means to prevent growth and attachment of the pathogen to surfaces in direct contact with food. Coating surfaces with nisin, an antagonistic starter culture (e.g., bacteriocin producer), egg white lysozyme, or bacteriophages was proposed to prevent bacterial attachment and colonization of surfaces [31,196]. Surfactants also decrease adhesion of the pathogen to surfaces [229].

SANITIZERS Cleaning the food-processing environment is generally a multistep process [308]. A first rinse removes loose soils. Detergents, followed by a second rinsing step, are then applied to eliminate residual soils. The washed surface is then sanitized to inactivate residual microbial contaminants.

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A final rinsing step with potable water removes the sanitizer and microbial residues. Sanitization is a critical step that decreases pathogenic and spoilage microorganisms in food-processing facilities and therefore prevents cross-contamination of food. Water may also disseminate microorganisms, unless a suitable sanitizer is added to the processing water. To be deemed effective, sanitizing agents must reduce at least 5 logs of a given test organism population within 30 sec of exposure at ambient temperatures [171]. Most sanitizing agents active against Listeria spp. belong to one of five categories: chlorine-containing compounds quaternary ammonium compounds acid sanitizers ozone iodophors Efficacy of sanitizers decreases with temperature [214], increasing surface porosity [214], and as an L. monocytogenes biofilm develops [247]. Plasmid-mediated resistance to multiple sanitizers is a problematic phenomenon because it can be transferred at high frequency (8 × 10–6 to 1 × 10–3 transconjugant CFU per one donor CFU) between Listeria spp. and S. aureus [194].

CHLORINE

AND

CHLORINATED COMPOUNDS

Chlorine and its derivatives are used extensively in food, water, and surface decontamination. Chlorinated compounds include hypochlorite solution, chloramines, and chlorine dioxide (gaseous or aqueous). According to the Grade-A Pasteurized Milk Ordinance, utensils and equipment are preferably sanitized with ≥ 50 ppm available chlorine at 24°C for ≥ 1 min [86]. A survivor plot of Listeria exposed to 1 ppm available chlorine follows a biphasic pattern. The pathogen population decreased rapidly during the first 2 min of exposure, whereas a subpopulation survived for > 60 min [90]. Listericidal efficacy of active chlorine is strongly impaired by organic loads, phosphate, temperatures close to 25°C, and pH > 6 [89,90,307]. Although a solution containing 100 ppm available chlorine was effective in broth [203], 200 ppm decreased populations only 0.6 log CFU on tomatoes after 3 h [25]; 1,000 ppm inactivated 2 logs when the pathogen formed a biofilm [243]. Sensitivity of L. monocytogenes to chlorine varies among strains [90] and with the physiological state of the Listeria population [89]. High chlorine concentrations may alter the taste of food and increase the risk of chlorine gas release into the processing environment, thus increasing safety hazards for workers, and necessitating the removal of chlorine residuals from wastewater. After considering chlorine’s advantages and adverse effects, 120 to 200 ppm active chlorine is recommended for treatment of fresh fruits and vegetables [25,307]. Addition of 10 to 100 ppm sodium hypochlorite to cheese brine will also inactivate Listeria in this product [191]. Addition of chlorine or hypochlorite to water generates hypochlorous acid (HOCl) and hypochlorite ion (OCl–) [86]: Cl2 + H2O → HOCl + H+ + Cl− Ca(OCl)2 + H2O → Ca2+ + H2O + 2 OCl– Ca(OCl)2 + 2 H2O → Ca(OH)2 + 2 HOCl HOCL
H + + OCl − The bactericidal activity of chlorine increases with the concentration of undissociated hypochlorous acid [86]; therefore, chlorine activity increases with decreasing pH. Its dissociated form (OCl–)

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has weak bactericidal activity [86]. Undissociated hypochlorous acid diffuses into the bacterial cell, where this acid induces formation of toxic oxidative species and combines with proteins. This leads to inhibition of mRNA, protein synthesis, and oxidative phosphorylation [86,271].

QUATERNARY AMMONIUM COMPOUNDS Quaternary ammonium compounds are noncorrosive cationic surfactants frequently used to sanitize equipment surfaces [208,318]. Application of 100 to 200 ppm active compounds on surfaces is listericidal [203]. Listeria monocytogenes is slightly more resistant to quaternary ammonium compounds than to other sanitizers [214]. However, when the pathogen was exposed to cleaning compounds (4°C for 30 min) before treatment with quaternary ammonium compounds (25°C for 30 sec), >7 logs were inactivated [308]. Quaternary ammonium compounds react with bacterial carboxylic groups. Consequently, the cytoplasmic membrane is permeabilized and the cytoplasmic content may coagulate [318]. Enhanced resistance (≤10-fold) of L. monocytogenes to sanitizers has been reported after 2-h exposure to sublethal concentrations of quaternary ammonium compounds [208]. Adaptation to quaternary ammonium compounds involves a decrease in efflux pump activity [318] and modifications in cell wall structure [228]. Cells adapted to benzalkonium chloride were more elongated and filamentous than unadapted cells [318]. Adapted cells doubled their contents of C17:0 (iso and anteiso) fatty acids in the membrane and increased activity of the efflux pump system. The multidrug efflux pump, MdrL, which prevents accumulation of chemicals in bacterial cells, was present in L. monocytogenes adapted to sanitizers [278]. The gene encoding for this pumping system in L. monocytogenes can be chromosomal and plasmid encoded [278]. The contribution of MdrL to adaptation to sanitizers, however, is unclear [228,278]. Adaptation to multiple sanitizers may contribute to persistence of the pathogen in food-processing environments. However, the minimal inhibitory concentration of sanitizers against L. monocytogenes adapted to quaternary ammonium compounds is below the concentration of sanitizers generally used in food-plant sanitation [208].

ACID SANITIZERS Acid sanitizers are nonvolatile agents that retain bactericidal activity at <100°C. They are frequently used in conjunction with rinsing agents in automated cleaning (i.e., clean in place) systems. Acetic, peracid, and peroctanoic acids, combined or not with other antimicrobials, are active against L. monocytogenes. When L. monocytogenes adhered for 24 h to stainless steel and then was treated with a combination of acetic acid (1%) and monolaurin (100 µg/mL) at 25°C, the population of the pathogen decreased > 5 logs in 25 min [247]. At 80 ppm, peracid and peroctanoic acids were more potent than chlorine against a Listeria biofilm in the presence of milk residues on stainless steel [108]. When fruits were inoculated with L. monocytogenes and washed with lactic acid (1.5%) and hydrogen peroxide (1.5%) at 40°C for 15 min, pathogen populations decreased ≥ 6 logs [327]. Mixtures of peroxyacetic acid and hydrogen peroxide (120 ppm) were proposed as alternatives to a chlorine wash for lettuce [307]. Inclusion of sodium lactate and sodium acetate in wiener or bratwurst formulations inhibited growth of L. monocytogenes during extended storage at refrigeration temperature [133]. Dipping these sausages in lactate–diacetate solution was less effective than including this mixture in product formulations [133].

OZONE Ozone (O3) has been used in European countries for decades and has recently been approved in the United States by the FDA for treatment, storage, and processing of foods, including meat and poultry, unless use is precluded by a standard of identity [119]. Aqueous ozone reduces the microbial load on surfaces of food, packaging material, and equipment [172]. Gaseous ozone minimized

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TABLE 6.7 Inactivation of L. monocytogenes in Suspensions or on Food Surfaces after Exposure to Ozonated Water Medium Water (pH 6.1) Soluble starch solutionb BSA solutionb,c Phosphate buffer (pH 5.9) Alfalfa sprouts Beef

Temperature (°C)

Ozone (ppm)

Time (min)

Inactivationa (log CFU/mL)

Ref.

20 20 20 25 4 4

0.2 0.2 0.2 1.8 23 3

2 2 2 0.5 2 5

5.0 5.0 3.7 7.0 0.9 1.1

275 275 275 173 329 244

a

Decrease in log count compared to control.

b

20 ppm, pH 6.0.

c

Bovine serum albumin.

growth of microbial contaminants during storage of fresh produce [172]. In pure cell suspensions, L. monocytogenes is inactivated rapidly by small concentrations of ozone. The pathogen population decreased ~7 logs in 50 mM phosphate buffer (pH 5.9) after 30-sec exposure to 1.8 ppm ozone at 25°C [173]. Inactivation of L. monocytogenes increases with ozone concentration [111]. Listericidal activity of ozone varies with temperature, medium composition, strain, and physiological status. At an equivalent ozone concentration and exposure time (0.25 ppm; 2-min exposure), the inactivation rate of the pathogen almost doubled as temperature decreased from 37 to 4°C [111]. Processing at refrigeration temperature is therefore desirable to optimize the listericidal activity of ozone. Organic compounds such as proteins react with ozone [275], thus reducing the concentration of active ozone available for microbial inactivation in food. Consequently, efficacy of ozone decreases when L. monocytogenes is present in food, compared to deionized water or phosphate buffer (Table 6.7). Listeria is more sensitive to ozone than are other bacteria [173,275]. Sensitivity to ozone varied among strains of L. monocytogenes [111]. Resistance to ozone increased as L. monocytogenes entered the stationary phase. Ozone decomposes very rapidly in an aqueous solution; this generates numerous free radical species, such as hydroxyl (⋅OH) and superoxide (⋅O2–) [172]. These reactive species have high oxidizing power and initiate the oxidation of organic compounds. As expected from these findings, ozone causes oxidative damage in bacteria. Cell wall, membrane, intracellular enzymes, and DNA are possible sites of damage by ozone. Catalase and superoxide dismutase may protect L. monocytogenes against ozone; superoxide dismutase is more important than catalase for this protection [111]. Sublethal treatment with ozone did not sensitize L. monocytogenes to mild heat, alkali, or 6% NaCl [244].

MISCELLANEOUS SANITIZING AGENTS Iodophors, such as octylphenoxy-polyethoxy ethanol iodine complexes, are used to sanitize surfaces in contact with food. Iodophors are polymeric organic molecules that complex iodine species (I–, I2, and I3–) [135]. Listericidal activity of 25 ppm available iodophor is equivalent to that of 200 ppm chlorine [203]. Listeria monocytogenes is ~10 times more resistant to iodophors when the pathogen is attached to the surface of polypropylene or rubber than to stainless steel or glass [214]. The activity of these sanitizers against L. monocytogenes attached to stainless steel was 2 to 13 times higher at 20°C than at 4°C [212]. No inactivation of Listeria, however, was detected when an iodophor (80 ppm I2) was used in the presence of milk or human serum [24].

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Iodophors cause multiple damage in bacterial cells. The bacterial cell wall is permeable to iodine, which interacts with double bonds of membrane phospholipids; oxidizes free sulfhydryl groups; and combines with tyrosine, histidine, cytosine, and uracil [135]. Therefore, death of cells treated with iodine was attributed to loss of membrane integrity, protein inactivation, and DNA denaturation [135]. The population of L. monocytogenes decreased >7 logs CFU on tomatoes when surfaceinoculated fruit was sprayed with a 0.5% calcinated calcium solution (HYCEA-S, Kaiho Ltd., Tokyo, Japan) [16]. Addition of 100 ppm hydrogen peroxide inactivated the pathogen in contaminated cheese brines [191]. Suspended in peptone water, the population of L. monocytogenes decreased >6 logs when incubated at 37°C for 8 h in the presence of a packaging material coated with nisin (188 µg/mL) and lauric acid (200 mg/mL) [145]. This type of active packaging successfully decreased L. monocytogenes by 1 log on the surface of contaminated turkey bologna, and no growth was reported for >21 days at 21°C [73]. Compounds that have profound listericidal efficacy on nonfood surfaces include sodium dichloroisocyanurate [24], glutaraldehyde [24], chitosan [179], carvacrol [179], monolaurin [325], and listeriophages [144]. Listeria monocytogenes is ubiquitous in nature and prevention of cross-contamination during handling of ready-to-eat food is essential. Regular use of listericidal hand sanitizers can contribute to prevention of cross-contamination with Listeria during food handling. Kerr et al. [168] estimated that 12 and 7% of food workers carried Listeria spp. and L. monocytogenes, respectively. Thimothe et al. [314] detected Listeria spp. on 5.1% of employee-contact surfaces (gloves and apron) in seafood-processing plants. Thorough hand rubbing for 30 sec with nonmedicated soap reduced the aerobic mesophilic flora <1 log on hands contaminated by raw chicken [137]. Medicated soap containing chlorhexidine achieved ~3 logs reduction under similar experimental conditions. A peroxide-based powder (Ultra-Kleen) effectively decontaminated gloves previously dipped in cooking water contaminated with Listeria spp. [221]. Image analysis of palm imprints made on agar plates (24.5 × 24.5 cm) can be used to predict hand hygiene status [137]. In this method, subjects placed the palm of each hand on the surface of agar plates for 15 sec, and imprints were allowed to dry at ambient temperature for 30 min before incubation at 33°C and 60% relative humidity for 48 h [57]. Photographs of the palm imprint were taken, scanned, and the degree of contamination of palms from food calculated by image analysis (Image-Pro Plus, version 4.1.0.12) [137].

ACTIVE PACKAGING Packaging materials containing agents with activity against L. monocytogenes are increasingly investigated as a means to improve food safety after processing. Listeriostatic or listericidal agents of active packaging are applied as coatings to surfaces in contact with food [71,145] or incorporated into packaging materials [36]. Immobilizing antimicrobial agents such as organic acids, bacteriocins, spice extracts, lysozyme, chitosan, listeriophages, or EDTA onto packaging helps prevent growth of Listeria on food surfaces [31,36,145]. Nisin (1 mg/mL) adsorbed on silanized silica inhibited some, but not all, strains of L. monocytogenes [31]. Effectiveness of adsorbed nisin increased with bacteriocin concentration [31]. Adsorbed antimicrobials are generally more effective against Listeria when used in combination rather than individually [145]. The listericidal activity of a nisin (4%)–lauric acid (8%) mixture, however, was dramatically reduced when the pathogen was at the surface of turkey bologna rather than suspended in 1% peptone water [73]. Organic acid anhydrides form an effective antimicrobial coating on nonpolar polymers such as low-density polyethylene [36]. The anhydride hydrolyzes in aqueous environments (e.g., moist food), and the resulting free acid migrates from the polymer surface into the food [36]. A mixture of 2.5% silver ion and 14% zinc ion zeolite (sodium aluminosilicate) on the surface of stainless steel inactivated >6 logs L. monocytogenes [71]. Similar listericidal activity was reported

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when this zeolite was wet-process or powder coated [71]. When coextruded in plastic film, silverion zeolite showed strong antimicrobial action, even though it was not tested specifically against Listeria [36]. Bactericidal activity of silver ion zeolite was associated with transfer of silver ions to the cell, which led to formation of reactive oxygen species [219].

MULTIPLE ANTIMICROBIAL TREATMENTS Controlling L. monocytogenes in food may require application of multiple hurdles. Thermal pasteurization alone is generally considered adequate for eliminating this pathogen. This heat treatment, however, may damage heat-sensitive nutrients and flavor compounds. Thermal treatments less than pasteurization require additional hurdles (i.e., treatment combinations) to achieve the desired lethality [193]. Treatments that are commonly combined include mild heating (normally, 55 to 75°C), addition of antimicrobial agents (e.g., nitrites), acidification, and nonthermal alternative processing (e.g., ultrahigh pressure). These treatments may be applied simultaneously or sequentially to maximize antimicrobial efficacy and retention of product quality. When milk and liquid egg were experimentally treated with mild heat, limited inactivation of Listeria spp. was observed. Combining heat with nisin resulted in greater listericidal action (Table 6.8) compared to heat alone. Hurdles with lethal or growth-inhibitory action against foodborne microorganisms may be combined, depending on food and processing conditions. Compatibility between hurdles is essential and a synergistic or additive antimicrobial action is expected. Synergistic action is most desirable; this is noticeable in combinations producing inhibition or lethality greater than the additive actions of individual hurdles [53]. Satisfactory hurdle treatments cause multiple cell injury, such as physical cell damage, disruption of cell homeostasis, or metabolic exhaustion [193]. Multiple hurdles are particularly useful in eliminating L. monocytogenes in ready-to-eat meat and poultry products [323]. Listeriosis is increasingly associated with consumption of these products;

TABLE 6.8 Inactivation of Listeria by Heat, Pulsed Electric Field (PEF), and High-Pressure Processing (HPP) in Combination with Nisin Food

Processing Parameters

Nisin (µg/mL)

Milk

Heat: 60°C, 2 min Heat: 60°C, 2 min PEF: 40 kV/cm, 64 µs, 28°C PEF: 40 kV/cm, 64 µs, 28°C No treatment Heat: 54°C, 16 min Heat: 54°C, 16 min HPP: 300 MPa, 20°C, 10 min + 0 day storage at 4°C + 0 day storage at 4°C + 18 days storage at 4°C + 18 days storage at 4°C PEF: 40 kV/cm, 64 µs, 31°C PEF: 40 kV/cm, 64 µs, 31°C No treatment + 0 day storage at 4°C + 18 days storage at 4°C

0 7 0 37 ≤ 37 0 10

0.5–0.7 2.2–4.0 2.0 3.0 0 1.9 3.0

215 215 54 54 54 178 178

0 5 0 5 0 100

0.8 0.7 0 3.5 2.1 4.7

268 268 268 268 53 53

100 100

0 0

291 291

Liquid whole egg

Log CFU/mL Decreased

Ref.

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recent recalls of contaminated batches have caused major economic losses to the industry [312]. Ready-to-eat meat and poultry products may be formulated to contain antilisterial agents (e.g., diacetate), are thermally treated, and occasionally may be stored in contact with antimicrobial packaging materials [36,73]. Increasing awareness of possible postprocessing contamination of ready-to-eat meat prompted processors to apply lethal treatments at late stages of production. These are referred to as “post-lethality treatments” because they are applied after the traditional critical control point, e.g., the main heating step during production of frankfurters [323]. In-package thermal pasteurization and high-pressure processing are examples of post-lethality treatments that target primarily environmental food-surface contaminants. Careful integration of hurdles is crucial to the success of the combined treatment. Consideration of hurdle–hurdle interactions is essential to avoid antagonism. Inactivation of L. monocytogenes increased when high-pressure treatment increased from 300 to 700 MPa [310]. Using pressures much lower in magnitude than those just described, Pagan et al. [248] observed some antagonism between pressure and ultrasonic treatments. When ultrasonic waves of 117 µm and pressurization were applied simultaneously, D40°C of L. monocytogenes decreased to a greater extent when pressure increased from 0 to 100 kPa than from 300 to 400 kPa. It is presumed that ultrasonic treatments inactivate bacteria through intracellular cavitation and high pressure counteracts this mechanism. Improper application of hurdles may have a negative impact on the safety of treated food. Treating L. monocytogenes with commonly used hurdles, such as mild heat and acid, induced stressadaptive responses and protected the pathogen against lethal preservation factors [17,206,345]. Simultaneous exposure to multiple hurdles, therefore, may be necessary to minimize the risk of stress hardening of foodborne pathogens. Unlike conventional preservation technologies, emerging alternative methods, such as high-pressure processing, target a limited number of loci in a microbial cell [189]. These technologies cause limited microbial lethality and considerable stress and injury. Adaptation of pathogens to stresses caused by application of these technologies may protect pathogens from subsequent lethal treatments and thus compromise the safety of food. Alternative processing technologies may benefit from the hurdle concept. Addition of bacteriocins, for example, improved the lethality of high-pressure processing against L. monocytogenes [268]. Treating L. monocytogenes with PEF decreased levels of molecular chaperones for a short period after treatment [188]. When heat was applied to PEF-treated cells to coincide with maximum depression in the level of chaperones, great lethality was observed. Applying heat before PEF processing resulted only in a limited additive lethal effect. Careful choice of treatment combinations, therefore, makes alternative technologies feasible in today’s food applications.

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Methods 7 Conventional to Detect and Isolate Listeria monocytogenes Catherine W. Donnelly and David G. Nyachuba CONTENTS Introduction ....................................................................................................................................216 Cold Enrichment ............................................................................................................................217 Selective Enrichment and Plating at 30 to 37°C...........................................................................219 Selective Agents ...................................................................................................................219 Potassium Tellurite ...................................................................................................219 Lithium Chloride/Phenylethanol ..............................................................................220 Nalidixic Acid...........................................................................................................221 Trypaflavine/Acriflavine ...........................................................................................221 Potassium Thiocyanate .............................................................................................222 Thallous Acetate .......................................................................................................222 Polymyxin B.............................................................................................................222 Moxalactam ..............................................................................................................223 Ceftazidime...............................................................................................................223 Selective Media for Enrichment and Isolation of Listeria............................................................223 Selective Enrichment Media ................................................................................................223 UVM Broth...............................................................................................................225 Fraser Broth ..............................................................................................................226 Isolation Media.....................................................................................................................227 McBride Listeria Agar (MLA).................................................................................227 LPM Agar .................................................................................................................228 Oxford Agar (OXA) and Modified Oxford Agar (MOX)........................................228 PALCAM Agar .........................................................................................................228 Other Selective Plating Media .................................................................................229 Comparative Evaluation of Direct Plating Media for Recovery of Listeria from Foods .............................................................................................230 Incubation Conditions ..............................................................................................232 Oblique Illumination ............................................................................................................232 β-Hemolysis ............................................................................................................233 Official Methods for Isolating L. monocytogenes from Food .............................................234 FDA Method.............................................................................................................234 International Dairy Federation Method ...................................................................238 USDA–FSIS Method................................................................................................239 The Netherlands Government Food Inspection Service..........................................243 Research Advances.........................................................................................................................244 215

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Considerations for Recovery of Injured Listeria ..........................................................................244 Conclusions ....................................................................................................................................248 References ......................................................................................................................................249

Die Methode ist alles. Ralovich, German proverb [122]

INTRODUCTION Listeria monocytogenes is a nonfastidious organism that can be subcultured on most common bacteriological media such as tryptose agar, nutrient agar, and blood agar. However, attempted isolation or re-isolation of Listeria from inoculated or naturally contaminated food and clinical specimens by use of nonselective media is often challenging. Difficulties encountered in isolating L. monocytogenes date back to initial characterization of this pathogen in 1926 when Murray and his coworkers [127] stated, “The isolation of the infecting organism is not easy and we found this to remain true even after we had established the cause of the disease.” Although efforts to isolate L. monocytogenes from blood and cerebrospinal fluid of infected patients have met with considerable success (mainly because of the presence of Listeria in pure culture), obvious difficulties arise when food and clinical specimens (tissue biopsies and autopsy specimens) contain small populations of L. monocytogenes in combination with large numbers of other organisms. The first isolation methods were generally based on direct inoculation of samples on simple agar media. Isolation was problematic in cases of low numbers of viable Listeria cells, and inoculation into test animals (for example, embryonated eggs) was recommended. Upon conducting experiments with guinea pigs and mice, Larsen concluded that this biological method could be of value for detecting small numbers of Listeria in samples without competitive microflora [104]. However, in the presence of other microorganisms, especially Gram-negative mixed flora, test animals died of septicemia. Direct plating, cold enrichment, selective enrichment, and several rapid methods can be used in various combinations to detect L. monocytogenes in food, clinical, and environmental samples. In 1948, Gray et al. [76] introduced the cold enrichment procedure as an alternative method to isolate L. monocytogenes from highly contaminated samples. Although this method has contributed much to present-day knowledge concerning the epidemiology of listeriosis, the prolonged incubation period necessary to obtain positive results is a serious disadvantage. Major improvements in selective enrichment and plating media have since decreased analysis times from several months to less than 1 week. Outbreaks of foodborne listeriosis coupled with the high mortality rates associated with sporadic cases of illness and the advent of mandatory hazard analysis critical control point (HACCP) programs have underscored the need for faster and more efficient methods to detect small numbers of Listeria in a wide range of foods. The purpose of this chapter is to review and update development of various enrichment broths, as well as plating media and methods, used to isolate Listeria spp. (including L. monocytogenes) from food, environmental, and clinical samples. Numerous enrichment broth and plating media formulations have been developed and used during the past five decades for selective cultivation of Listeria. Detection and isolation of Listeria remain complicated tasks. Unfortunately, researchers have not yet been able to identify a single procedure sensitive enough to detect L. monocytogenes in all types of foods within a reasonable time. Furthermore, many selective enrichment broths and plating media fail to allow repair or growth of sublethally injured Listeria frequently present in semiprocessed and processed foods [32] or food-processing environments.

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Despite these inherent shortcomings, research efforts in response to foodborne listeriosis outbreaks have led to development of numerous regulatory procedures, including the U.S. Department of Agriculture Food Safety and Inspection Service (USDA–FSIS) and the U.S. Food and Drug Administration (FDA) procedures [86,88,91,178]. Both of these methods have been adopted in the United States as “standard methods” to isolate L. monocytogenes from a wide variety of foods and food-processing environments. However, in an effort to detect more rapidly and reliably healthy and sublethally injured Listeria in the wide range of foods currently examined, these methods and others have proved to be somewhat insensitive and do undoubtedly require further modifications and refinement.

COLD ENRICHMENT Difficulties in detecting and isolating L. monocytogenes typically arise when small numbers of Listeria are present in environmental and clinical or food samples containing large numbers of indigenous microorganisms. Hence, numbers of Listeria must be increased significantly, relative to that of the background flora, before the bacterium can be detected. Thirteen years after the first description of L. monocytogenes by Murray et al. [127], Biester and Schwarte [17] observed that Listerella (Listeria) could be frequently isolated from naturally infected sheep organs that were held refrigerated in 50% glycerol for several months. Although the organism was only rarely isolated after initial plating of diluted specimens, Biester and Schwarte failed to comment on the significance of cold storage. Following similar chance observations, a young graduate student, M. L. Gray, recognized the benefits of low-temperature incubation for recovering L. monocytogenes from clinical specimens. In 1948, Gray et al. [76] reported that in three of five bovine listeriosis cases, L. monocytogenes was only isolated after brain tissue was diluted in tryptose broth, stored for 5 to 13 weeks at 4°C and then plated on tryptose agar. Although a few Listeria colonies were observed after directly plating the remaining two brain tissue samples on tryptose agar, the bacterium was more readily isolated following cold enrichment. These results clearly demonstrated the ability of L. monocytogenes to multiply to detectable levels in the presence of other microbial contaminants during extended storage at refrigeration temperature (4°C). Gray’s cold enrichment method, in which samples homogenized in tryptose broth were incubated at 4°C and plated weekly or biweekly on tryptose agar during 3 months of storage, was soon adopted as the standard procedure for recovering L. monocytogenes. Normally only a few weeks of cold enrichment are required before Listeria can be detected; however, in one instance [74], 6 months of refrigerated storage was necessary before L. monocytogenes could be isolated from calf brains. Although the cold enrichment procedure is clearly slow and laborious, this method greatly enhances the likelihood of isolating Listeria (if present) from a variety of specimens, including food. In 13 studies summarized by Bojsen-Møller [21], Listeria was identified in 995 tissue and organ specimens from naturally and experimentally infected domestic animals. Using direct plating and cold enrichment procedures, Listeria was isolated from 684 of 995 (68.7%) specimens, whereas 307 of 995 (30.8%) specimens required cold enrichment before the bacterium could be detected. Furthermore, cold enrichment failed to detect Listeria in only 4 of 684 (0.6%) samples that were previously positive by direct plating. A study by Ryser et al. [156] stressed the importance of cold enrichment for recovery of L. monocytogenes from cottage cheese manufactured from milk inoculated with this pathogen. Using direct plating, L. monocytogenes was recovered from 43 of 112 (38.4%) cottage cheese samples stored at 3°C for up to 28 days, whereas cold enrichment of the same samples in tryptose broth for up to 8 weeks yielded Listeria in 59 of 112 (52.7%) samples. Thus, cold enrichment was necessary to detect this pathogen in 16 of 112 (14.3%) cheese samples. Ryser and Marth also found cold enrichment to be of great value in detecting low levels of L. monocytogenes in cheddar [157], Camembert [158], and brick cheese [160] manufactured from pasteurized milk inoculated with the bacterium.

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Lewis and Corry [109] compared cold enrichment and the FDA method [86] for isolating L. monocytogenes and Listeria spp. from ready-to-eat (RTE) foods at retail in the United Kingdom. Of the 57 food samples examined using cold enrichment, 5 yielded L. monocytogenes, and 2 L. innocua while the FDA method yielded 3 samples positive for L. monocytogenes only. Despite the proven success of cold enrichment, the mechanism by which numbers of L. monocytogenes are enhanced during prolonged incubation at 4°C is not fully understood. Although cold enrichment exploits the psychrotrophic nature of L. monocytogenes and simultaneously suppresses growth of indigenous nonpsychrotrophic organisms, Gray and Killinger [74] indicated that, at times, growth of Listeria was too rapid to attribute enhanced growth of this pathogen to mere multiplication. When this procedure was first described in 1948, Gray et al. [76] suggested possible involvement of an inhibitory factor in bovine brain tissue that suppressed growth of competing organisms. However, this theory has since been dispelled by subsequent studies that demonstrated enhanced growth of Listeria during cold enrichment of such diverse samples as mouse liver [144], oat silage [73], feces [139], sewage [56], cabbage [78], raw milk [170], and cheese [156–160]. A more plausible explanation is that, in many clinical specimens, Listeria may exist within monocytes, macrophages, or other phagocytic cells, with cold storage facilitating release of the intracellular organism. More recent research on the role of cold-shock proteins, cold-acclimating proteins, and other mechanisms that enable psychrotrophic growth of L. monocytogenes may help further explain the preferential growth of Listeria during cold enrichment [11,98]. For instance, anteiso-C15:0 fatty acid reportedly plays a critical role in adaptation of L. monocytogenes to cold temperatures [4]; mutants deficient in this fatty acid have been shown to be cold sensitive. As previously reported by Ryser and Marth [161], over 20 media formulations have been successfully used to cold enrich a diverse group of samples naturally or artificially contaminated with L. monocytogenes. Incubation at 4°C is partially selective for growth of L. monocytogenes, so nonselective broths such as tryptose broth and Oxoid nutrient broth no. 2 (ONB2) rapidly emerged as media of choice; tryptose broth was generally recognized as superior. In earlier studies, cold enrichment was used as the sole enrichment procedure and was followed by plating a portion of the enriched sample on tryptose agar at intervals during 2 to 12 months [164]. Following incubation, plates were examined under oblique lighting for typical bluish green, Listeria-like colonies. Cassiday and Brackett also have briefly reviewed the methods and media used by various researchers [22a,35,36]. Although growth of L. monocytogenes is favored at 4°C, other organisms, including Proteus, Hafnia, Pseudomonas, enterococci, and certain lactic acid bacteria are also able to multiply in nonselective media at refrigeration temperatures [2], thus making detection of Listeria more difficult. To prevent overgrowth by non-Listeria organisms, investigators began adding selective agents to various nonselective cold enrichment broths in an effort to inhibit non-Listeria microbes. In 1972, Bojsen-Møller [21] recognized that supplementing tryptose phosphate broth with polymyxin B substantially reduced populations of Gram-negative rods (i.e., Escherichia coli, Pseudomonas aeruginosa, and Proteus spp.) and enterococci while at the same time allowing rapid growth of L. monocytogenes. Unfortunately, certain species of lactic acid bacteria resistant to polymyxin B can ferment lactose to lactic acid and reduce the pH to the point where L. monocytogenes fails to grow at 4°C. Attempts at maintaining a pH of 7.2 by adding 0.1 M MOPS (3-N-morpholino propane sulfonic acid) to cold-enriched raw milk samples were unsuccessful [80]. Recovery of L. monocytogenes is enhanced when cold enrichment is used as a secondary enrichment following selective primary enrichment at 30 to 37°C. Bannerman and Bille [10] subjected numerous cheese and cheese factory environmental samples to secondary cold enrichment in the FDA Listeria enrichment broth (LEB) following primary enrichment at 30°C for 48 h. After plating enrichments on two selective agars, 34 and 62 of 96 isolates were obtained using warm and cold enrichment, respectively. Thus, cold enrichment for 28 days resulted in a 29.2% (28 of 96) increase in recovery of L. monocytogenes from cheese and cheese factory samples. However, with the advent of improved selective media and methods, most investigators have concluded that

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cold enrichment offers no advantages over selective enrichment [82]. In addition, the lengthy incubation period necessary for cold enrichment makes this procedure impractical for routine regulatory analysis of foods that most often require quick reporting of results.

SELECTIVE ENRICHMENT AND PLATING AT 30 TO 37 °C The principle of enrichment at elevated temperatures (30 to 37°C) is based on selective inhibition of indigenous microflora through addition of inhibitory agents while at the same time allowing unhindered growth of Listeria. Given the many months required for cold enrichment, the scientific community soon became aware of the need for a shorter incubation period. In 1950, Gray et al. [75] isolated L. monocytogenes from contaminated material that was inoculated into nutrient broth containing 0.05% potassium tellurite and incubated at 37°C for 6 to 8 h before being plated on tryptose agar with or without 0.05% potassium tellurite. Even though subsequent studies showed both potassium tellurite-containing media to be partially inhibitory to Listeria [96,108,133,145], Gray and his colleagues can still be credited with introducing the first cold enrichment procedure and the first warm enrichment media for selective isolation of L. monocytogenes. Since 1950, various combinations of selective agents have been added to basal media (i.e., tryptose broth, ONB2, and tryptose phosphate broth) to obtain media suitable for selective enrichment of Listeria at 30 to 37°C. Mavrothalassitis [120] reported an optimum incubation temperature of 30°C for enrichment of L. monocytogenes from heavily contaminated samples. Results from at least two additional studies [41,130] also showed that laboratory cultures of L. monocytogenes, L. seeligeri, or L. ivanovii were more susceptible to commonly used Listeria selective agents (i.e., ceftazidime, cefotetan, laxamoxef, and fosfomycin) when incubated at 37 rather than 30°C. Hence, most Listeria enrichments are done at 30°C. Ryser and Marth [161] previously reviewed the wide range of media formulations developed for selective enrichment of L. monocytogenes from environmental, clinical, and food specimens.

SELECTIVE AGENTS Modest, nonspecific nutritional requirements of L. monocytogenes have led to difficulties in formulating media that enhance growth of this pathogen. Consequently, efforts have primarily focused on inhibition of the indigenous bacterial flora by taking advantage of the resistance of L. monocytogenes to various selective agents, including chemicals, antimicrobials, and dyes. The major advances that have contributed to present-day ability to isolate Listeria from heavily contaminated environments are shown in Table 7.1. Although many inhibitory agents have proven to be at least somewhat useful for selective isolation of L. monocytogenes from naturally and artificially contaminated biological specimens, others have demonstrated very little value when added to basal media, as previously reviewed by Ryser and Marth [161]. Throughout the following discussion of selective agents, one must keep in mind that formulating media that are selective and now differential for L. monocytogenes is not a straightforward process; many selective agents may partially or completely inhibit growth of this pathogen, particularly when the organism is sublethally injured [32,46,131,167]. Potassium Tellurite Many selective media, including the early formulation by Gray et al. [75], contain inhibitory substances that are now of questionable value. As previously described, in 1950, Gray et al. [75] examined the potential usefulness of potassium tellurite and sodium azide in Listeria-selective media. Sodium azide prevented growth of L. monocytogenes in tryptose broth, whereas potassium tellurite was quite selective for the pathogen. However, shortly after these findings were published, Olson et al. [133] observed that potassium tellurite prevented growth of numerous L. monocytogenes

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TABLE 7.1 Recognition of Selective Agents Useful in Isolation of Listeria Year

Compound

1950

Potassium tellurite

1960

Lithium chloride/phenylethanol

1966

Nalidixic acid

1971

Acriflavin(e)/trypaflavin(e)

1971

Polymyxin B

1986

Moxalactam

1988

Ceftazidime

Role in Selective Media Selective/differential for Listeria that reduces tellurite to tellurium, producing black colonies Amplification of Listeria in the presence of Gramnegative bacteria Inhibitory to Gram-negative bacteria through interference with DNA gyrase Inhibitory to Gram-positive cocci Prevents growth of Gramnegative rods and streptococci Broad spectrum; inhibitory to many Gram-positive and Gram-negative contaminants, including Staphylococcus, Proteus, and Pseudomonas Broad-spectrum cephalosporin antibiotic

Ref. 24,75,96,100,108,121,133,145,171

47,59,77,78,80,106,113,121,157,158,170

1,20,52,55,71,92,96,134,135,147,166

3,19,44,51,52,57,77,89,93,94,134,135, 145,146,148,149,152 21,43,47,113,134,152,168

86,106,124,134

10,114,117,130

strains. Other investigators [96,100,108,121,145] have substantiated these findings and discouraged the use of potassium tellurite as a selective agent. The advantage of adding potassium tellurite to selective media is that the resulting Listeria colonies appear black from reduction of potassium tellurite to tellurium. Unlike the typical black-yellowish and gray colonies produced by Gram-positive cocci, the marginal zone of Listeria colonies appears green when the organism is grown on media containing potassium tellurite and viewed with oblique illumination [164]. A modification of Vogel Johnson agar (MVJA) was evaluated by Buchanan et al. [24] for isolating Listeria from foods. Selective agents, including moxalactam, nalidixic acid, bacitracin, and potassium tellurite, permitted growth of Listeria while suppressing background contaminants. Furthermore, the ability to distinguish colonies readily was not predicated on the need for obliquely transmitted light. Buchanan et al. [27] also found that lithium chloride-phenylethanol-moxalactam agar (LPM) and MVJA generally gave comparable recovery of Listeria from naturally contaminated samples of fresh meat, cured meat, poultry, fish, and shellfish. Adding tellurite and mannitol to MVJA greatly aided in differentiating Listeria colonies from those formed by naturally occurring contaminants, including various species of enterococci and staphylococci. However, Smith and Archer [171] reported that potassium tellurite prevented repair of heat-injured L. monocytogenes. Lithium Chloride/Phenylethanol Using the combination of phenylethanol and lithium chloride, McBride and Girard [121] succeeded in amplifying numbers of L. monocytogenes in the presence of Gram-negative bacteria. The usefulness of phenylethanol and lithium chloride as Listeria-selective agents has since been

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confirmed by other investigators, resulting in the earlier widespread use and acceptance of McBride Listeria agar (MLA) as a plating medium for L. monocytogenes [38,59,77,78,80,106, 113,121,157,158,170]. A modification of MLA (omission of sheep blood and addition of cycloheximide as an antifungal agent) was once recommended by the FDA for analyzing food samples suspected of harboring Listeria [113,114]. Ryser and Marth [159,160] and Yousef and Marth [186] reported that increasing the lithium chloride concentration to 0.5% (0.05% lithium chloride in the original formulation [121]) increased selectivity of the medium without appreciably decreasing recovery of healthy Listeria [159,160,183]. Nalidixic Acid Beerens and Tahon-Castel [14] were first to report the usefulness of nalidixic acid in isolating L. monocytogenes from heavily contaminated pathological specimens. Increased isolation of Listeria using media containing nalidixic acid primarily resulted from inhibition of indigenous Gram-negative bacteria [71]. The benefits of adding nalidixic acid to otherwise noninhibitory media were soon confirmed in many laboratories [20,52,92,96,134,135,166]. After discovering the benefits of adding nalidixic acid to enrichment broth [14], Ralovich et al. [147] effectively used serum agar containing nalidixic acid to isolate L. monocytogenes from feces, organs, and other clinical specimens. Although the microbial background flora was largely inhibited on this medium, streptococci and other nalidixic acid-resistant organisms occasionally persisted. Nalidixic acid was eventually recognized as one of the most important selective agents, and it is now used alone or more commonly in combination with other selective agents for isolating L. monocytogenes from food and clinical specimens. Farber et al. [55] developed an improved Listeria-selective plating medium by combining the positive attributes of McBride Listeria agar and LPM agar. In their formula for Farber Listeria agar, oxolinic acid was substituted for nalidixic acid. Both agents function by interfering with the activity of DNA gyrase, an enzyme needed to maintain proper DNA structure and resealing of chromosomal nicks [71]. Trypaflavine/Acriflavine Despite successful use of nalidixic acid, Ralovich et al. [148,149] found that growth of certain Gram-positive cocci and Gram-negative rods in the presence of this selective agent complicated the isolation of Listeria. Such difficulties led to inclusion of trypaflavine, a known inhibitor of Gram-positive cocci, in media containing nalidixic acid. This medium soon became known as trypaflavine nalidixic acid serum agar (TNSA). The end result was the selective inhibition of virtually all other bacteria; growth of L. monocytogenes was only slightly decreased [19,133]. Following successful use of this medium in many European studies [19,93,134,135,148], Ralovich et al. [145] endorsed TNSA as the plating medium of choice for isolating L. monocytogenes from contaminated materials. Additional work revealed that contaminating organisms, predominantly streptococci, grew infrequently on clear media containing both antibiotics and were generally discernible from L. monocytogenes with the naked eye. In 1972, Seeliger [165] reported that combined use of acriflavine and nalidixic acid greatly suppressed Gram-negative organisms and fecal streptococci without apparently affecting recovery of L. monocytogenes. These findings were subsequently confirmed by Bockemühl et al. [20], who reported easy recovery of L. monocytogenes from enriched fecal samples using an agar medium that contained nalidixic acid and acridine dye. Confirmation of these findings in other European laboratories [52,57,77,94] led to widespread use of trypaflavine/nalidixic acid as Listeria-selective agents. In 1974, Hofer [89] proposed using a medium prepared from tryptose agar containing nalidixic acid, trypaflavine, and thallous acetate. Trypaflavine can be replaced by other acridine dyes, including xanthacridine, acriflavine, or proflavinehemisulfate [146]. According to Gregorio et al. [77], use of nalidixic acid together with

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acriflavine or trypaflavine gave rise to media that were equally inhibitory to background microflora, suggesting that similar results can be obtained by substituting acriflavine for trypaflavine. Based on results from European laboratories [44,51,93,146], a serum agar- or blood agar-based medium containing trypaflavine, acriflavine, and nalidixic acid appeared to be satisfactory for selective isolation of L. monocytogenes from samples containing a mixed microbial flora. In 1984, Rodriguez et al. [152] developed a blood agar medium containing acriflavine and nalidixic acid (Rodriguez isolation medium [RIM]) that was far superior to the earlier formulations of Ralovich et al. [147,149]. During the last decade, numerous media containing acriflavine and nalidixic acid with or without other antibiotics have been developed for selective isolation/enrichment of Listeria from food and environmental samples, including Merck Listeria agar [22,79], which is commercially available in Europe. Potassium Thiocyanate In 1961, Fuzi and Pillis [65] proposed a medium containing 0.35% potassium thiocyanate for selective enrichment of L. monocytogenes. Although it was reported useful by some researchers [52,107,166], others found that potassium thiocyanate inhibited L. monocytogenes [100,108,148]. Despite these reports, several studies demonstrated that an enrichment broth containing this selective agent in combination with nalidixic acid was useful in isolating L. monocytogenes from cabbage [78], milk [80,170], and other dairy products [102]. In 1972, Ralovich et al. [148] endorsed Levinthal’s broth and Holman's medium, which contain nalidixic acid and trypaflavine, for selective enrichment of Listeria. Results obtained by Slade and Collins-Thompson [170] demonstrated that growth of L. monocytogenes in ONB2 containing nalidixic acid and potassium thiocyanate can be improved by adding acriflavine. Thallous Acetate During the early 1950s, thallous acetate was employed as a selective agent for lactic acid bacteria; however, it was not until 1969 that Kramer and Jones [100] recommended the combined use of thallous acetate and nalidixic acid in Listeria-selective media. Three years later, Khan et al. [96] found that, unlike potassium tellurite, thallous acetate used alone or together with nalidixic acid did not adversely affect recovery of L. monocytogenes from biological specimens and silage samples. In 1979, Leighton [108] demonstrated that the combined use of thallous acetate and nalidixic acid completely suppressed growth of E. coli strains previously resistant to nalidixic acid. Greater inhibition of Gram-positive bacteria also occurred when both selective agents were used together rather than separately. Although Leighton [108] recommended a medium composed of tryptose phosphate broth, thallous acetate, and nalidixic acid for recovery of L. monocytogenes from mixed bacterial populations, thallous acetate (as well as potassium thiocyanate, potassium tellurite, and lithium chloride) altered the colonial morphology of L. monocytogenes from the smooth to rough form. In view of this experience, most currently used formulations of Listeria-selective media omit thallous acetate. Polymyxin B In 1971, Despierres [43] reported that the combination of polymyxin B and nalidixic acid was useful for recovering L. monocytogenes from feces and that these antibiotics prevented growth of many background organisms, including Enterococcus faecalis. That same year, Ortel [134] proposed another medium containing polymyxin B and bacitracin to isolate L. monocytogenes from stool samples. According to Bojsen-Møller [21], Gram-negative rods and enterococci failed to grow in tryptose phosphate broth containing polymyxin B, but growth of L. monocytogenes was relatively unaffected. After examining six different enrichment and isolation media, Rodriguez et al. [152] concluded that little if any benefit was gained by adding polymyxin B to media already containing nalidixic acid and acriflavine.

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Doyle and Schoeni [47] successfully isolated L. monocytogenes from milk and clinical and fecal samples after enrichment in a selective broth containing polymyxin B, acriflavine, and nalidixic acid that resembled isolation medium II developed by Rodriguez et al. [152]. Although the selective enrichment broth developed by Doyle and Schoeni gained some attention [113], the necessity for polymyxin B in this medium remains somewhat questionable. Siragusa and Johnson [168] successfully isolated L. monocytogenes from yogurt using a medium containing polymyxin B, nalidixic acid, and acriflavine. Their medium reportedly prevented growth of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, thus making it particularly suitable for isolating L. monocytogenes from certain fermented dairy products. Moxalactam Results from antibiotic susceptibility tests [134] led Lee and McClain [106] to add moxalactam (a broad-spectrum antibiotic that is inhibitory to many Gram-positive and Gram-negative bacteria, including Staphylococcus, Proteus, and Pseudomonas) to MLA containing 0.25% phenylethanol and 0.5% lithium chloride. The result was a highly selective medium for recovery of L. monocytogenes from raw beef and many other foods. This medium, LPM agar, is recommended by the USDA–FSIS for isolating L. monocytogenes from raw meat and poultry [124] and also has been incorporated into the current FDA procedure as a second selective plating medium [86]. Ceftazidime Bannerman and Bille [10] used Columbia agar base in combination with acriflavine and ceftazidime (AC agar), a broad-spectrum cephalosporin antibiotic, to isolate L. monocytogenes from cheese samples. AC agar was superior to FDA-modified McBride Listeria agar (MMLA) [114,117]; it recovered approximately 50% more L. monocytogenes isolates from soft cheese and cheese manufacturing environments than did FDA-MMLA. Except for a few enterococci, the combination of acriflavine and ceftazidime inhibited all other non-Listeria organisms, including yeasts and molds. However, van Netten et al. [130] reported that PALCAM agar, which contains polymyxin B and lithium chloride along with half or less the concentration of acriflavine and ceftazidime found in AC agar, was superior to the latter medium. After comparing 13 different plating media, these authors also concluded that media containing ceftazidime and 1.5% lithium chloride afforded more selectivity than did phenylethanol alone. However, increased selectivity results in decreased recovery of stressed or sublethally injured cells that are frequently present in foods.

SELECTIVE MEDIA FOR ENRICHMENT AND ISOLATION OF LISTERIA SELECTIVE ENRICHMENT MEDIA Frequent outbreaks of foodborne illness caused by L. monocytogenes in the recent past and the high mortality rate associated with listeriosis have highlighted the need for more sensitive, reliable, and rapid detection methods for the pathogen. The logical approach was to use some of the previously described enrichment broths containing selective agents and to incubate samples at an elevated temperature, generally 30°C. In response to numerous requests from the food industry, several enrichment schemes have been developed that include primary or secondary selective enrichments. An outbreak of listeriosis epidemiologically linked to consumption of pasteurized milk [63] led Hayes et al. [80] to develop a two-stage enrichment procedure for isolating L. monocytogenes from raw milk. Primary cold enrichment in ONB2 followed by secondary enrichment at 35°C in ONB2 containing potassium thiocyanate (KSCN) and nalidixic acid and plating on GBNA yielded the highest number of positive milk samples. No statistically significant difference in recovery of Listeria was observed using Stuart transport medium or selective enrichment broth containing

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potassium thiocyanate and nalidixic acid. Although 15 milk samples were positive when plated on GBNA medium as compared with 11 on MLA2 without blood, the difference was not statistically significant. The authors concluded that primary cold enrichment in ONB2 followed by secondary selective enrichment at 35°C and plating on GBNA medium were the most useful for identifying positive raw milk samples. Slade and Collins-Thompson [170] developed a somewhat shorter two-stage enrichment procedure to isolate Listeria from foods. Their method was tested using raw milk inoculated to contain approximately 100 L. monocytogenes CFU/mL. Results showed that tryptose broth was superior to ONB2 as a primary cold enrichment medium. In addition, diluting milk samples 1:10, rather than 1:5, increased the number of Listeria isolations on selective media. The more dilute samples probably maintained a higher pH (≥6) during cold enrichment as a result of fewer lactic acid bacteria and little lactose being present; this in turn led to faster growth and increased detection of Listeria on solid media. Original MLA without blood was the only medium tested that proved to be useful for plating primary cold enrichments because tryptose agar and trypaflavine nalidixic acid agar were typically overgrown by competing microflora. Favorable results were, however, obtained using tryptose agar after secondary enrichment at 37°C. Addition of acriflavine to thiocyanate nalidixic acid broth proved beneficial for recovery of L. monocytogenes. Thus, following 7 to 14 days of cold enrichment in tryptose broth, L. monocytogenes was most frequently isolated after plating samples enriched in thiocyanate nalidixic acid broth on MLA with blood or tryptose agar. A “shortened” enrichment procedure and a two-stage cold/selective enrichment procedure were developed in Canada by Farber et al. [54] for isolating Listeria spp. from raw milk. In the shortened enrichment procedure, milk samples underwent primary and secondary enrichment at 30°C as well as primary cold enrichment in two selective media (FDA enrichment broth and University of Vermont medium [UVM]). Although no single step within the procedure was completely satisfactory for isolating Listeria from raw milk, the two steps that were most helpful involved surface plating the primary FDA enrichment broth culture on MLA2 with blood after 1 day of incubation at 30°C, and surface plating the 30-day-old cold enriched FDA enrichment broth culture (initially incubated 7 days at 30°C) on MLA2 with blood. Collectively, these steps detected Listeria spp. in 31 of 51 (60.8%) positive raw milk samples. Although 11 isolations were made after 1 day but not 7 days of primary selective enrichment at 30°C, 6 isolations were only possible after 7 days of primary selective enrichment. Thus, incubating the primary selective enrichment at 30°C for 7 days before plating on MLA2 with blood markedly enhanced recovery of Listeria from raw milk. The two-stage cold/warm enrichment method, which was the second of two procedures developed by Farber et al. [54], also detected Listeria spp. in raw milk samples. Using this procedure, Listeria spp. were isolated from 12 samples that tested negative using the shortened enrichment procedure. Similarly, 10 samples that tested positive for Listeria spp. using the shortened enrichment procedure were negative with the two-stage cold/warm enrichment method. Thus, when used alone, neither procedure detected Listeria in all positive samples. Following cold enrichment, similar numbers of samples were positive for Listeria spp. after enrichment in FDA enrichment broth and UVM. However, eight raw milk samples were only positive after 2 weeks of cold enrichment as compared with three samples in which Listeria was only detected after 4 weeks of cold enrichment. These results are similar to those of Doyle and Schoeni [47], who also observed that Listeria spp. could be more readily isolated from raw milk and soft, surface-ripened cheese [48] during the first 2 weeks of cold enrichment. Food-associated outbreaks of listeriosis along with discovery of L. monocytogenes in many European varieties of soft- and smear-ripened cheese prompted two Swiss investigators, Bannerman and Bille [10], to develop a two-stage selective/cold enrichment procedure to recover Listeria spp. from cheese and dairy plant surfaces. Their isolation method is similar to the shortened enrichment

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procedure just described [54] with the exception that the secondary selective enrichment step has been eliminated and AC agar has been included as an additional selective plating medium. Using this method, Listeria spp. were isolated from 157 of 1,099 (14.3%) cheese and environmental samples. A total of 99 samples were positive for Listeria using both plating media. Following selective enrichment, 56 of 99 (57%) and 35 of 99 (35%) samples were positive after surfaceplating enrichment cultures on AC agar and FDA-MMLA, respectively. Increased selectivity of AC agar was presumably responsible for detection of approximately 50% more Listeria isolates as compared with FDA-MMLA. Important information concerning presence of Listeria spp. in food and environmental samples can be gained using the three procedures just described as well as procedures developed by Hayes et al. [80] and Slade and Collins-Thompson [170]; however, the need for cold enrichment in these procedures increased the length of analysis to 30 to 40 days. Hence, the time constraints of this method negate its use in any isolation procedure that is to be adopted by the food industry as a “standard” enrichment method. Rodriguez et al. [154] developed a complicated scheme to isolate Listeria from raw milk that more importantly paved the way for subsequent development of several widely used enrichment media, including UVM enrichment broth [33,45]. Their protocol included three noninhibitory collection (primary enrichment) media, three selective (secondary) enrichment media, and one selective plating medium, RIM III, all of which were previously described by Rodriguez et al. [152]. The three selective enrichment media used in this protocol contained nalidixic acid and trypan blue with or without polymyxin B; nalidixic acid and acriflavine were used as selective agents in the plating medium. Milk was added to all three collection media, with collection medium B streaked onto RIM III after 7 and 15 days of storage at 4°C. Collection medium A was incubated at 4°C for 24 h, subcultured in all three secondary enrichment media, which were incubated at 22°C until a color change occurred, and then samples were streaked onto plates of RIM II. A portion of collection medium A also was diluted in collection medium C, which was streaked on to RIM III following 7 and 15 days at 4°C. According to these authors, 11 L. monocytogenes isolates were obtained after primary cold enrichment, with collection medium C accounting for 9 of 11 isolations. Although results for collection medium C appear impressive, the increased number of isolations using this medium may have resulted from a more dilute sample: approximately 1:40 as compared with approximately 1:8 in collection media A and B. Under these conditions, collection medium C should have maintained a higher pH during cold enrichment because fewer lactic acid bacteria and less lactose were likely present, thereby enhancing the growth environment for L. monocytogenes. In contrast to cold enrichment, 49 L. monocytogenes isolates were obtained following secondary enrichment at 22°C with 16, 32, and 1 colonies originating from Rodriguez enrichment media 1, 2, and 3, respectively. Recovery of only one Listeria isolate using Rodriguez enrichment medium 3 is not surprising considering that collection medium A was diluted approximately 1:68 in collection medium C after only 24 h of enrichment at 4°C. Transfer of the culture after 24 h of cold enrichment provides little opportunity for appreciable growth of L. monocytogenes, so the organism was likely diluted out of the sample. Overall, primary cold enrichment of milk samples diluted approximately 1:8 followed by secondary enrichment in Rodriguez enrichment media 1 and 2 at 22°C and plating on an isolation medium containing nalidixic acid and acriflavine provided the best opportunity for detecting L. monocytogenes in raw milk. UVM Broth Selective media originally recommended by the FDA [114,117] and USDA–FSIS [123,124] for enrichment of food samples containing L. monocytogenes were modifications of media proposed by Ralovich et al. [149] and Rodriguez et al. [152] as modified by Donnelly and Baigent [45], respectively.

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Donnelly and Baigent [45] explored the use of several selective enrichment media to inhibit growth of raw milk contaminants and select for L. monocytogenes. The most successful medium for this application was a modification of Rodriguez enrichment medium III [152]. This medium, designated LEB by Donnelly and Baigent [45], consisted of proteose peptone (5.0 g/L), tryptone (5.0 g/L), Lab-Lemco powder (5.0 g/L), yeast extract (5.0 g/L), sodium chloride (20.0 g/L), disodium phosphate2-hydrate (12.0 g/L), potassium phosphate monobasic (1.35 g/L), esculin (1.0 g/L), nalidixic acid (40 mg/L), and acriflavine HCl (12 mg/L). McClain and Lee [123] modified this formula to contain 20 mg/L nalidixic acid, and this formulation was known as USDA LEB I. These authors further modified LEB I to contain 25 mg/L acriflavine and used this medium, LEB II, for secondary enrichment of meat and poultry samples. USDA–FSIS currently recommends use of UVM broth (LEB I) for primary enrichment of meat, poultry, egg, and environmental samples [33,91,178]. Fraser Broth Fraser broth [64] is a modification of USDA LEB II which contains lithium chloride (3.0 g/L) and ferric ammonium citrate (0.5 g/L). This medium reportedly was advantageous for detecting Listeria spp. in enriched food and environmental samples. Because Listeria will turn Fraser broth black from esculin hydrolysis within 48 h of incubation [23], this broth has now replaced USDA LEB II in the USDA protocol as the preferred secondary enrichment medium for meat, poultry, and environmental samples [91,178]. In 1986, Doyle and Schoeni [47] used the microaerophilic nature of L. monocytogenes in developing a shortened one-step enrichment procedure to isolate this organism from milk as well as fecal and biological specimens. In their protocol, the sample was placed inside an Erlenmeyer flask equipped with a side arm and then diluted 1:5 in Doyle and Schoeni selective enrichment broth (DSSEB). Following 24 h of incubation at 37°C in an atmosphere of 5% O2:10% CO2:85% N2, a portion of the sample was streaked onto plates of MLA (original formulation with blood), which were similarly incubated under microaerobic conditions. Using DSSEB, L. monocytogenes was consistently isolated from raw milk samples inoculated to contain 10 L. monocytogenes CFU/mL. In addition, about two and five times as many L. monocytogenes isolates were recovered from fecal and biological specimens, respectively, using DSSEB rather than cold enrichment and direct plating. Another enrichment procedure, which is partially based on microaerobic incubation, was developed by Skovgaard and Morgen [169] to isolate Listeria spp. from heavily contaminated samples, including feces, silage, minced meat, and poultry. In this two-step enrichment procedure, microaerobic incubation (24 h/30°C/95% air: 5% CO2) of the sample in USDA LEB I is followed by aerobic secondary selective enrichment in USDA LEB II, after which untreated and KOH-treated samples are surface plated on LPM agar. Using this isolation scheme, which, with the exception of microaerobic incubation, closely resembles the original USDA procedure, numerous fecal, silage, minced beef, and poultry samples were positive for Listeria spp., including L. monocytogenes. Based on these results, the authors concluded that their method was suitable for detecting Listeria in heavily contaminated materials, including samples of raw ground beef and poultry. Although both procedures just described decrease the Listeria detection time to approximately 3 days, incubating enrichment cultures under microaerobic conditions is particularly awkward and not feasible for large-scale testing programs. A large listeriosis outbreak in which coleslaw was implicated as the vehicle of infection prompted Hao et al. [78] to compare various media and methods to detect L. monocytogenes in cabbage. Preliminary results clearly demonstrated a need for some type of enrichment procedure before L. monocytogenes could be isolated from inoculated samples. After comparing results from various plating and enrichment media, these investigators proposed a two-step enrichment procedure for isolating L. monocytogenes from cabbage. A cold enrichment period of 14 or 30 days at 5°C in ONB2 or brain heart infusion broth (BHI) led to increased recovery of Listeria from cabbage

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following secondary enrichment (30°C/48 h) in FDA enrichment broth or ONB2 containing potassium thiocyanate and nalidixic acid. A comparison of nine selective plating media, with and without an additional 5 mg of Fe3+/L, led to the recommendation of modified Doyle/Schoeni selective agar II and MLA with glycine anhydride rather than glycine (MLA2) for isolating L. monocytogenes from cabbage. Both media contained 5% sheep blood, which was beneficial for picking Listeria-like colonies. As was true for the cold enrichment broths, several popular plating media, including FDA-MMLA and LPM agar, were not examined in this study. However, their efficacy in isolating L. monocytogenes from cabbage and other vegetables needs to be determined before recommending this procedure for use in routine analysis of such products. Despite repeated efforts toward developing an effective enrichment medium for recovery of L. monocytogenes, no one single selective enrichment broth has proven to be totally reliable for analysis of food products containing Listeria. Nevertheless, several enrichment broths have moved to the forefront, including the FDA enrichment broth [86], UVM broth [91,178], and Fraser broth [91], all of which are commercially available from BBL, Difco Laboratories, and other manufacturers. Truscott and McNab [174] developed a selective enrichment medium called Listeria test broth (LTB) as an alternative to UVM broth for detecting L. monocytogenes in meat products. After primary and/or secondary enrichment of 50 frozen ground beef samples in both enrichment broths, L. monocytogenes was detected in 19 of 50 (38%) and 16 of 50 (32%) samples using UVM and LTB, respectively. Although Listeria recovery rates for these two broths are not appreciably different, neither medium alone was able to detect the pathogen in all 29 samples that were positive. In addition, LPALCAMY broth, which was developed by van Netten et al. [130], has shown superior results to USDA LEBs I and II as well as the tryptose broth-based antibiotic medium of Beckers et al. [13] for detecting L. monocytogenes in naturally contaminated cheese, minced meat, fermented sausage, raw chicken, and mushrooms. However, given wide variations in the type and the number of naturally occurring microbial contaminants in the food supply, development of a single enrichment broth for truly optimal recovery of Listeria from all types of food appears improbable.

ISOLATION MEDIA McBride Listeria Agar (MLA) MLA was the first widely used plating medium for selective isolation of L. monocytogenes. This medium, introduced by McBride and Girard [121] in 1960, is prepared from phenylethanol agar to which lithium chloride, glycine, and sheep blood are added. At least seven subsequent changes in the original formulation of MLA have led to considerable confusion as to the exact composition of this medium. Ironically, the first reported modification of MLA by Bearns and Girard [12] dates back to 1959, nearly 1 year before the original formulation appeared in the literature [121]. This medium, named modified McBride medium (MLA2) by the authors and known today as one of several modified MLAs, is similar to the original formulation except that sheep blood is omitted and glycine anhydride is substituted for glycine [106]. In most instances, MLA2 was more Listeria selective than nalidixic acid agar [59,134], acriflavine nalidixic acid agar [170], or acridine nalidixic acid agar [59]. The selectivity of MLA2 can be further improved, without affecting recovery of Listeria, by increasing the lithium chloride content to 0.5%. With the further addition of sheep blood, this medium became partially differential and hence was better suited than MLA2 for recovering L. monocytogenes from brick [160], feta [137], and blue cheese [138], as well as cold-pack cheese food [159]. Following an earlier report in which glycine was found to partially inhibit L. monocytogenes [106], many individuals began to replace glycine with glycine anhydride, which is far less inhibitory to Listeria. Nevertheless, two widely used formulations of the original MLA containing

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glycine have been commercially available since 1985 from Difco Laboratories and BBL (now Becton Dickinson). Although addition of blood provides one means of identifying possible L. monocytogenes colonies (virtually all are at least somewhat β-hemolytic) and enhances growth of the pathogen in certain B vitamin- and/or amino acid-deficient media, many workers preferred to omit blood from the various formulations of MLA and examine the plates under oblique illumination for blue to bluish green Listeria-like colonies. In 1987, Lovett et al. [117] added cycloheximide to blood-free MLA2 and named this particularly useful medium FDA-modified McBride Listeria agar (FDA-MMLA). Although one earlier study claimed that TNSA was superior to MLA2, subsequent data indicated that FDA-MMLA [113,114,115,117] and MLA2 [69,78,80,117, 149,170], which contain glycine anhydride, were the MLA formulations of choice for isolating Listeria spp. from foods, particularly dairy, vegetable, and seafood products. The FDA formulation previously served as one of two plating media (the other is LPM agar) in the FDA procedure [116]. LPM Agar In 1986, Lee and McClain [106] added 4.5 g of lithium chloride and 20 mg of moxalactam to MLA2 and named their new medium lithium chloride-phenylethanol-moxalactam (LPM) agar. Although this selective medium (commercially available from Becton Dickinson is particularly well suited for isolating Listeria from raw meat and poultry—as evidenced by its inclusion as the medium of choice in an earlier version of the USDA procedure—LPM agar has since been replaced by modified Oxford agar [33], which produces black Listeria colonies, each with a black halo following 24 h of incubation. However, LPM plus esculin and ferric iron is still used as one of the selective isolation agars in the FDA procedure [88]. Oxford Agar (OXA) and Modified Oxford Agar (MOX) In 1989, Curtis et al. [42] developed an agar medium that eliminated the need for oblique illumination. Their medium, Oxford agar (OXA), was prepared from Columbia agar base to which several selective agents, including colistin sulfate (20 mg/L), fosfomycin (10 mg/L), cefotetan (2 mg/L), cycloheximide (400 mg/L), lithium chloride (15 g/L), and acriflavine (5 mg/L), were added. Esculin and ferric ammonium citrate also were added as differential agents to produce black Listeria colonies from esculin hydrolysis. This medium was slightly modified by McClain and Lee by incorporating moxalactam; this new medium was designated modified Oxford agar (MOX) [33]. In May 1989, the USDA–FSIS procedure was changed to incorporate MOX as the recommended plating medium. Late in 1990, the FDA modified its procedure by replacing FDAMMLA with Oxford agar (OXA). In the present version of the FDA method [88], one of the following selective media must now be used: PALCAM, OXA, MOX, or LPM fortified with esculin and Fe3+. PALCAM Agar In 1988, van Netten et al. [129] reported that RAPAMY agar, a modification of TNSA developed by Ralovich et al. [149] that includes acriflavine, phenylethanol, esculin, mannitol, and egg yolk emulsion, was suitable for enumerating Listeria spp. Virtually identical populations were observed when overnight broth cultures of L. monocytogenes, L. seeligeri, and L. ivanovii were surfaceplated on RAPAMY and nonselective agar; growth of all non-Listeria organisms tested, except Enterococcus faecalis and Enterococcus faecium, was completely inhibited on the selective medium. Like OXA [42], RAPAMY agar also produced distinctive black Listeria colonies surrounded by a dense black halo from esculin hydrolysis. Although such characteristic colonies were present

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against a deep red background (inability to utilize mannitol) on RAPAMY agar, E. faecalis and E. faecium generally produced colonies with blue-green halos. Although attempts to eliminate growth of these two species of enterococci by adding cefoxitin (moxalactam) to this medium failed, results suggested that RAPAMY agar could be used to quantify Listeria spp. in thermally processed and dried foods with total aerobic plate counts of ≤106 CFU/g and Enterococcus counts of ≤102 CFU/g. However, as might be expected, high populations of enterococci severely hampered detection of Listeria spp. in chicken, minced meat, and mold-ripened cheese. Further attempts by van Netten et al. [128] to eliminate growth of enterococci by adding fosfomycin (20 mg/L) to RAPAMY agar met with only limited success. Addition of lithium chloride (1.5%) to RAPAMY agar inhibited many Listeria spp.; however, an improved selective and differential medium was obtained by adding lithium chloride to RAPAMY agar and omitting nalidixic acid. The resultant medium was named ALPAMY agar because it contains acriflavine, lithium chloride, phenylethanol, esculin, mannitol, and egg yolk emulsion agar. In a study with pure cultures, ALPAMY agar allowed uninhibited growth of all 10 L. monocytogenes strains tested but completely prevented growth of single strains of L. seeligeri and L. ivanovii. Selectivity tests showed that ALPAMY agar supported growth of only 2 of 41 non-Listeria organisms—one strain each of Staphylococcus aureus and Micrococcus spp., both of which were readily differentiated from Listeria colonies. Subsequent studies indicate that ALPAMY agar is far superior to RAPAMY agar for detecting Listeria in raw milk and soft cheeses manufactured from raw milk, as well as in raw vegetables and chicken. This medium is the forerunner to PALCAM agar [130], which contains polymyxin B and lithium chloride along with half or less the concentration of acriflavine and ceftazidime found in AC agar. It is recommended that PALCAM agar plates be incubated for 40 to 48 h at 30°C under microaerobic conditions (5% oxygen, 7.5% carbon dioxide, 7.5% hydrogen, and 80% nitrogen). This medium, along with L-PALCAMY enrichment broth, is the basis for the Netherlands Government Food Inspection Service (NGFIS) method for Listeria detection and isolation. Other Selective Plating Media Interest in foodborne listeriosis during the 1980s led to development of many additional Listeriaselective media for examining milk and dairy products. In 1984, Martin et al. [119] developed gum base nalidixic acid medium (GBNA), a synthetic agar-free solid medium superior to the MMLA of Bearns and Girard [12] for isolating L. monocytogenes from raw milk [80]. Bailey et al. [8] also found that a modified version of this medium containing lithium chloride and moxalactam was suitable for isolating L. monocytogenes from raw chicken. A selective agar medium [78] based on the enrichment broth of Doyle and Schoeni [47], from which acriflavine was omitted and Fe3+ was added, compared favorably with the original formulation of MLA [121]. Supplementation of selective [78] and nonselective [38] media with Fe3+ enhances growth of L. monocytogenes and may be beneficial for isolating sublethally injured cells from food samples containing a mixed microbial flora. Attempts to isolate L. monocytogenes from food products have focused on enhancing the selectivity of currently available blood-free plating media, as well as development of media that incorporate differential agents other than blood to aid microbiologists in differentiating Listeria and L. monocytogenes colonies in mixed cultures. In 1987, Buchanan et al. [24] found the combination of moxalactam, nalidixic acid, and bacitracin to be effective in allowing growth of Listeria spp. while preventing growth of most other foodborne organisms, including micrococci and streptococci. These selective agents were used to formulate MVJ on which L. monocytogenes colonies appear entirely black (reduction of tellurite) on a red background (due to the microbe’s inability to use mannitol). Thus, suspect Listeria colonies could be readily identified on MVJ without using oblique illumination. Adding the same three selective agents to the MMLA of Bearns and Girard [12] resulted in Agricultural Research Service-modified McBride Listeria agar (ARS-MMLA) which could be used in conjunction with oblique lighting to quantitate Listeria in a wide range of dairy and meat products.

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In a subsequent study, Buchanan et al. [26] found that MVJ was slightly superior to ARS-MMLA for recovery of L. monocytogenes from inoculated samples of milk, dairy products, meat, and coleslaw. Although ARS-MMLA was more selective than MVJ, the black Listeria-like colonies that appeared on MVJ were more readily discernible. Initial comparisons of ARS-MMLA and MVJ with LPM agar indicated that both of the new media functioned well. In a follow-up study, Buchanan et al. [25] assessed the ability of MVJ and LPM Agar to detect Listeria in retail samples of raw meat, fish, and shellfish. Listeria populations were generally too low to be detected by direct plating on either medium. However, using USDA Listeria enrichment broth I (USDA LEB I) in a three-tube/24-h most probable number (MPN) method, comparable isolation rates were obtained for MVJ and LPM agar. The differential capability of MVJ was again extremely useful in selecting presumptive Listeria colonies. Comparative Evaluation of Direct Plating Media for Recovery of Listeria from Foods The need for reliable media in routine food analysis precipitated several studies to identify the most suitable direct plating media. Rijpens and Herman [151] conducted a study to compare selective and nonselective primary enrichments for detection of L. monocytogenes in cheese. A completely selective enrichment procedure was compared with two partially nonselective protocols. After enrichment for approximately 48 h, the enrichment media were streaked on selective agars and presumptive Listeria colonies were confirmed using PCR. In some instances, PCR was also done directly on the enrichment broth. The conventional, completely selective enrichment procedure was not always the best choice to detect stressed L. monocytogenes in cheeses. The methods that incorporated a nonselective enrichment step gave better results than the completely selective method. However, for mold-ripened soft cheeses, best results were obtained using the completely selective enrichment procedure. In another study by Johansson et al. [90], enrichment in half-Fraser broth for 24 h at 30°C, followed by plating onto L. monocytogenes blood agar (LMBA) and PALCAM medium combined with additional streaking proved to be the most rapid and specific method to detect indigenous L. monocytogenes populations from soft mold-ripened cheese in comparison with standard methods. With a high sensitivity (93%) and a low detection limit (1 to 10 CFU/25 g–1), this procedure provided negative and presumptive positive results within 2 to 3 days. Differences among LMBA, PALCAM, and Oxford medium were highly significant (at 99% significance level). Overall, plating on LMBA after standard enrichment protocols gave the best results. An improvement in detection was also obtained by modifying the confirmation procedure. A loopful of culture (an additional streak) from PALCAM or Oxford medium was streaked on a nonselective medium in addition to streaking only separate colonies as specified in the standards. A year-long survey of two Northern Ireland milk-processing plants for L. monocytogenes was carried out by Kells and Gilmour [95]. Sample sites included the milk-processing environment (walls, floors, drains, and steps), processing equipment, and raw and pasteurized milk. The FDA Listeria-selective enrichment procedure was used to process samples and an additional agar medium, LMBA, was utilized as part of the isolation procedure to compare its performance to that of the recommended Oxford and PALCAM agars. LMBA was able to isolate L. monocytogenes from 94.1% of sites compared to isolation rates of 76.5 and 79.4% using Oxford and PALCAM agars, respectively. Duarte et al. [50] examined four secondary enrichment protocols (conventional methods: UVM II, Fraser 24 h and Fraser 48 h; and an impedimetric method: Listeria electrical detection medium) for their ability to detect Listeria spp. and L. monocytogenes in fish and environmental samples collected along the processing chain of cold-smoked fish. From all methods, Listeria spp. and L. monocytogenes were present respectively in 56 and 34 of the 315 samples analyzed. Fraser broth incubated for 48 h gave the fewest false-negative Listeria spp. results (4/56; [7.1%]), but concurrently

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only 15/34 (44.1%) samples were correctly identified as containing L. monocytogenes. Listeria electrical detection (LED) medium detected only 36/56 (64.3%) Listeria spp.-positive samples. Despite this lower isolation rate, LED identified 20/34 (58.8%) L. monocytogenes-positive samples correctly and gave fewer false positive results. The overall conclusion of these authors, similar to those of many others, was that more than one isolation method is needed to estimate L. monocytogenes contamination rates accurately. Golden et al. [68], Hao et al. [78], and Cassiday et al. [34] collectively compared 20 selective plating media for their ability to recover uninjured cells of L. monocytogenes from samples of pasteurized milk, Brie cheese, ice cream mix, raw cabbage, dry-cured/country-cured ham, and/or raw oysters inoculated to contain approximately 102, 104, and 106 L. monocytogenes colony-forming units per gram or milliliter. Gum base nalidixic acid tryptose soya medium (GBNTSM), MLA2, FDA-MMLA, and modified Despierres agar (MDA) were consistently superior to nine other media used by Golden et al. [68] for enumerating all three inoculum levels of Listeria in samples of pasteurized milk and ice cream mix. Ability to recover low levels of Listeria from both products was facilitated by the lack of significant levels of non-Listeria contaminants. Five of fourteen plating media used in this study failed to recover L. monocytogenes from inoculated samples of pasteurized milk as well as Brie cheese and were therefore omitted for analysis of ice cream and raw cabbage. Examination of Brie cheese containing approximately 102 and 104 L. monocytogenes CFU/g indicated that none of the nine remaining direct plating media was sufficiently selective to prevent overgrowth of Listeria by molds, yeasts, and Gram-positive cocci. Despite these inherent difficulties in detecting small numbers of Listeria, modified Rodriguez isolation medium III (MRIM III), MLA2, FDA-MMLA, and MDA were judged to be satisfactory when Brie cheese contained ≥106 Listeria CFU/g. However, subsequent results from the same laboratory [35] indicate that LPM agar was superior to these four media for isolating L. monocytogenes from Brie cheese. With raw cabbage, enumeration of Listeria was a problem only at the lowest inoculum level where large populations of microbial contaminants (i.e., Gram-positive and Gram-negative rods as well as Gram-positive cocci) typically interfered with recovery. At the two higher inoculum levels, L. monocytogenes was readily quantitated by direct plating on MDA, GBNTSM, and MLA2. However, this same investigative team [35] later obtained even better results using LPM agar. One year earlier, Hao et al. [78] successfully recovered L. monocytogenes from inoculated samples of cabbage using GBNA, Doyle and Schoeni selective enrichment agar (DSSEA), DSSEA + ferric citrate, DSSEA + acriflavine + ferric citrate, thiocyanate nalidixic acid agar (TNAA) + glucose + ferric citrate, and MLA2, but concluded that DSSEA + acriflavine + ferric citrate and MLA2 outperformed the other media tested. When results from the previous three studies are combined, LPM agar, GBNTSM, MLA2, FDA-MMLA, and MDA generally emerged as the plating media of choice for detecting uninjured Listeria in dairy and vegetable products. Overall, these findings agree with those of at least four other studies [66,84,102,110] in which LPM agar outperformed other popular plating media, including FDA-MMLA, RIM III, and/or MVJ for recovery of L. monocytogenes from raw milk, ice cream, yogurt, soft cheese, and/or vegetables inoculated with the pathogen. In addition, Rodriguez et al. [153] found that RIM III containing 6 rather than 12 g of acriflavine hydrochloride was superior to the original formulation of MLA for isolating L. monocytogenes from artificially contaminated raw milk and hard cheese. Although the best media for recovering Listeria from dairy products and vegetables remain to be defined, OXA, MOX, LPM, and PALCAM agar appear to be the present plating media of choice in the United States for selective isolation of Listeria from such products as evidenced by their inclusion in the FDA and USDA procedures [83,86,91,114,115]. Given the inherent differences that exist between the natural microflora found in various foods, one can easily surmise that Listeria-selective plating media best suited for dairy products and vegetables might be somewhat less than ideal for analysis of meat, poultry, and seafood. Consequently, Cassiday et al. [34] evaluated 10 selective plating media for their ability to enumerate

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L. monocytogenes in artificially contaminated dry- and country-cured ham as well as raw oysters. According to their results, MDA, FDA-MMLA, and LPM agar recovered approximately equal numbers of uninjured Listeria from dry-cured ham. However, ease in differentiating L. monocytogenes colonies from those formed by background contaminants led these authors to recommend LPM agar for analysis of dry-cured ham. Not surprisingly, LPM agar also was equal or superior to three other plating media (i.e., MRIM III, MVJ, and UVM) deemed acceptable for isolating Listeria from country-cured ham. Unlike both types of ham, high populations of indigenous microflora in raw oysters greatly complicated detection of Listeria on virtually all 10 plating media. Although MRIM III and MVJ supported less growth of Listeria than other marginally acceptable plating media (including MLA2, FDAMMLA, and GBNTSM), MRIM III and MVJ were somewhat more reliable for differentiating L. monocytogenes from background contaminants. Therefore, these authors hesitantly recommended MRIM III and MVJ for examination of raw oysters. Several less extensive studies also have dealt with the ability of various plating media to recover Listeria from meat, poultry, and seafood. According to a 1988 report by Loessner et al. [110], recognition of L. monocytogenes in inoculated samples of raw ground beef and scallops was only possible using LPM agar. Among the three other plating media tested, RIM III and the original formulation of MLA proved to be insufficiently selective, whereas MVJ was inhibitory to the L. monocytogenes strain tested. Unlike these findings, Garayzabel and Genigeorgis [66] indicated that LPM agar and RIM III were acceptable for detecting Listeria in raw meat and both media were superior to FDA-MMLA. Bailey et al. [8] found that LPM agar and GBNA fortified with lithium chloride and moxalactam were superior to unfortified GBNA and MLA for recovering L. monocytogenes as well as other Listeria spp. from naturally contaminated raw poultry. Incubation Conditions Most plating media used to isolate Listeria are normally incubated aerobically at 30 to 37°C. Plates containing popular selective media such as LPM agar or MOX agar normally are incubated for 48 h; plates containing pure or near-pure cultures of Listeria on nonselective media can generally be examined after 24 h. Because growth of L. monocytogenes is reportedly enhanced under conditions of reduced oxygen [164], inoculated plates [47,128,129,156–158,160] as well as selective enrichment broths [38] have been incubated under microaerobic conditions (5% O2:10% CO2:85% N2). Microaerobic conditions are especially recommended when using PALCAM agar.

OBLIQUE ILLUMINATION Except for plating media that contain esculin, xylose, mannitol, or other differential agents, most formulations of Listeria-selective plating media can be classified into one of two categories based on presence or absence of blood. Recognition of Listeria-like colonies on blood-free media such as MMLA, TNSA, and GBNA is greatly facilitated when colonies are observed under oblique illumination with a binocular scanning microscope. When the Henry technique [85], in which plates are examined under obliquely transmitted white light at an angle of 45° (Figure 7.1), is used, Listeria colonies are small, round, finely textured, bluish green to bluish gray with an entire margin. In 1984, Martin et al. [119] compared the appearance of L. monocytogenes on nalidixic acid agar and tryptone soya gum base nalidixic acid medium and found that the uniformly transparent nature of the gum-base medium greatly enhanced the bluish-green color of Listeria colonies when observed under oblique illumination, as described by Henry [85]. Noting that the angle of transmission in the Henry method is 135°, Lachica [101] found that the bluish-green hue of Listeria colonies was more easily observed if plates were viewed from the backside at an angle of 45° with a 5× magnification hand lens while colonies were directly illuminated with a high-intensity beam of light that traveled perpendicular to the bench surface (Figure 7.2). This latter method has

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FIGURE 7.1 Oblique illumination technique developed by Henry. Angles of reflected light (β) and transillumination (α) equal 45 and 135°, respectively. (From Henry, B. S. 1933. J. Infect. Dis. 52:374–402.)

eliminated many of the problems (i.e., reproducibility and convenience) associated with the classical technique developed by Henry [85] more than 70 years ago. Given enough experience, either of these two lighting techniques can be used easily to differentiate probable Listeria colonies from background organisms, even on heavily contaminated plates. However, these procedures are time consuming and not readily adaptable for routine use in large testing laboratories. β-Hemolysis Addition of blood to solid media also can be used to differentiate Listeria, including L. monocytogenes, from other microorganisms. When grown on media containing blood, such as MLA, L. monocytogenes colonies are typically surrounded by a narrow zone of β-hemolysis. In some instances, β-hemolytic activity is so weak that the clearing zone cannot be observed until the colony is gently removed from the agar surface.

FIGURE 7.2 Modified Henry technique developed by Lachica [101]. Angle of transillumination (α) equals 135°. (From Lachica, R. V. 1989. Annu. Meeting, Soc. Ind. Microbiol., Seattle, Washington, August 13–18, Abstr. P-44.)

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In 1989, Blanco et al. [18] proposed overlaying previously inoculated plates of blood-free Listeria selective agar with a thin layer of blood agar so that the β-hemolytic activity associated with pathogenic Listeria could be directly observed after reincubation. According to these authors, hemolysis was more readily observed using this procedure than when blood was incorporated into plating media before incubation. However, further work using highly contaminated samples such as raw milk showed that the success of this procedure primarily depended on selectivity of the initial plating medium, with highly selective media yielding the best results.

OFFICIAL METHODS

FOR ISOLATING

L.

MONOCYTOGENES FROM

FOOD

Heightened worldwide interest in foodborne listeriosis coupled with the advent of mandatory HACCP programs for meat and seafood products in the United States has led to development of more reliable commercial screening methods for Listeria. Two protocols developed in the United States by the FDA and USDA–FSIS have emerged as “standard methods” to isolate L. monocytogenes from dairy foods, seafoods, vegetables and meat, poultry, and egg products. Despite widespread use of these methods in the United States, Canada, and Western Europe, both procedures are still plagued with difficulties that include the inability to isolate Listeria from all positive samples as well as difficulties in recovering sublethally injured cells. In response to these concerns, the USDA–FSIS and FDA protocols have been modified to enhance their ability to recover injured Listeria. Working in cooperation with the International Dairy Federation (IDF), other official European agencies have developed somewhat similar protocols partially based on current FDA methodology. In this section, positive and negative aspects of the most widely used Listeria testing protocols will be discussed, along with identification of some of the most critical steps involved in isolating L. monocytogenes from different foods. FDA Method The FDA method, originally developed by Lovett et al. [114,117], is the most frequently used procedure in the United States for detecting and enumerating L. monocytogenes in milk, milk products (particularly ice cream and cheese), seafood, and vegetables. The original protocol [114] has been modified several times since it was developed [86,88,114,117,177]. The most current standard FDA methodology [88] and permitted alternative rapid methodologies recommended to detect and isolate L. monocytogenes from foods are as follows. Presumptive contaminated food lots are sampled. Generally, subsamples are composited, if required, according to FDA field laboratory instructions. Analytical portions (25 g) are preenriched for Listeria species at 30°C for 4 h in buffered Listeria enrichment broth (BLEB M52), equivalent [87] to AOAC/IDF dairy products enrichment broth [5,175] base containing sodium pyruvate. Four hours after nonselective incubation, the selective agents (acriflavin, 10 mg/L; sodium nalidixate, 40 mg/L; optional antifungal, e.g., cycloheximide 50 mg/L) are added. Incubation for selective enrichment is continued at 30°C for a total of 48 h. The enrichment culture is streaked at 24 and 48 h on one of the prescribed differential selective agars to isolate Listeria species or L. monocytogenes. Surveillance enumeration of L. monocytogenes levels in contaminated food is now required for regulatory samples that test positive for the pathogen [88]. Detection may be done first and if contamination is detected, a reserve sample portion can be tested for numbers. This is probably the preferable method because, generally, only a small percentage of samples can be expected to be positive and most often at low levels approaching 1 CFU/25 g. However, the option of combining regulatory detection and enumeration is permitted. Enumeration of L. monocytogenes in positive samples is performed on reserve samples by colony count on L. monocytogenes differential selective agar in conjunction with MPN enumeration using selective enrichment in BLEB with subsequent plating on ALOA (a diagnostic, chromogenic

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isolation medium) [179a] or BCM [50,167] differential selective agar. Most samples are likely to be negative for Listeria and most positive samples will only contain a few colony-forming units per 25 g. Hence, it is efficient to delay enumeration of reserve samples until the Listeria detection stage is completed. Nevertheless, it may sometimes be more convenient to detect and enumerate simultaneously. To accomplish this, the enrichment homogenate is prepared as described earlier and 0.1 mL is immediately spread on ALOA, BCM, or an equivalent L. monocytogenes selective agar. Plates are incubated at 35°C for 24 to 48 h. The combined minimal method will allow the cell number of presumptive L. monocytogenes to be categorized as <0.04 CFU/g, 0.04 to 100 CFU/g, 100 to 25,000 CFU/g, or >25,000 CFU/g. More replica plates and more decimal dilutions in trypticase soy broth with 0.6% yeast extract (TSBye M157) are optional to obtain more precise enumeration. Five representative colonies are tested for ability to ferment L-rhamnose by the conventional fermentation method, by the BCM rhamnose confirmatory agar, or by a rapid L. monocytogenes identification kit to rule out definitively the uncommon occurrence of L. ivanovii in foods. Alternatively, prescribed rapid detection kits with their respective enrichment media may be conditionally used to screen for presence of Listeria contaminants. Putative Listeria isolates on selective agars from standard or screen-positive enrichments are purified on nonselective agar and confirmed by conventional identification tests or by a battery of such tests in kit form. Isolates may be rapidly confirmed as L. monocytogenes (or not) by using specific test kits. Subtyping of L. monocytogenes isolates is optional except for FDA isolates, which must be typed serologically, and strain typed by pulsed-field gel electrophoresis (PFGE) and ribotyping. Nonobligatory pathogenicity testing of L. monocytogenes isolates is described. The major changes in the revised FDA methodology (Figure 7.3) include: Certain prescribed rapid detection kits and their enrichments are now authorized screening alternatives to the standard selective enrichment. It is now necessary to use only one instead of two of the several prescribed selective isolation agars (Oxford agar, PALCAM, LPM plus esculin and ferric iron, MOX). Oxford agar is still the preferred standard selective isolation medium. MOX has been added to the list of prescribed selective agars and LPM without added esculin and ferric iron has been removed. Use of the new chromogenic differential selective agars, like BCM, ALOA, CHROMagar Listeria, and Rapid L’mono, is encouraged as long as it is in parallel with one of the prescribed selective agars. The new agar media differentiate L. monocytogenes and L. ivanovii colonies from those of other Listeria spp. and will greatly facilitate choosing L. monocytogenes colonies when colonies of more than one species are present on a plate. The Henry illumination technique is de-emphasized because only differential selective isolation agars are prescribed. The current enrichment medium, which resembles the step-1 enrichment of the internationally harmonized method proposed by Asperger et al. [6], is basically unchanged. However, pimaricin (natamycin), a much less toxic compound than cycloheximide, is introduced as the alternative antifungal compound in the Listeria enrichment medium. If L. monocytogenes is detected in a food sample, enumeration of the level of contamination in the food is required. The isolation procedure involves streaking BLEB culture onto one of the following esculincontaining selective isolation agars: OXA or PALCAM or MOX or LPM fortified with esculin and Fe3+. These esculin-containing media are listed in order of preferred use, subject to their availability. OXA, PALCAM, or MOX plates are incubated at 35°C for 24 to 48 h and fortified LPM plates at 30°C for 24 to 48 h. It is strongly recommended that one of the L. monocytogenes–L. ivanovii differential selective agars, such as BCM, ALOA, RapidL’mono, or CHROMagar Listeria be streaked at 48 h (optionally at 24 h) in addition to the chosen esculin-containing selective agar. This will reduce the problem of masking of L. monocytogenes by L. innocua.

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Add 25 g or 25 mL sample to 225 mL BLEB

Stomach or blend

Screening with rapid detection kits

Incubate 24–48 h at 35°C

Spread 0.1 mL on ALOA M10a, BCM M17a or an equivalent L. monocytogenes selective agar, and if enumeration is required, more replica plates and more decimal dilutions in TSBye

Streak onto

OXA/MOX and LPM (with or w/o Esculin & Fe3+) or PALCAM

OXA/MOX 35° C 24–48 h

LPM 30° C 24–48 h

PALCAM 35° C 24–48 h

Examine Listeria-like colonies

FIGURE 7.3 FDA procedure for isolating L. monocytogenes from foods. (From Hitchins, A. D. 2003. In Food and Drug Administration bacteriological analytical manual, 8th ed. AOAC International, pp. 10.01–10.13.)

BCM has been collaboratively validated by FDA [87]. An ISO TC34 SC9 comparative validation showed that all the media (and a selective blood agar—LMBA, Sifin, Germany) inhibited Listeria competitors more or less equally well. ALOA was preferred only because its formulation is public. Another differential selective medium, chromogenic Listeria agar (M40b) is now available. Listeria colonies are black with a black halo on esculin-containing media. Certain other bacteria can form weakly brownish-black colonies, but color development takes longer than 2 days. Five or more typical colonies are transferred from OXA and PALCAM or modified LPM or MOX to TSAye, streaking for purity and typical isolated colonies. If BCM plates are streaked as recommended previously and blue colonies are observed, they are presumptive L. monocytogenes colonies because L. ivanovii is not often reported in foods. L. monocytogenes and L. ivanovii colonies on ALOA are blue and have a zone of lipolysis around them. Purification on TSAye is a mandatory step in the conventional analysis because isolated colonies on selective agar media may still be in contact with an invisible weak background of partially inhibited competitors. At least five isolates are necessary because more than one species of Listeria may be isolated from the same sample. BCM and ALOA plates are used to help in reducing the number of colonies that need to be picked. L. monocytogenes and L. ivanovii can be distinguished using a commercial confirmatory medium (Biosynth International, Inc.) or by conventional

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TABLE 7.2 Serotypes of Listeria Species L. monocytogenes L. L. L. L. a

ivanovii innocua welshimeri seeligeri

1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b 4c, 4d, 4e, “7” 5 4ab, 6a, 6b, Una 6a, 6b 1/2b, 4c, 4d, 6b, Un

Un = undefined.

Source: von Koenig, C. H. et al. 1983. Infect. Immun. 40:1170–1177.

rhamnose/xylose fermentation broths or agars. TSAye plates are incubated at 30°C for 24 to 48 h. The plates may be incubated at 35°C if colonies will not be used for wet-mount motility observations (one of the many classical identification tests). For the approved rapid methods [88], the selective isolation agar recommended by the manufacturer must be used but auxiliary use of the new L. monocytogenes–L. ivanovii differential agars is also recommended. Isolates should be typed serologically and genetically. For serological typing, commercial sera are used to characterize isolates as type 1 or 4 or not type 1 or 4 (types 3, 5, 6, etc.) [88]. Table 7.2 exhibits the serological relationships of Listeria spp. Most L. monocytogenes isolates obtained from patients and the environment are type 1 or 4. More than 90% of L. monocytogenes isolates can be serotyped with commercially available sera. All nonpathogenic species, except L. welshimeri, share one or more somatic antigens with L. monocytogenes. Serotyping alone without thorough characterization, therefore, is not adequate for identification of L. monocytogenes. The BCM L. monocytogenes detection system (LMDS), mentioned earlier [50,167], consists of a selective pre-enrichment broth (LMPEB), selective enrichment broth (LMSEB), selective/ differential plating medium (LMPM), and identification on a confirmatory plating medium (LMCM). Restaino et al. [150] explored the efficacy of the BCM LMDS using pure cultures and naturally and artificially contaminated environmental sponges. The BCM LMPEB allowed growth of Listeria and resuscitation of heat-injured L. monocytogenes. The BCM LMSEB, which contains the fluorogenic substrate 4-methylumbelliferyl-myo-inositol-1-phosphate and detects phosphatidylinositol phospholipase C (PI-PLC) activity, provided a presumptive positive test for the presence of pathogenic Listeria (L. monocytogenes and L. ivanovii) after 24 h at 35°C. An initial inoculum of 10 to 100 CFU/mL of L. monocytogenes in BCM LMSEB yielded a fluorogenic response after 24 h. On BCM LMPM, L. monocytogenes and L. ivanovii were the two Listeria species forming turquoise convex colonies (1.0 to 2.5 mm in diameter) from PI-PLC activity on the chromogenic substrate, 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate. L. monocytogenes was distinguished from L. ivanovii by its fluorescence on BCM LMCM or acid production from rhamnose. False-positive organisms (Bacillus cereus, Staphylococcus aureus, Bacillus thuringiensis, and yeasts) were eliminated by at least one of the media in the BCM LMDS. Using a pure culture system, the BCM LMDS detected one to two L. monocytogenes cells from a sponge rehydrated in 10 mL of DE neutralizing broth. In an analysis of 162 environmental sponges from facilities inspected by the USDA, the values for identification of L. monocytogenes by BCM LMDS and the USDA method were 30 and 14 sites, respectively, with sensitivity and specificity values of 85.7 and 100.0% with BCM LMDS vs. 40.0 and 66.1% with the USDA method. No false-positive organisms were isolated by BCM LMDS, whereas 26.5% of the sponges tested by the USDA method produced false-positive results.

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Serology is useful when epidemiological considerations are crucial. A TSBye culture is used to inoculate tryptose broth. This is incubated for 24 h at 35°C, the temperature at which flagella (H) antigen expression is reduced. The culture is transferred to tryptose agar slants and incubated for 24 h at 35°C. Both slants are washed in a total of 3 mL Difco fluorescent antibody (FA) buffer and transferred to a sterile 16- × 125-mm screw-cap tube. These slants are heated in a water bath at 80°C for 1 h. Cells are sedimented by centrifugation at 1600 g for 30 min, 2.2 to 2.3 mL of supernatant fluid are removed, and the pellet is resuspended in the remaining buffer. The manufacturers usually provide recommendations for sera dilution and agglutination procedures to be followed. Genetic subtyping involves submission of data from pulsed-field gel electrophoresis (PFGE) of DNA restriction fragments of FDA isolates to PulseNet (CDC, Atlanta, Georgia). Isolates should also be ribotyped or sent to a ribotyping laboratory [184,185]. Present versions of the FDA procedure have greatly shortened and simplified isolation of Listeria spp. from many foods compared with earlier methods developed to detect the pathogen in clinical specimens; revised procedures have afforded many improvements over the original FDA protocol [114,117]. In 1987, Doyle and Schoeni [48] compared the original FDA classic cold enrichment and shortened enrichment procedures for their ability to recover L. monocytogenes from 90 samples of commercially produced, soft, surface-ripened cheese that was previously identified as likely to contain L. monocytogenes. Although L. monocytogenes was isolated from 41 of 90 (46%) cheeses, no single procedure detected the pathogen in all positive samples. A total of 21 samples were positive after cold enrichment as compared with only 16 and 13 samples that were positive using the FDA and shortened enrichment procedures, respectively. Thus, the latter two protocols failed to recover L. monocytogenes from 5 of 21 (23.8%) and 8 of 21 (38.1%) samples that were positive following cold enrichment. Furthermore, because Listeria was never isolated from the same positive sample by all three protocols, it appears that the original FDA method was inferior to cold enrichment. Similar results were obtained by Doyle et al. [49] when these same three enrichment procedures were used to isolate L. monocytogenes from milk samples after HTST pasteurization. Researchers in Canada [55] and England [141] found negligible differences between numbers of Listeria recovered from naturally contaminated samples of raw milk and soft cheeses analyzed by the FDA and cold enrichment procedures, although both methods again failed to detect Listeria in all positive samples. These variable findings for the original FDA and cold enrichment procedures have been attributed to nonuniform distribution of Listeria within samples. However, Doyle and Schoeni [48,49] found cold enrichment superior to the FDA method for analysis of soft, surface-ripened cheese, when nonuniform distribution of Listeria is expected, as well as in pasteurized milk. Hence, variations in the ability of the FDA and cold enrichment procedures to detect Listeria in dairy products probably result from inherent differences between the two methods (media, incubation conditions) and/or presence of microbial competitors rather than nonuniform distribution of Listeria in the product. Although these results indicate that cold enrichment was generally superior to the original FDA protocol, the time-consuming nature of cold enrichment makes this procedure unacceptable as a commercial screening method for L. monocytogenes. International Dairy Federation Method Using the original FDA method as a starting point, the IDF initiated development of a “reference” method in 1988 [173] to recover L. monocytogenes from dairy products. Development of the IDF method essentially followed that of the FDA protocol as previously reviewed by Ryser and Marth [161] with the eventual elimination of pre-enrichment (for detecting sublethally injured Listeria) and the KOH treatment of the enrichment broths before plating on Listeria-selective media. The present IDF method [7] received AOAC approval in 1993 based on results from an AOAC

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Add 25 g or 25 mL into 225 mL IDF Selective Enrichment Medium

Blend or stomach for about 2 min

If necessary, adjust pH 48 h/30°C Streak onto Oxford Agar 48 h/37°C

Pick 5 presumptive Listeria colonies (black with black halos) for confirmation

FIGURE 7.4 IDF procedure for isolating L. monocytogenes from milk and dairy products. (From Association of Official Analytical Chemists. 1996. In Official methods of analysis of the association of official analytical chemists. Gaithersburg, Md.: AOAC International, 17.10.01.)

collaborative study [175] assessing the ability of this method to recover L. monocytogenes from inoculated samples of raw milk, ice cream, Camembert cheese, Limburger cheese, and skim milk powder. The AOAC-approved IDF method (Figure 7.4) closely resembles the FDA protocol (see Figure 7.3); the sample is enriched in IDF enrichment broth that contains the same concentrations of selective agents found in LEB [88]. Following 48 h of incubation at 30°C, enrichments are plated on Oxford agar as opposed to the FDA procedure, which calls for Oxford agar and LPM without esculin/Fe3+ or PALCAM. This method requires a minimum of 4 days to obtain presumptive results and continues to be popular in Europe for detecting Listeria in dairy products. USDA–FSIS Method The USDA–FSIS devised a method for detecting L. monocytogenes in meat and poultry products (Figure 7.5) [91,176,178]. The original USDA protocol developed in 1986 by Lee and McClain [106,124] differs from the original and revised FDA procedures in that primary and secondary enrichment steps are included for detecting Listeria. The original USDA procedure enabled Listeria detection within 3 days compared with 9 to 11 or 5 to 6 days using the original and revised FDA methods, respectively. The original USDA–FSIS procedure was revised in May 1989 [33] and differed from the original method in that LEB II was replaced by Fraser broth [64] as the secondary enrichment medium; LPM agar was replaced by MOX; and the regulatory sample size was increased to 25 g. Fraser broth and modified Oxford agar will blacken during incubation because Listeria spp. and other contaminants can hydrolyze esculin; colonies of Listeria will exhibit black halos on modified Oxford agar following 24 to 48 h of incubation. However, MOX is more selective than LPM or Oxford agar [42], and staphylococci and streptococci are generally unable to grow on it.

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Add 25 g meat sample to 225 mL UVM broth, stomach 2 min

Incubate at 30°C

0.1 mL + 10 mL Fraser broth

Incubate 26 ±2 h 35° C

Incubate 48 h 35°C

Streak onto MOX

Examine for black colonies (presumptive Listeria)

If negative

Streak onto MOX

Incubate 35°C, 48 h

Examine for black colonies

FIGURE 7.5 USDA procedure for isolating L. monocytogenes from red meat, poultry, egg products, and environmental samples. (From USDA/FSIS. 2002. In Microbiology laboratory guidebook, 3rd ed., revision 3, chapter 8.)

Reported inadequacies in the previous [33] USDA–FSIS procedure were related to use of Fraser broth for secondary enrichment. False-negative results caused by reliance on Fraser broth darkening and a 24-h secondary enrichment have been reported by several laboratories [9,99]. Kornacki et al. [99] compared recovery of L. monocytogenes from Fraser broth incubated for 26 vs. 48 h. L. monocytogenes was isolated from 60 of 1,088 meat product and environmental swab samples from meat and dairy plants. False-negative rates as high as 6.7% were attributed to the inability of L. monocytogenes to be detected in Fraser broth at 26 h but not at 48 h, and to the failure of Fraser broth to blacken. Furthermore, investigators failed to detect L. monocytogenes in eight Fraser broth enrichments that were positive by primary enrichment. These findings clearly stress the importance of incubating Fraser broth enrichments for 48 h. The USDA–FSIS has recommended several modifications to its original procedure. The latest USDA–FSIS method to isolate and identify L. monocytogenes from red meat, poultry, egg, and environmental samples has been in effect since April 2002 [178]. This revised method includes use of rapid screening tests and has a sensitivity of <1 CFU/g in a 25-g sample. In this revised protocol, media required for enrichment, plating, and preliminary confirmation tests include modified University of Vermont broth (UVM, also known as UVM1), Fraser broth (FB), modified Oxford agar (MOX), horse blood overlay agar (HL, also known as HBO), trypticase soy agar with 5% sheep

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blood (TS-SBA, also known as CAMP test agar), and BHI broth. Additional media for environmental samples include Dey–Engley (D/E) neutralizing broth (e.g., Nasco cat# B01256WA or equivalent) and an optional medium, trypticase soy agar-yeast extract (TSA-YE). At least one L. monocytogenes-positive control strain and one L. innocua-negative culture are required; other Listeria spp., such as L. seeligeri, L. grayi, and L. ivanovii, may be necessary as controls for additional confirmatory testing. If the β-lysin CAMP factor test is not employed, Staphylococcus aureus and Rhodococcus equi are required to do the traditional CAMP test. A 25- ± 1.0-g portion is used for raw and processed red meat, poultry, and egg product testing. Primary enrichment in UVM broth is done as follows: For all meat, poultry, and egg product samples, 225 ± 5 mL of UVM broth is added to 25± 1.0-g test portion, stomached or blended for 2 min, and then incubated at 30 ± 2°C for 22 ± 2 h. For environmental sponge samples, 200 ± 5 mL of UVM broth are added to each bagged sponge sample, stomached 2 ± 0.2 min, and incubated at 30 ± 2°C for 22 ± 2 h. For environmental aqueous chilling solutions (which may include water, brine, and propylene glycol solutions) and surface rinse solutions, about 100 mL of sample solution is filtered through one or more 0.45-µm hydrophobic grid membrane filters. Using sterile forceps, the membrane is aseptically removed from the plastic housing and transferred to a stomacher bag. About 200 mL or additional volume of UVM broth sufficient to cover the filters completely is added, stomached 2 min, and incubated at 30 ± 2°C for 22 ± 2 h. Secondary enrichment is achieved by transferring 0.1 ± 0.02 mL of the UVM enrichment to 10 ± 0.5 mL of FB (containing appropriate supplements, per media preparation instructions). Inoculated FB tubes are incubated at 35 ± 2°C for 22 ± 2 h. During secondary enrichment, a loopful or a drop approximating 0.1 mL of UVM is streaked over the surface of a MOX plate. Alternatively, a sterile cotton-tipped applicator or equivalent is dipped into the UVM and 25 to 50% of the surface of a MOX plate swabbed. A loop is then used for isolation from the swabbed area onto the remainder of the plate. The MOX plate is incubated at 35 ± 2°C for 26 ± 2 h. UVM-streaked MOX plates are examined for colonies having morphology typical of Listeria spp. At 24 h, suspect colonies are typically small (ca. 1 mm) and are surrounded by a zone of darkening from esculin hydrolysis. If suspect colonies are present on MOX, they are transferred to HL agar (description follows). If no suspect colonies are evident, the MOX plate is reincubated until a total incubation time of 48 ± 2 h has been achieved. Then the FB is examined after 26 ± 2 h of incubation for the potential presence of L. monocytogenes by visual examination of the broth for darkening from esculin hydrolysis. If any degree of FB darkening is evident, 0.1 mL of FB is aseptically dispensed onto a MOX plate. About 25 to 40% of the surface of MOX is swabbed or streaked with the FB inoculum and a loop is used to streak for isolation from the initial swab/streak quadrant onto the remainder of the plate. The MOX plate is incubated at 35 ± 2°C for a minimum of 24 h. Upon incubation, suspect colonies are examined and selected from any MOX agar plate pending analysis (i.e., MOX plates streaked from 26 ± 2 h FB and/or UVM) as described earlier. If no FB darkening is evident, the FB is reincubated at 35 ± 2°C and re-examined for evidence of darkening until a total incubation time of 48 ± 2 h has been achieved. If any degree of darkening is evident, a MOX plate is swabbed, streaked, and incubated as already described. If no darkening is evident and no suspect MOX and/or HL colonies have been demonstrated, the sample is considered negative for L. monocytogenes. Isolation and purification of presumptive Listeria in the current USDA–FSIS [178] method is accomplished as follows. If suspect colonies are present on MOX from any source, a loop or equivalent sterile device is used to contact a minimum of 20 (if available) suspect colonies that are collectively streaked for isolation on one or more HL plates. The streaked HL plates are incubated at 35°C for 22 ± 4 h. After incubation, the HL plates are examined against backlight for translucent

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colonies surrounded by a small zone of β-hemolysis. If at least one suspect colony is clearly isolated, one should proceed to confirmatory testing, while holding all HL plates containing suspect colonies (at room temperature or refrigerated) until confirmatory testing is complete. If suspect colonies or β-hemolytic growth are present on HL but not clearly isolated, representative suspect colonies/growth should be restreaked onto one or more fresh HL plates and incubated at 35°C for 22 ± 4 h. If, however, no suspect isolates are present on HL, follow-up of MOX and/or HL isolates from other branches of analysis should be done (e.g., FB follow-up vs. UVM direct streak follow-up). If no branch of analysis produces suspect β-hemolytic colonies on HL, the sample may be reported as negative for L. monocytogenes. Confirmatory identification of L. monocytogenes consists of preliminary confirmation tests such as the tumbling motility test followed by biochemical tests. The CAMP/CAMP factor test and genetic tests may be required in certain circumstances. To facilitate rapid screening of processed meat and poultry products, USDA–FSIS has approved the use of a commercial PCR-based procedure, L. monocytogenes BAX screening test [178]. All samples identified as presumptively positive for L. monocytogenes by these tests are subject to cultural confirmation. For this method, L. monocytogenes detection limits are determined to be <1 CFU/g in a 25-g sample. Sample preparation and enrichment are similar to those described for the USDA–FSIS isolation and identification of L. monocytogenes from red meat, poultry, egg, and environmental samples [178]. Secondary enrichment involves transferring 0.1 mL of the UVM enrichment to 10 mL to MOPS–BLEB (prepared using a complete powdered LEB medium [DIFCO–BBL] to which the MOPS free acid and sodium salt are added before autoclaving) and incubating at 35°C for 18 to 24 h. A MOX plate is streaked with a loopful of UVM over the surface and a loop is used to streak for isolation from the swabbed area onto the remainder of the plate; the plate is incubated at 35 ± 2°C for 26 ± 2 h. The BAX screening test is then done according to the current BAX User’s Guide. Cultural confirmation of presumptive positive samples is obtained by streaking a MOX plate using a loopful of the MOPS–BLEB, incubating the MOX at 35 ± 2°C for a minimum of 24 h before proceeding with all isolation and purification procedures as described earlier in the USDA–FSIS method. In their study on comparative evaluation of culture- and BAX polymerase chain reaction PCRbased detection methods for Listeria spp. and L. monocytogenes in environmental and raw fish samples, Hoffman and Wiedmann [89a] evaluated two commercial PCR-based Listeria detection systems, the BAX for screening L. monocytogenes and the BAX for screening genus Listeria, and a culture-based detection system, the Biosynth L. monocytogenes detection system (LMDS), for their ability to detect L. monocytogenes and Listeria spp. in raw ingredients and the processing environment. For detection of L. monocytogenes from raw fish, enrichment was done in Listeria enrichment broth (LEB), followed by plating on OXA and LMDS L. monocytogenes plating medium (LMPM). Detection of Listeria and L. monocytogenes from environmental samples was done using LMDS enrichment medium, followed by plating on OXA and LMPM. A total of 512 environmental and 315 raw fish samples were taken from two smoked-fish processing facilities and screened using these molecular and cultural Listeria detection methods. The BAX method for L. monocytogenes was used to screen raw fish and was 84.8% sensitive and 100% specific. The BAX method for the genus Listeria was evaluated on environmental samples and had 94.7% sensitivity and 97.4% specificity. In conjunction with enrichment in LEB, LMPM had a sensitivity and specificity of 97.8 and 100%, respectively, for detection of L. monocytogenes in raw fish. Use of LMDS enrichment medium followed by plating on LMPM yielded sensitivity and specificity rates of 94.8 and 100%, respectively, for detection of L. monocytogenes from environmental samples. This shows that both BAX systems and LMPM allow for reliable and rapid detection of Listeria spp. and L. monocytogenes. However, BAX systems are slightly faster; they provide screening results in about 3 days compared to 4 to 5 days when LMPM is used.

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A study by Lammerding and Doyle [103] found that the USDA protocol yielded better recoveries of L. monocytogenes from dairy products than did the FDA method. Warburton [181] reported that, with a limited number of samples, the USDA–FSIS method proved to be slightly more efficient in isolating L. monocytogenes than the FDA method in a comparative study of the FDA and USDA–FSIS methods for detection of this organism in foods and environmental samples. Nineteen laboratories across Canada took part in this study. Similarly, Ferron and Michard [58] compared the FDA and USDA–FSIS enrichment procedures using 300 pastry samples provided by 100 different suppliers in western France. The USDA procedure was superior, detecting 69% of all positive samples compared with the FDA procedure, which detected only 34%. The USDA–FSIS method appears to be more efficient in quantitative recovery of L. monocytogenes from contaminated food and environmental samples. The Netherlands Government Food Inspection Service Using the NGFIS protocol, food samples are enriched in L-PALCAMY enrichment broth for 48 h at 30°C. After 24 and 48 h, 0.1 mL of L-PALCAMY enrichment broth is streaked onto PALCAM agar. Plates are incubated at 30°C for 48 h under microaerophilic conditions (5% oxygen, 7.5% carbon dioxide, 7.5% hydrogen, and 80% nitrogen) [109], after which presumptive Listeria colonies are black and surrounded by a dense black hole from esculin hydrolysis. Lund et al. [118] examined 300 raw milk samples for presence of Listeria using three primary enrichment media. A total of 84 positive samples were identified by one or more of these media. PALCAMY was the most effective medium, identifying 50 of 84 positive samples, followed by UVM and LEB, which identified 46 and 42 Listeria-positive samples, respectively. Given that the best of these primary enrichment broths identified only 50 of 84 (59.5%) Listeria-positive samples, the use of two or more primary enrichment broths identified an additional 34 samples and increased the overall incidence of Listeria by almost 41%. These results once again highlight the inadequacy of relying on a single primary enrichment broth for Listeria detection. Noah et al. [132] evaluated the impact of more than one test procedure on recovery of Listeria species from naturally contaminated seafood and seafood products. A total of 211 samples were evaluated using five different protocols. The FDA procedure [116] was used as a control against which the efficacy of the other procedures was evaluated. A total of 60 samples were identified as Listeria-positive by at least one of the procedures. Of these samples, the FDA procedure missed seven samples that were subsequently found to harbor Listeria when other procedures were employed. The overall incidence of Listeria increased 11.7% using more than one testing procedure. Hayes et al. [81] assessed the USDA–FSIS and cold enrichment procedures for recovery of L. monocytogenes from suspect food samples. Both procedures identified L. monocytogenes in 28 of 51 positive samples. The USDA–FSIS procedure identified 21 samples missed by cold enrichment, whereas the cold enrichment procedure identified an additional 2 samples that the USDA–FSIS procedure missed. Three enrichment methods were also compared by Hayes et al. [82] during an examination of foods obtained from the refrigerators of patients with active clinical cases of listeriosis. Of 2,229 food samples examined in this study, 11% were positive for L. monocytogenes. Overall, the USDA–FSIS [33], FDA [116], and NGFIS [130] methods were not statistically different in their ability to isolate Listeria from 899 samples included in the comparative evaluation. The FDA procedure [116] identified 65% of all L. monocytogenes-positive foods, whereas the USDA–FSIS and NGFIS procedures detected L. monocytogenes in 74% of foods shown to be positive. Although none of these widely used Listeria detection methods proved to be highly sensitive when used independently, use of any two methods improved detectability from 65 to 74% for individual protocols to 87 to 91% for combined protocols.

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RESEARCH ADVANCES Peng and Sheref [140] have developed a simple, automated method to detect Listeria in foods. It consists of a 6-h pre-enrichment step followed by overnight incubation in selective Listeria broth (SLB) at 35°C. Changes in light transmittance in the selective broth are registered continuously by an optical sensor of the BioSys instrument, now Soleris (Neogen Corp., Lansing, MI) and recorded in the computer. Esculin hydrolysis by Listeria results in black coloration of the medium that causes a sharp drop in light transmittance, whereas transmittance in negative samples remains unchanged. Confirmation of L. monocytogenes is carried out only on esculin-positive samples and is completed within 6 h. This method is able to detect as few as 10 to 50 cells of Listeria in 25 g of food.

CONSIDERATIONS FOR RECOVERY OF INJURED LISTERIA Most conventional and rapid detection procedures for Listeria use highly selective enrichment media to facilitate growth over competitive background flora. However, these procedures will not generally recover sublethally injured Listeria, which could exist in various heated, frozen, or acidified foods or within heated, frozen, and sanitized areas/surfaces of food-processing facilities. Sublethal injury in Listeria as a result of heating, freezing, drying, irradiation, or exposure to chemicals (i.e., sanitizers, preservatives, acids) has been extensively studied [1,15,30–32,34,39,41, 66,69,70,122,155,157,163] and was reviewed in Chapter 6. Under ideal conditions, such injury is reversible; Listeria is capable of repairing sublethal damage in foods where growth conditions are favorable. Repair of heat-injured L. monocytogenes has been reported in whole and 2% milk stored at 4°C [126]. Ngutter and Donnelly [131] have documented nitrite injury of L. monocytogenes in frankfurters. The injury was completely reversible or growth of uninjured Listeria occurred in LRB when injury was between 98.5 and 98.7%. However, total recovery was not observed in Listeria repair broth (LRB) when injury exceeded 99%. Modified UVM was unable to reverse the effects of nitrite-injured L. monocytogenes. Generally, LRB was found to be consistently superior to UVM at recovering L. monocytogenes from frankfurters. Several investigators have attempted to improve the sensitivity of current detection systems by focusing on recovery of injured Listeria that may be present in food products and food-processing environments. Most research is focusing on RTE foods because they usually do not undergo any further treatment sufficient to kill Listeria before consumption. All current detection procedures, with the exception of cold enrichment, involve selective enrichment and/or selective plating. The main limitation of selective enrichment or selective plating is that they often do not allow for growth of sublethally injured Listeria [46]. Cold enrichment, despite its superiority in recovering injured Listeria, is not feasible for routine testing because several months of incubation may be necessary to obtain positive results. By failing to consider recovery of injured Listeria, current methodologies undoubtedly underestimate the true incidence of this organism. Several previous studies have reported on the ability of commonly used plating media to recover injured Listeria. Among the most commonly used selective agents examined, phenylethanol, acriflavine, polymyxin, and sodium chloride were found to inhibit recovery of thermally stressed and nonstressed Listeria [39,105,171,180,181,183]. Furthermore, when examined for ability to quantitatively recover thermally stressed Listeria, LEB agar, modified McBride’s agar (MMA), LPM agar [106], and FDA enrichment broth agar showed significantly impaired recovery [26]. Warburton et al. [182] examined the ability of the modified FDA and USDA–FSIS methods to recover stressed cells and low levels of L. monocytogenes in food and environmental samples. Although the modified FDA and USDA–FSIS methods were comparable in their abilities to isolate stressed and low-level populations of L. monocytogenes, these authors failed to assess the extent of injury within bacterial populations following exposure to sublethal stress. The percentage of injury existing

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within a population of bacterial cells can profoundly affect comparative results of media performance. Thus, it is difficult to determine whether valid conclusions can be drawn from such studies. Busch and Donnelly [32] developed an enrichment medium capable of resuscitating heat-injured Listeria. This medium, LRB, permits complete repair of injured Listeria within 5 h at 37°C after which selective agents can be added to inhibit the growth of competing microflora during continued incubation. In studies comparing the efficacy of LRB in promoting repair/enrichment of heat-injured Listeria with that of existing selective enrichment media, repair was not observed in FDA enrichment broth [116], phosphate-buffered Listeria enrichment broth (PEB; Gene-Trak Systems, Framingham, Massachusetts), or UVM enrichment broth [103]. Final Listeria populations in selective enrichment media after 24 h of incubation at 30°C were 1.7 × 108 to 9.1 × 108 CFU/mL compared with populations in LRB, which consistently averaged 2.5 × 1011 to 8.2 × 1011 CFU/mL [32]. Knabel [97] developed a one-step, recovery-enrichment broth—optimized Penn State University (oPSU) broth—that consistently detected low levels of injured and uninjured L. monocytogenes cells in RTE foods. The oPSU broth contains special selective agents that inhibit growth of background flora without inhibiting recovery of injured Listeria cells. After recovery in the anaerobic section of oPSU broth, Listeria cells migrate to the surface, forming a black zone. This migration separates viable from nonviable cells and the food matrix. The high Listeria-to-background ratio in the black zone results in consistent detection of low levels of L. monocytogenes in pasteurized foods by cultural and molecular methods, greatly reducing false-negative and false-positive results. One other advantage of oPSU broth is that it does not require transfer to a secondary enrichment broth; this makes it less laborious and less subject to external contamination than two-step enrichment methods. Studies with LRB were extended to examine the potential for repair of freeze-injured and sanitizer-injured L. monocytogenes [60,163]. Although variation in susceptibility of L. monocytogenes to freeze injury was recorded, in general, L. monocytogenes is not severely injured by freezing [53,69,136]. Percentage of injury ranged from only 40 to 60% after Listeria populations were frozen at –9 to –11°C for 24 h [60]. As storage time increased, the percentage of injury increased to a maximum of only 70 to 80%. To examine reversibility of freeze injury, low-level populations of freeze-injured L. monocytogenes cells were added to UVM enrichment broth, FDA enrichment broth, and LRB. Repair of freeze-injured populations occurred quickly, probably because of the low initial degree of injury; the pathogen again attained high populations in LRB. Sallam and Donnelly [163] examined the ability of four commonly used dairy plant sanitizers to induce injury in L. monocytogenes when exposed to sublethal concentrations. UVM broth failed to support growth of sanitizer-injured cells, whereas LRB permitted their recovery. Flanders et al. [62] examined the efficacy of using a repair step to increase recovery of injured Listeria from environmental sponge samples obtained from dairy-processing plant environments. The USDA–FSIS Listeria isolation protocol using UVM-modified Listeria enrichment broth was compared with a modified USDA–FSIS format that utilized LRB as the primary enrichment medium. UVM and LRB broths also were used in conjunction with a rapid DNA hybridization (Gene-Trak) and ELISA (Organon Teknika, Durham, North Carolina) assay. Of 80 sites found positive by any method, UVM and LRB showed similar recovery rates (87.5 and 88.8%, respectively). However, combining the cultural methods with either rapid method for each broth increased detection to 97.5 to 98.8% [62]. Flanders et al. [61] also evaluated the abilities of LRB, LRB containing ceftazidime (LRBC), and UVM to enhance recovery of Listeria from dairy plant environmental samples. Although no single broth could detect all Listeria-positive sites, LRBC identified 67 of 89 positive sites (75.3%), and LRB and UVM each detected 60 of 90 positive sites (66.7%). Combining results from any two broths increased recovery from 66.7–75.3% to 82.2–94.4%. The combination of LRBC and UVM detected 94.4% of positive samples, whereas LRB and LRBC identified 91.1% of positive samples. Pritchard et al. [144] also compared the ability of UVM, LRB, and LRBC to isolate Listeria from dairy plant environments. Of 80 positive samples identified, 54 samples came from UVM

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medium, 56 were from LRB, and 57 came from LRBC. A total of 26 samples (32.5% of positive samples) were identified by LRB or LRBC but not by UVM media. Combining UVM with LRB or LRBC again substantially increased the number of positive samples identified. When results from UVM and LRB were combined, 65 to 80 (81.3%) positive samples were identified. Using UVM and LRBC, 74 of 80 (92.5%) positive samples were identified. These results illustrate the severe limitations associated with current regulatory procedures used to assure absence of Listeria in foods and food-processing environments. Ryser et al. [162] evaluated the ability of UVM and LRB to recover different strain-specific ribotypes of L. monocytogenes from meat and poultry products. Forty-five paired 25-g retail samples of ground beef, pork sausage, ground turkey, and chicken underwent primary enrichment in UVM and LRB (30°C/24 h) followed by secondary enrichment in Fraser broth (35°C/24 h) and plating on modified Oxford agar. A 3-h nonselective enrichment period at 30°C was used with LRB to allow repair of injured Listeria before adding selective agents. Listeria spp. were detected in 73.8 and 69.4% of the 180 meat and poultry samples tested using LRB and UVM, respectively. Although these differences were not statistically significant, combining UVM and LRB results increased overall Listeria recovery rates to 83.3%. Thus, enrichment in LRB for repair of injured cells in conjunction with the USDA–FSIS method has potential to improve recovery of Listeria from meat and poultry products. In this study, following 24 h of incubation at 35°C, Listeria colonies were biochemically confirmed and selected isolates were ribotyped using the automated Riboprinter Microbial Characterization System, (E.I. du Pont de Nemours and Co., Inc., Wilmington, DE). A total of 36 different Listeria strains comprising 16 L. monocytogenes (including 4 known clinical ribotypes), 12 L. innocua, and 8 L. welshimeri ribotypes were identified from selected positive samples (15 samples of each product type, 2 UVM and 2 LRB isolates/sample). Using both UVM and LRB, 26 of 36 (13 L. monocytogenes) ribotypes were detected; whereas 3 of 36 (1 L. monocytogenes) and 7 of 36 (3 L. monocytogenes) Listeria ribotypes were observed using only UVM or LRB, respectively. Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. monocytogenes), 21 (12 L. monocytogenes), 20 (9 L. monocytogenes), and 19 (11 L. monocytogenes) different Listeria ribotypes, respectively; some Listeria ribotypes were confined to a particular product. More importantly, striking differences in the number and distribution of Listeria ribotypes, including previously recognized clinical and nonclinical ribotypes of L. monocytogenes, were observed when 10 UVM and 10 LRB isolates from five samples of each product were examined. When a third set of six samples per product type was examined from which two Listeria isolates were obtained using only one of the two primary enrichment media, UVM and LRB failed to detect L. monocytogenes (clinical and nonclinical ribotypes) in two and four samples, respectively (Table 7.3). These findings stress the complex microbial ecology of Listeria in foods and limitations of existing detection procedures to fully characterize the total Listeria population. Furthermore, two of the L. monocytogenes ribotypes missed using UVM were known clinical ribotypes linked to sporadic and epidemic cases of human listeriosis in England and Scotland [125]. Continuing work [143] on enrichment of dairy environmental samples in UVM and LRB has shown that combining these two primary enrichment media into a single tube of Fraser broth for secondary enrichment yields a significantly higher (P < 0.05) percentage of Listeria-positive samples than when LRB or UVM is used alone. These findings, combined with reports of L. innocua being able to outgrow L. monocytogenes in UVM (and Fraser broth) [40,142] suggest that different ribotypes of L. monocytogenes may vary somewhat in nutritional requirements or their ability to compete with other ribotypes of L. monocytogenes or other Listeria spp. Refinement of existing Listeria recovery methods should consider the nutritional needs associated with those specific genetic types widely distributed in foods. Silk et al. [167] examined five selective (Food and Drug Administration Bacteriological Analytical Manual Listeria enrichment broth base with selective agents [BAMS]), BCM Listeria

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TABLE 7.3 Ribotypes of Listeria spp. Recovered from 10 Samples of Raw Chicken Following Primary Enrichment in UVM or LRB and Secondary Enrichment in Fraser Broth Ribotype 1-909-3 5-418-3 5-415-4 5-413-2 2-864-3 1-916-1a 5-408-1 1-909-4 1-910-7 5-426-1 1-923-1a 5-408-4 1-907-1a 1-919-2 1-864-7 1-915-7

Listeria spp. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L.

innocua monocytogenes innocua monocytogenes welshimeri monocytogenes monocytogenes innocua innocua innocua monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes

No. of Isolates UVM

LRB

0 2 4 2 2 3 2 5 0 0 0 0 0 0 0 0

1 0 0 0 0 3 0 5 1 1 3 2 1 1 1 1

a

Recognized clinical ribotypes associated with known epidemic or sporadic cases of human listeriosis.

Source: Adapted from Ryser, E. T. et al. 1996. Appl. Environ. Microbiol. 62:1781–1787.

pre-enrichment broth [BCM, Biosynth International, Inc., Naperville, Illinois], Fraser broth, Listeria repair broth with selective agents [LRBS], and University of Vermont-modified Listeria enrichment broth) and three nonselective (Food and Drug Administration Bacteriological Analytical Manual Listeria enrichment broth base [BAM], Listeria repair broth [LRB], and trypticase soy broth [TSB]) media with respect to the growth of healthy and repair of heat-injured L. monocytogenes (strain F5069 [ATCC 51414]). After inoculation of approximately 2 × 102 CFU healthy cells/mL, each test broth was incubated at 30°C for 72 h. During the incubation period, 12 samples concentrated in the growth transition regions of lag-log-stationary phase were removed from the broth, diluted in Butterfield phosphate buffer, plated onto trypticase phosphate agar (TPA; trypticase phosphate broth [TPB] with 1.5% agar; Difco) and incubated at 35°C for 48 h before enumeration. L. monocytogenes was injured by placing 200 mL of 56°C preheated TPB in a 500-mL Erlenmeyer flask followed by addition of a 1:100 dilution of an 18-h-old culture of L. monocytogenes F5069. This suspension was heated for 20 min in a shaking water bath before being transferred into a 250-mL centrifuge bottle and spun at 10,000 × g for 10 min at 25°C. The supernatant fluid was discarded and the pellet washed with test medium. The test broths were incubated at 30°C for 72 h and sampled as described earlier for healthy cells. Heat-injured cells were plated onto TPA and TPA supplemented with 4% NaCl (TPAN) and incubated at 35°C for 48 h before enumeration. The percentage of injury was calculated using the following equation: (1 – [counts of TPAN/counts on TPA]) × 100. These researchers also modeled growth of this strain of L. monocytogenes with the Gompertz equation [29,37,67] using a nonlinear regression procedure (PROCLIN) of the SA system for

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Windows version 8.2 (SAS Institute Inc., Cary, North Carolina). Gompertz parameter values were then used to calculate the exponential growth rate (EGR), lag-phase duration (LPD), generation time (GT), and maximum population density (MPD) [28]. For heat-injured cell populations, the Gompertz equation was used to model L. monocytogenes growth on TPA and TPAN. The time required for the repair was determined by graphing the models and determining the time point at which bacterial counts on nonselective (TPA) and selective (TPAN) media were equal [37]. Growth curves were generated using healthy and heat-injured L. monocytogenes strain F5069 in the three nonselective and five selective enrichment broths mentioned previously. The Gompertz equation was successfully used to model the growth of L. monocytogenes. Gompertz parameters were used to calculate EGR, LPD, GT, MPD, and time required for repair of injured cells. Statistical differences (P < 0.05) in broth performance were noted for LPD and MPD when healthy and injured cells were inoculated into the broths. With the exception of Fraser broth, no significant differences were found in the time required for repair of injured cells. These results suggest that the distinction between selective and nonselective broths in their ability to grow healthy Listeria and to repair sublethally injured cells is not solely an elementary issue of presence or absence of selective agents. Heat shock proteins produced by Listeria during heating [172], for example, may be conferring some cross-protective properties [112], making the injured cells less sensitive to selective agents than they were previously thought to be. Roth and Donnelly [155] assessed survival of acid-injured L. monocytogenes in four different acidic foods and also examined the efficacy of LRB and UVM to recover acid-injured Listeria from such foods. L. monocytogenes was injured in lactic (pH 3.0) and acetic (pH 3.5) acids. Two levels of injury were produced and monitored: one population with 99.99% injury and the second with approximately 95% injury. The four acidic food systems studied at 4 and 30°C included fresh apple cider (pH 3.3), plain nonfat yogurt (pH 4.2), fresh coleslaw (pH 4.4), and fresh salsa (pH 3.9). Acid-injured Listeria was added to each acidic food and monitored by selective and nonselective plating. Simultaneously, samples were enriched in LRB and UVM followed by standard isolation/identification procedures with survival of healthy L. monocytogenes also monitored. Although acid-injured cells failed to repair in the acidic foods tested, the pathogen did survive for more than a week. Storage temperatures did affect the survival rate of acid-injured cells; storage at 4°C had a bacteriostatic effect and at 30°C was bacteriocidal. Parameters involved in survival of acid-injured Listeria include the degree to which the bacterial population is injured (percentage of injury), storage temperature, and the pH of the food. At time points where differences were detected, LRB proved to be superior (22 of 54) in its ability to detect injured Listeria compared with UVM (3/54). Hence, use of LRB is recommended when examining acidic foods for L. monocytogenes.

CONCLUSIONS Standard enrichment and plating procedures for Listeria detection will continue to enjoy widespread use because they are economical when compared to rapid methods. Disadvantages of standard procedures include their time-consuming and cumbersome nature, as well as inability to detect injured Listeria in processed RTE foods. Advances in media development and formulation have increased recovery and decreased potential for false positive results. The ability to recover a broad diversity of Listeria spp. from foods is critical in epidemiological investigations where traceback to contaminating foods needs to be done rapidly and accurately. Outbreaks of foodborne listeriosis coupled with the high mortality rates associated with sporadic cases of illness and the advent of mandatory HACCP programs have underscored the need for faster and more efficient methods to detect small numbers of Listeria in a wide range of foods. There is no doubt that continued work to refine optimal enrichment and detection procedures for Listeria in foods will occur in the future.

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Methods for Detection 8 Rapid of Listeria Byron F. Brehm-Stecher and Eric A. Johnson CONTENTS Introduction ....................................................................................................................................257 The “Ideal” Detection Method.............................................................................................258 Traditional Methods for Detection of Listeria ....................................................................258 Rapid Methods for Detection of Listeria.............................................................................258 Commercially Available Rapid Test Kits and Systems .......................................................258 Automation ...........................................................................................................................259 Detection of Generic Listeria versus Listeria monocytogenes ............................................259 Diagnostic Targets ................................................................................................................259 Nucleic Acid–Based Methods........................................................................................................259 The Polymerase Chain Reaction..........................................................................................260 Potential Pitfalls of PCR ......................................................................................................261 DNA Microarrays .................................................................................................................262 Fluorescence in Situ Hybridization ......................................................................................263 Recombinant Bacteriophage.................................................................................................264 Antibody-Based Methods ..............................................................................................................264 Potential Pitfalls of Antibody-Based Methods ....................................................................265 Advances in Antibody-Based Technologies.........................................................................265 Cell Separation and Concentration ......................................................................................266 Additional Methods........................................................................................................................267 Flow Cytometry....................................................................................................................267 Biosensors.............................................................................................................................269 Chip-Based Microanalytical Systems ..................................................................................271 Spectroscopic Methods.........................................................................................................271 Conclusions ....................................................................................................................................274 Acknowledgments ..........................................................................................................................274 References ......................................................................................................................................275

INTRODUCTION Members of the genus Listeria are found in association with soil, water, and vegetation and can grow at refrigeration temperatures. Foods involved in outbreaks of listeriosis include ready-toeat (RTE) products such as milk, soft-ripened cheeses, coleslaw, and vacuum-packaged meats [117]. Although listeriosis is relatively rare, it is characterized by a high mortality rate (~25–30%). The seriousness of this disease, and the efficiency and volume of today’s food production and distribution networks highlight the need to develop rapid methods for detecting L. monocytogenes. This chapter provides a broad overview of methods and technologies for rapid detection of 257

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Listeria or L. monocytogenes that have been reported in the literature. Some methods, such as those employed by commercially available test kits, have received relatively wide acceptance and are used routinely in laboratory analyses. Other methods described here are not yet widely used and may be considered “emerging technologies.” The benefits and drawbacks of each approach for rapid detection, and in some instances, characterization, of Listeria or L. monocytogenes will be considered.

THE “IDEAL” DETECTION METHOD Although no ideal detection method exists for Listeria (or for any analyte), consideration of characteristics that such a system would have may be useful in evaluation of existing methods or in development of new ones. The ideal detection method would be: specific for the target analyte (Listeria or L. monocytogenes), sensitive (able to detect 1 CFU in a 25-g sample), rapid (substantially faster than cultural methods alone), reproducible, simple to use (with easily interpreted results), capable of direct detection in foods with minimal or no interference from the food matrix, able to distinguish between live and dead (or injured) cells, inexpensive (relative to expenses associated with traditional methods of detection), validated against standard techniques, automatable, and scalable according to testing needs. Additional properties may include continuous online operation and operation over a wide dynamic range. Although no single method fulfills all of these criteria, food processors can prioritize which characteristics of the ideal are required for their specific testing needs and choose an available method that best meets these needs.

TRADITIONAL METHODS

FOR

DETECTION

OF

LISTERIA

“Classical” or culture-based methods for the detection of Listeria are limited primarily by their heavy demands on time and labor [9,33,99]. As an extreme example, the original cold-enrichment method described by Gray and colleagues [47] used incubation periods ranging from several weeks to months, a time frame clearly at odds with today’s rapid pace of food processing and distribution. A wide variety of selective enrichment broths and agars have since become available for detection of Listeria or for differentiation of L. monocytogenes from other members of the genus [9,111]. Additional formulations have been described for the recovery of thermally or chemically injured listeriae [32,127]. Still, at least four sequential steps are generally required for cultural methods of detection: pre-enrichment, selective enrichment, selective plating, and biochemical screening. As a result, positive detection of Listeria in food or environmental samples using cultural methods alone may take up to 5–7 days [99].

RAPID METHODS

FOR

DETECTION

OF

LISTERIA

Using the time required for cultural methods as a benchmark, a rapid method can be broadly defined as any approach yielding comparable results in less time. The potential benefits of rapid methods include reduced likelihood that contaminated product will be released for sale, reduction in costs associated with media, labor, or storage of product pending microbiological results, and increased time on shelves for products cleared for sale. The ability to rapidly detect listeriae in foods and in the food-processing environment may also enable more timely monitoring of critical control points, contributing to our ability to control L. monocytogenes in these environments and, ultimately, the incidence of disease [150].

COMMERCIALLY AVAILABLE RAPID TEST KITS

AND

SYSTEMS

The benefits of commercially supported rapid test kits or systems include test availability, standardization, independent validation, simplicity, cost-effectiveness, and the availability of technical support [99]. Test kits and systems for rapid detection of both generic Listeria and L. monocytogenes are

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commercially available, and many of these have received AOAC approval. Assay formats include colorimetric DNA probe, latex bead-based lateral flow immunoassay, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofluorescence assay (ELFA), immunomagnetic separation (IMS), fluorescence in situ hybridization (FISH), and polymerase chain reaction (PCR). Most methods require selective enrichment for up to 48 h. One exception may be IMS-based methods, which can be used to selectively concentrate generic Listeria or L. monocytogenes from a sample without enrichment.

AUTOMATION A drawback of many assay formats is that they are relatively complex and may involve repetitive steps, which can lead to operator fatigue and error. A number of rapid methods are amenable to automation. Benefits of assay automation include reduced labor, increased throughput, better reproducibility and faster time-to-results [99,100]. Automated methods reported for rapid detection or characterization of Listeria include enzyme-linked immunoassays, DNA or RNA probe assays, ribotyping, biochemical screening, and impedance testing [28,37,67,83,100,104,105,137].

DETECTION

OF

GENERIC LISTERIA

VERSUS

LISTERIA

MONOCYTOGENES

The decision to test for generic Listeria or for L. monocytogenes has legal, ethical, and practical implications. This decision is further complicated by limited knowledge of exactly which factors contribute to the pathogenicity of L. monocytogenes [101,149]. Apart from a few isolated cases, most human cases of listeriosis are caused by L. monocytogenes, arguing for use of tests that are specific for this species. However, testing for generic Listeria has been suggested as a more robust means of environmental monitoring than testing for L. monocytogenes alone [130]. Additionally, recent regulatory directives now require certain sectors of the food industry to conduct routine environmental testing for generic Listeria [4]. It is likely that this new regulatory environment will lead to an increased demand for rapid methods to detect generic Listeria.

DIAGNOSTIC TARGETS A diagnostic target is any unique molecule whose detection signals the presence of a specific organism in a sample. Potential diagnostic targets for Listeria or L. monocytogenes include evolutionarily distinct nucleic acid sequences found in rRNA, mRNA, or chromosomal DNA. Additional targets may include structural components such as flagellar, somatic, or capsular antigens, or proteinaceous virulence factors such as ß-hemolysin or phospholipase C [10,110,136]. The presence of these targets may be detected using a number of techniques, including PCR, DNA, or RNA hybridization, antibody-based approaches, or phenotypically, using diagnostic media. As a practical matter, it may not be possible to demonstrate that a given sequence, molecule, or enzymatic activity is truly unique among all microorganisms. However, for it to be diagnostically useful, a target must only be unique within a certain environmental niche. If necessary, microorganisms that could potentially lead to false-positive results may also be excluded from the sample before testing using selective enrichment. In choosing a diagnostic target, any potential pitfalls regarding its use must be identified. For example, expression of diagnostic epitopes may depend on media used to grow cells, and virulence factor mRNAs may be expressed only at certain temperatures [63,93,98].

NUCLEIC-ACID-BASED METHODS Availability of complete genome sequences of Listeria monocytogenes (serotypes 1/2a, 4b, 6a) and L. innocua has provided new insights into molecular features contributing to the pathogenesis of L. monocytogenes [16,44,145] (http://www.tigr.org/tdb/mdb/mdbcomplete.html). In addition to providing a greater understanding of the pathogenesis of L. monocytogenes, these efforts may also yield new diagnostic targets for detection of this organism. Several nucleic-acid-based approaches

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are available to detect L. monocytogenes and other Listeria spp., including amplification-based strategies such as the ligase chain reaction [148], nucleic-acid-sequence-based amplification (NASBA) [11], and PCR. PCR is discussed in more detail in the following subsection.

THE POLYMERASE CHAIN REACTION PCR provides a method for exponential amplification of specific DNA sequences present in a sample. Key components of the buffered PCR reaction mixture include oligonucleotide primers, deoxyribonucleotide triphosphates, the DNA template to be amplified, and a thermostable DNA polymerase. The primers are designed to hybridize on either side of the target sequence, defining the region to be amplified [99,116,153]. Additional components such as enzyme cofactors (e.g., Mg2+) or additives intended to increase the specificity of the reaction or the thermal stability of the enzyme may also be present. PCR reactions are characterized by a three-step cycle involving (1) denaturation of the double-stranded template, (2) hybridization or annealing of primers to complementary regions of the target template, and (3) primer-directed synthesis of new DNA, also termed extension [99,116,153]. DNA fragments generated in each cycle serve as templates for subsequent rounds of amplification and the number of templates present doubles with each cycle, leading to an exponential accumulation of the product [99,116,153]. Typically, denaturation is carried out at 95°C, annealing at 55°C, and extension at 72°C, the temperature optimum for Taq polymerase. However, extension will still occur at nonoptimal temperatures, and annealing and extension can sometimes be combined into a single step [6]. PCR is by far the most widely reported rapid method for detection of Listeria, particularly L. monocytogenes, and a number of authoritative reviews have been written on this topic [76,99,153]. Once available only to a handful of specialist laboratories, PCR has since become accessible to a much wider user base. Factors responsible for easing PCR further into the mainstream testing environment include the commercial availability of prepackaged reagents and advances in automated detection of PCR products, which obviate the need for labor-intensive postamplification handling steps. Still, some barriers exist to routine adoption of PCR by the food-testing community, including the relatively high cost of instrumentation and a lack of standardized and validated methods for PCR in foods [85]. Several primers available for PCR-based detection of L. monocytogenes have been tabulated in recent reviews [76,153]. Commonly targeted genes in L. monocytogenes include the listeriolysin O gene, hly [5,6,14,20,54,56,59,69,96,112,146], and the gene for the invasion-associated protein p60, iap [14,15,22,53,69,118]. Other targets include genes for aminopeptidase C [152], internalins A and B [65], phospholipase C [24], a fibronectin-binding protein [42], and lmaA (also referred to as Dth-18), whose product is responsible for delayed-type hypersensitivity reactions in L. monocytogenes-immune mice [76,115]. Genus or species-specific 16S rRNA [48,142,143,146] and 23S rRNA [59,112] sequences have also been targeted. Apart from detection, several PCRbased methods have also been developed for characterization or typing of L. monocytogenes isolates [38,62,69,140]. Real-time PCR represents an important development in the field of PCR technology. Real-time refers to the ability to detect a PCR product as it forms from cycle to cycle, in contrast to endpoint detection approaches such as gel electrophoresis. Advantages of real-time methods include their wide dynamic ranges, quantitative nature, and the fact that both amplification and detection take place in the same closed tube, reducing the chance of contamination between reactions [56,98]. Because detection of PCR products is automated, the need for time-consuming postreaction handling is eliminated [98]. Several different chemistries are available for real-time PCR, including probe-based methods such as the 5′ nuclease (TaqMan) reaction and the use of fluorescence resonance energy transfer (FRET) probes or molecular beacons. These approaches use detection probes that hybridize to unique sequences within target amplicons. Advantages of probe-based methods include the ability to detect target amplicons in the presence of nonspecific PCR products and simultaneous

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detection of multiple amplicons in the same reaction. Cycle-to-cycle production of PCR products can also be monitored using fluorescent dyes that stain double-stranded DNA [64]. Although this approach is less expensive, it lacks the specificity of probe-based systems. In the 5′ nuclease reaction, the detection probe is labeled at the 5′ end with a reporter dye and at the 3′ end with a quencher molecule. During the extension step of PCR, Taq DNA polymerase displaces the detection probe from its target on the template and digests it, using the inherent 5′ to 3′ exonuclease activity of this enzyme [25,96]. In an intact probe, fluorescence from the reporter dye is effectively quenched. Upon digestion of the probe, the reporter dye is released into solution, away from the quencher, increasing the overall fluorescence of the system [25,96]. Signal generation in the 5′ nuclease reaction is very specific, as only those probes that are hybridized to their targets will be available for digestion by Taq polymerase. Design and operational factors affecting the performance of 5′ nuclease assays have been systematically examined using L. monocytogenes as a model target organism [82]. Use of more than one reporter dye enables multiplexing of the 5′ nuclease reaction. An example is the assay for the simultaneous and quantitative detection of both L. monocytogenes and generic Listeria developed by Rodríguez-Lázaro et al. [112]. Food or beverage systems in which L. monocytogenes–specific 5′ nuclease assays have been applied include water, cabbage, and various dairy products, including raw or pasteurized milk, yogurt, cottage cheese, and ice cream [25,96,56]. Reported sensitivities, per 25-g sample of food, range from 5 CFU after 20-h enrichment [25] to 1.42 × 102 CFU after direct extraction of DNA from spiked samples [56]. A non-real-time (endpoint) 5′ nuclease assay using an “asymmetric” set of singly labeled probes has also been suggested as a less expensive alternative to real-time, dual-probe approaches for detection of L. monocytogenes [69].

POTENTIAL PITFALLS

OF

PCR

Errors that can affect the specificity of the final test can be introduced at various points during PCR assay development. These include inaccuracies in the original sequences used for alignments during primer design and use of misidentified reference strains to validate assay performance [5]. Additionally, target sequences must be carefully chosen to ensure they are present only in the group to be detected [76,99]. Primer specificity should also be validated experimentally against an extensive panel of well-characterized reference strains consisting of both target and nontarget organisms [5,99]. Foods contain a number of components that can interfere with the PCR reaction. These PCR inhibitors include proteins, fats, polyphenolic compounds, target or primer-degrading nucleases, and competitors of Mg2+ [99,116]. Additionally, certain components of selective media used to enrich for Listeria may also have inhibitory activity, including acriflavin, bile salts, esculin, and ferric ammonium citrate [116]. Apart from dilution, approaches for removal of PCR inhibitors include centrifugation, filtration, immunomagnetic capture of target cells, adsorption of cells to hydroxyapatite or metal hydroxides, surface adhesion of cells to polycarbonate membranes, spotting of food washes onto filters impregnated with chelators and denaturants, and chaotropic precipitation of DNA [8,33,59,72,84]. The extreme sensitivity of PCR can become a liability when amplicons produced in previous reactions are present as contaminants. One approach for reducing false-positives from such carryover contamination involves substitution of dTTP with dUTP in the reaction mix. A short incubation with the enzyme uracil-N-glycosylase (UNG) at the beginning of each new reaction leads to degradation of contaminant amplicons (e.g., those containing uracil residues). Ideally, this enzyme would be inactivated during PCR cycling. However, residual activity after cycling has been reported for UNG isolated from Escherichia coli. Such activity could result in unwanted degradation of uracil-containing products before endpoint analysis. Use of a more heat-labile UNG, such as that described from a psychrophilic marine bacterium, may help circumvent this potential problem [120].

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The inability of PCR to distinguish between live and dead cells has often been cited as a key drawback of this method. Knowing whether a positive result derives from live cells or from “environmental” DNA is of significant practical value to food processors. At least two strategies have been devised for the PCR-based detection of live L. monocytogenes cells. One approach, reverse transcription-PCR (RT-PCR), takes advantage of the inherent lability of mRNA, which has a half-life on the order of a few minutes [68]. Because mRNA should be quickly degraded following cell death, its detection can be used as a sensitive indicator of the presence of viable cells [68,98]. Klein and Juneja [68] examined the suitability of three mRNA targets for RT-PCR. Using Southern hybridization to digoxigenin-labeled probes, these authors were able to detect an iap-specific product from between 10 and 15 L. monocytogenes cells inoculated from pure culture into nonselective media, with a total assay time of 54 h. Detection of hly or prfA messages was substantially less sensitive. Using iap-specific RT-PCR, low numbers of L. monocytogenes were also detected in an artificially contaminated cooked meat system after nonselective enrichment [68]. Norton and Batt [98] adapted the 5′ nuclease assay to detect hlyA mRNA, raising the possibility of automated detection of viable L. monocytogenes by real-time RT-PCR. Drawbacks of these mRNA-based approaches include difficulties in extracting target RNA and interference from DNA [68,98]. A second approach to PCR-based detection of viable cells is based on use of the DNA-binding dye ethidium monoazide (EMA), a reporter of membrane integrity [97,114]. This dye is excluded from intact (e.g., viable) cells, but readily enters those having damaged cell membranes. Once inside the cytoplasm, EMA binds to cellular DNA. Subsequent exposure to visible light results in photolytic activation of bound EMA and its covalent linkage to DNA. Unbound dye also undergoes photolysis and is no longer available for covalent binding. Nogva et al. [97] found that covalent linkage of EMA to pure DNA inhibited the 5′ nuclease reaction, suggesting the use of this approach for PCR-based discrimination of cell viability. An optimized EMA treatment gave good differentiation between live and dead cells for isopropanol-killed L. monocytogenes. EMA-mediated reductions in PCR signals also correlated well with plate counts when other methods of preparing killed cells (heat, benzalkonium chloride) were used [97]. Further work is needed to determine how well the EMA method will work with natural populations and if it can be applied in food systems.

DNA MICROARRAYS DNA microarrays are powerful tools for global genetic analyses of microorganisms. A single highdensity microarray can be used to simultaneously measure gene expression and identify speciesspecific polymorphisms for every gene in an organism. This enables researchers to identify genes associated with virulence or to examine physiological responses to stress and changes in the environment [27]. Alternatively, a single microarray could be used to detect multiple pathogens, and contain several pathogen-specific probes per organism [119,145]. In the analysis of Listeria, microarrays have been used to assess the genetic diversity among different strains of L. monocytogenes, enabling identification of lineage and serotype-specific differences in the genome content of these strains [17,114,155]. Host cell responses to infection with L. monocytogenes have also been characterized using microarray technology [23]. Apart from these more sophisticated applications, DNA microarrays have also found practical application as a means of endpoint detection in PCR [18,114,138]. In this latter application, DNA microarrays provide an alternative to gel electrophoresis, allowing the unambiguous detection of target amplicons [138]. This involves using them essentially as dense arrays of dot-blots against which PCR products are hybridized [18]. In conventional PCR, successful amplification is visualized by electrophoresis, with the appearance of a band correlated to the expected size of the fragment. Resolution of similarly sized fragments requires use of more complicated techniques such as denaturing gradient gel electrophoresis (DGGE) [22]. Using microarrays, a specific product can be demonstrated on the basis of its hybridization to the array. The high density of microarrays enables simultaneous detection of several genetic markers in a single experiment, even in the

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presence of nonspecific PCR products that might confound gel-based analyses [18,138]. In addition to planar arrays, microsphere-based arrays have also been developed for multiplex detection of pathogen-specific amplicons, including those from L. monocytogenes [34].

FLUORESCENCE

IN

SITU HYBRIDIZATION

First described for bacteria in 1989 [29], Fluorescence in situ hybridization (FISH) is a rapid nucleic acid–based method for identification of specific cells. In this technique, fluorescently labeled nucleic acid probes are hybridized to complementary rRNA sequences located on ribosomes within whole, permeabilized cells. Actively growing cells may contain several thousand ribosomes and the aggregate signal arising from multiple probe/ribosome binding events leads to sequence-specific fluorescence of target cells [2]. A typical FISH assay involves at least four different steps: fixation, hybridization, removal of nonspecifically bound probe by washing, and detection [3]. Although fixation alone is enough to permeabilize some cell types to probe entry, the thick cell wall of Listeria requires additional enzymatic or chemical digestion before hybridization with DNA-based probes [139]. FISH probes may be designed against either the 16S or the 23S ribosomal subunits. The 23S subunit is larger and contains several potentially useful variable regions, but the 16S subunit has been more thoroughly characterized, and is therefore more frequently used [2]. A number of reports have described development of Listeria or L. monocytogenes–specific probes targeting the 16S rRNA [13,48,88,121,142]. However, these probes were developed for PCR or membrane-based assays, rather than for detection of whole cells. The three-dimensional structure of the ribosome is complex, and potentially diagnostic sequences may be “buried” within this structure or located under protein binding sites. As a result, not all rRNA-targeted probes will bind to their complementary targets in whole bacterial cells. Two FISH-suitable DNA probes targeting the 16S rRNA of Listeria spp. have recently been described: Lis-637, which reacts with all Listeria species except for L. grayi, and Lis-1255, which reacts with all six Listeria species as well as with members of the closely related genus Brochothrix [118]. The specificities of these probes reflect the difficulty of designing FISH-suitable probes that react with all six species, yet are restricted to the genus Listeria. A FISH-based testing kit containing probes said to be specific for Listeria spp. or for L. monocytogenes is now commercially available (VIT-Listeria, vermicon, A.G., Munich). According to the manufacturer, Listeria spp. and L. monocytogenes can be detected in a single test with this slide-based kit. The test is said to require about 30 min of preparative time, and to be complete within 3 h. In addition to the sample, a positive and a negative control are done with each test. After hybridization, L. monocytogenes is visually differentiated from other Listeria spp. according to the color of probe-conferred fluorescence, with L. monocytogenes fluorescing red and Listeria spp. fluorescing green. This probe system was evaluated against cultural methods for detection of Listeria spp. and L. monocytogenes in 195 food samples (mainly minced meat) and 103 swabs from food-processing environments [124]. For standard cultural detection, samples were pre-enriched for 24 h in Half Fraser broth, transferred to Fraser broth for another 24 h, plated onto PALCAM or Oxford agars, and incubated for 48 h before biochemical testing. For rapid FISH-based detection, FISH analysis was done after the second liquid enrichment. For detection of L. monocytogenes, the rapid method yielded the same results as culture, but 48 h earlier. Detection of generic Listeria was also as rapid, but in some samples, strong green autofluorescence from nontarget bacteria reduced the sensitivity of the test [124]. Although most reports of FISH-based detection of Listeria spp. have involved DNA-based methods, peptide nucleic acid (PNA) probes have been shown to have substantial practical and functional advantages for detection of this genus [12]. PNAs are synthetic DNA mimics made by attachment of natural or modified nucleobases onto a repeating backbone of amide-linked N-(2-aminoethyl) glycine units [95]. As with DNA probes, PNAs hybridize to complementary DNA or RNA sequences through Watson–Crick base pairing, but they also exhibit several advantageous

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properties that can be ascribed to the uncharged, hydrophobic nature of the PNA backbone [123]. Some of these properties include accelerated hybridization kinetics, intrinsic resistance to nucleases and proteases, and the ability to penetrate recalcitrant biological structures such as the Gram-positive cell wall [123]. PNA probes are also able to bind their target sequences under high-stringency, denaturing conditions (e.g., low salt, high temperature, high pH). As a result, PNA probes are able to bind to portions of the ribosome that are inaccessible to DNA probes because of the higherorder structure of the ribosome [123]. Together, these properties make PNA probes uniquely suited for use as FISH probes, with important applications in detection of Listeria spp. (see later section titled “Flow Cytometry”). FISH has been used to great effect for cultivation-independent characterization of complex microbial communities of interest to environmental microbiologists [2]. Similar approaches may also be useful to describe microbial ecosystems occurring in foods [43]. For example, methods have recently been described for the FISH-based analyses of thinly sectioned, resin-embedded cheeses, including Stilton and Brie [36]. In this way, valuable information may be collected on the spatial arrangements and physical interactions of different cell types occurring within these foods, including pathogens such as L. monocytogenes.

RECOMBINANT BACTERIOPHAGE Phage-based diagnostic schemes take advantage of host specificities displayed by these viruses. Phage-bacterium recognition is mediated by specific receptors displayed on the host cell surface. For L. monocytogenes, known phage receptors include teichoic-acid-associated carbohydrate residues and peptidoglycan [73,79,132]. Infection begins with recognition and attachment to host surface receptors, followed by injection of phage nucleic acids into the host cell. As with other viruses, the machinery of the host cell is then subverted to make more virus particles [87]. The efficiency of phage-mediated delivery of nucleic acids to specific host cells forms the basis of their use as diagnostic agents. Development and application of a recombinant phage-based method for identification of Listeria spp. has been described [79,80]. In this work, a recombinant derivative of the Listeria-specific bacteriophage A511 was constructed. The recombinant phage, A511::luxAB, carried the gene for a Vibrio harveyi LuxAB fusion protein immediately downstream from the gene for the major capsid protein (cps) [79]. Bacterial luciferase catalyzes a light-producing reaction involving oxidation of reduced flavin mononucleotide (FMNH2) and long-chain aliphatic aldehydes by molecular oxygen. An aldehyde substrate (nonanal) was added to the system and postinfection transcription of the luxAB fusion within target cells resulted in Listeria-specific luminescence [79]. Because the reducing power needed to regenerate FMNH2 is derived from cellular metabolism and because dead cells do not support phage replication, only live host cells are detected with this method [91,125]. This phage-based detection approach was comparable in sensitivity to standard plating methods for Listeria, yet detection of L. monocytogenes was possible in food and environmental samples after 20 h of enrichment, compared to the 4 days required for the culture method [80]. Apart from selective identification of live target cells, an advantage of phage-based detection methods is that phage can be produced in relatively large quantities, an attractive feature for any diagnostic reagent [87]. Drawbacks include the inability to detect phage-resistant strains and the potential for contamination of certain foods (e.g., seafood) with naturally bioluminescent microflora that might confound the analysis [80,87,132].

ANTIBODY-BASED METHODS Antibodies play several essential roles in host defense against intracellular pathogens such as L. monocytogenes. These include preventing entry into host cells through binding of key surface molecules and neutralization of toxins and opsonization, leading to activation of the complement

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cascade [35]. The molecular selectivity that antibodies display in these natural roles also forms the basis for their use as diagnostic reagents. Antibodies are versatile and can be used to detect both whole cells and isolated cellular components [75]. A comprehensive review of L. monocytogenesspecific antigens and antibodies reported for their detection has been published [10]. Apart from detection, antibodies can also be used as capture agents for selective concentration of target cells (see later section titled “Cell Separation and Concentration”). Certain properties make antibodies uniquely suitable for detecting live target cells. These include the ability of some antibodies to bind epitopes present only in living cells [122,131] and their capacity to bind under mild, physiologically compatible conditions. Additionally, antibodies directed against surface antigens do not require cell permeabilization to reach their targets, and can therefore be used on unfixed (living) cells. These properties enable antibody-based labeling to be combined with cell analysis and sorting techniques such as flow cytometry, with subsequent cultural analyses of sorted cells.

POTENTIAL PITFALLS

OF

ANTIBODY-BASED METHODS

In comparison to amplification-based detection methods such as PCR, antibody-based methods are relatively insensitive. Typical detection limits for ELISA are ~105 to 106 CFU/mL [93,99]. Antibody concentrations can be varied to saturate available target molecules, but in some assay formats high concentrations can lead to deleterious effects such as cell clumping and precipitation [30]. Antibodies may also suffer from substantial cross-reactivity, and expression of antigens may vary under different cultural conditions or as a result of environmental stress [31,40,93]. Finally, genes coding for antigenic determinants can be acquired from closely related species through lateral transfer. As an example of this, strains of L. innocua expressing cell surface antigens characteristic of L. monocytogenes serogroup 4 have recently been isolated [73].

ADVANCES

IN

ANTIBODY-BASED TECHNOLOGIES

Typical methods of generating antibodies include immunization of animals for production of polyclonal antisera or use of monoclonal-producing hybridoma technology [60]. With the advent of phage display techniques, recombinant antibody fragments can now be selected from highly diverse libraries consisting of billions of clones [106]. In a phage display library, a large repertoire of single-chain or other antibody fragments is displayed on the surface of filamentous phage such as M13, fused to the phage’s coat proteins. Phage displaying antibody fragments that bind to a given cell type can be isolated from the library through sequential affinity selection or “biopanning.” In this process, phage are mixed in vitro with whole target cells, which are bound to a solid support. Phage having an affinity for target cells are captured, and nonbinding phage are washed away. Bound phage can then be eluted, propagated in a susceptible host, and subjected to further rounds of affinity selection [7,60,102,106]. Subtractive panning against cells of closely-related species may be used to remove phage that cross-react with nontarget cells [102]. The same overall approach may also be followed using a purified antigen instead of whole cells. Multiple panning iterations can result in selection of antibody fragments that are highly specific for target cells or antigen. Advantages of this technique include its speed, simplicity, and ability to detect antigens in their native (e.g., nondenatured) form [7,60,106]. Recently, Paoli et al. [102] isolated single-chain antibodies (scFvs) that react with L. monocytogenes, but not with other Listeria spp. Benhar et al. [7] used the same library (“Griffin.1”) to isolate scFvs for use in a Listeria-specific biosensor. Biopanning against a phage display library of llama single-domain antibodies has also been reported for selection of antilisteriolysin binding fragments, although affinities of these fragments were reported to be low [21]. An interesting variation on the immunoassay format used a superconducting quantum interference device (SQUID) for label-free detection of L. monocytogenes in solution [49]. Cells of

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L. monocytogenes were reacted with antibody-labeled magnetic spheres, and a pulsed magnetic field was applied. Differences in the magnetic relaxation signals of bound or unbound magnetic spheres, as measured with a SQUID microscope, allowed indirect detection of L. monocytogenes in solution. This technology is still in the early developmental stage and the reported sensitivity (5.6 × 106 L. monocytogenes cells per mL) was low. However, this approach differs from a typical immunoassay in that it is label free, and there is no need for immobilization of the target cells or for removal of unbound microspheres before taking measurements [49]. Through molecular imprinting (MIP), artificial antibodies or biological receptor mimics can be formed within a polymer matrix. Imprinted polymers capable of recognizing specific molecular substrates, viruses, or even whole cells have been demonstrated [51,52]. Some of the earliest work on whole-cell imprinting was done using L. monocytogenes as a template organism. In this work, chemically functional (lectin-reactive) “lithographic prints” of this organism were made on surfaces of polymer beads [1]. Future improvements in MIP-based recognition of whole cells may lead to new Listeria-specific bioaffinity surfaces for use in biosensing applications. Unlike natural molecular recognition elements (e.g., antibodies), molecularly imprinted polymers can be designed to have high physical and chemical stabilities, can bind target molecules under nonaqueous conditions, and may be reused multiple times [107].

CELL SEPARATION

AND

CONCENTRATION

Separation of low numbers of target cells from food matrices represents a major obstacle to rapid detection of specific foodborne bacteria, including Listeria [94]. Contamination can occur in a wide variety of foods, and may occur unevenly within a given food, resulting in “hot spots” or localized regions of higher bacterial concentration [33]. Effective methods for capture and concentration of cells from foods could potentially shorten enrichment times or even eliminate the need for growthbased enrichment completely [144]. Concentration techniques may also be useful for removal of food components that could interfere with assay performance. Examples include removal of particulate matter that could interfere with light scattering in flow cytometry, or removal of PCR inhibitors. Common physical means of separating cells from food matrices include filtration and centrifugation [8,69]. Methods available for nonspecific capture of bacteria based on cell surface properties include phase partitioning using aqueous polymers, surface adhesion to polycarbonate membranes, and immobilization on hydroxyapatite or metal hydroxide particles [8,81,103]. Dielectrophoresis provides a means for effective separation of bacteria from other biological particles having different dielectric properties [57] (see later section titled “Chip-Based Microanalytical Systems”). Immunomagnetic separation (IMS) enables specific capture and concentration of Listeria spp. directly from foods or environmental samples [59,90,96,134]. This approach uses commercially available paramagnetic beads whose surfaces are functionalized with Listeria-specific antibodies. Typically, samples are homogenized in buffer and large food particles are removed via centrifugation. Antibody-coated beads are then combined with the resulting supernatant liquid, and the sample is incubated with gentle mixing to facilitate cell binding [59]. Once cells are bound to the surface of the beads, a magnetic field is applied, trapping the beads in place. At this point, the supernatant liquid may be removed and the beads washed to allow more complete removal of food components. Immunomagnetically isolated cells may then be added to liquid or solid media for further growth or enumeration, or processed for molecular analyses. Other variations of the method include application of IMS after an initial enrichment, or the use of DNA-reactive beads to isolate DNA before PCR [66,96]. Problems reported for this technique include reduced recovery of beads from high-fat foods, and poor cell recovery when multiple wash steps were used [33,59]. The relatively small fraction of the original sample typically subjected to IMS has also been cited as a potential source of reduced assay sensitivity [66]. Use of less expensive nonmagnetic beads, combined with centrifugal recovery, has been suggested as an economically feasible approach to scaling up the immunoseparation of L. monocytogenes from foods to larger sample volumes [66].

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As another approach to this problem, one commercially available IMS-based system (PATHATRIX, Matrix MicroScience, Ltd., Cambridgeshire, UK) operates by continuously circulating an entire 1:10 dilution (225-mL + 25-g food sample) over a magnetic bead-based antibody “capture phase.” Heating of the sample during circulation allows simultaneous capture and enrichment, increasing the sensitivity of the method. Once cells are captured and washed, the magnet is removed and the beads are eluted for further cultural or molecular analyses. The system’s test for Listeria spp. is designated as an AOAC “Performance Tested” method. Another immunocapture system that addresses the need for concentration of low numbers of Listeria spp. and other target bacteria from large sample volumes has been described [94,144]. This system employs a fluidized bed of relatively large (~ 3-mm diameter) antibody-coated glass beads through which a liquefied food or environmental sample is passed at flow rates exceeding 100 mL/min. After capture, bound cells are detected via bead-based ELISA or with molecular methods. The system is able to detect as few as 100 CFU/mL in as little as 30 min and is volume independent, being able to process samples ranging from milliliters to several liters.

ADDITIONAL METHODS FLOW CYTOMETRY Flow cytometry is a basic diagnostic tool that facilitates the rapid analysis of complex cell populations according to single cell fluorescence characteristics. Data on multiple cellular parameters may be collected simultaneously, including information on cell count, size, or response to diagnostic fluorescent probes. Fluorescent probes interacting with a variety of cellular targets are commercially available, and range from nucleic acid–binding dyes and respiratory substrates to fluorescently labeled antibodies or rRNA-targeted nucleic acid probes. In flow cytometry, cells suspended in a liquid sample are passed individually through an intense light source, such as a laser or an arc lamp. Data on cellular characteristics, such as light scattering and probe-conferred fluorescence, are collected and saved as a computer file for subsequent offline analysis. The information-rich nature of these files enables detailed and quantitative analyses of specific populations and subpopulations of interest. Software for data analysis is available commercially or as freeware and provides flexible options for data presentation. Different types of data displays can be used to highlight particular features of the dataset. In general, data collected on several thousand events are displayed as dot plots, contour plots, or histograms, using a four-decade log scale. Until recently, most commercially available flow cytometers were large, expensive machines optimized for immunological studies on mammalian cells. However, a handful of smaller, less expensive machines, some of which are designed specifically for microbial analysis are now available. These include “turnkey” or “pushbutton” systems incorporating automated reagentdispensing and sample-handling capabilities. Many of these systems also offer precise, metered sample injection, enabling the absolute enumeration of target cells as a function of sample volume. Combined with commercial availability of a wide variety of fluorescent stains and probes, microbial cytometry is now more accessible than ever before. As a general tool, flow cytometry can be used in a wide variety of fluorescence-based applications. This method has been used to study several aspects of the biology of L. monocytogenes, including its uptake and survival in peripheral blood leukocytes [109], identification of potential virulence genes induced during the infection process [151], characterization of bacteriocin-mediated cell damage [108,126], and fluorescence-based viability assessment of both L. monocytogenes and other Listeria spp. [61,108]. However, very little has been published regarding use of flow cytometry for rapid detection of L. monocytogenes or other Listeria spp. In their pioneering work, Donnelly et al. combined use of fluorescent antibodies and nucleic acid staining with flow cytometry to detect L. monocytogenes in raw milk [30,31]. This work demonstrated the potential of flow cytometry for detecting specific microorganisms in foods. Because the antibodies used in this study showed

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FIGURE 8.1 Combined PNA-FISH and flow cytometry for rapid detection of Listeria spp. in hot dogs. Commercially purchased hot dogs were contaminated with L. monocytogenes, vacuum-packaged, and stored for 1 week under conditions of mild temperature abuse (8°C). Samples were then opened, washed with a small amount of a dilute, nonselective nutrient medium (1/4-strength MRS broth), and either processed immediately for hybridization and cytometry or incubated for up to 70 min at 35°C before processing. Whereas L. monocytogenes was detected directly in washes from contaminated samples without incubation (panel B), brief exposure to dilute nutrient media allowed further growth of the cells, leading to brighter hybridizations with the rRNA-targeted PNA probes (panel D). Panels A and C represent noninoculated controls without incubation, or with 70-min exposure to dilute nutrient media, respectively. Samples were analyzed using a Becton Dickinson FACSCalibur.

substantial cross-reactivity, especially with Staphylococcus aureus, a selective enrichment step was done before cytometric analysis. After enrichment, it was possible to differentiate Listeria from nontarget microflora on the combined basis of light scatter, nucleic acid staining, and immunofluorescence characteristics [31]. In recent work, we have combined PNA-FISH with flow cytometry for rapid detection of Listeria spp. in media or in washes from vacuum-packaged RTE meats (Figure 8.1). Using this approach, L. monocytogenes was detected directly from contaminated packages of hot dogs held for up to a week under conditions of mild temperature abuse (Figure 8.1B). Brief postsampling incubation in dilute nutrient media yielded further cell growth and rRNA production, leading to brighter hybridizations with the rRNA-targeted probes (Figure 8.1D). Compared to hot dogs, sliced turkey samples contained high levels of particulate matter, likely caused by enhanced microbial degradation of their open surfaces, with associated release of meat fragments and production of fat micelles. However, detection of L. monocytogenes in these samples was still possible even without use of additional methods for sample cleanup, such as filtration (Figure 8.2). For both meat systems, hybridizations were very brief (10–15 min) and cell fixation and permeabilization steps

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FIGURE 8.2 Combined PNA-FISH and flow cytometry for rapid detection of Listeria spp. in sliced turkey. Commercially purchased sliced turkey meat was contaminated, processed, and analyzed as described for hot dogs in Figure 8.1. Background noise from particulate matter (population A) was high in these unfiltered washes, but did not interfere with detection of L. monocytogenes. In this figure, two discrete subpopulations of L. monocytogenes can be seen: the main population (B) and a smaller, more active population containing higher amounts of rRNA (C).

were accomplished simultaneously with resuspension of pelleted wash material in 50% ethanol. For RTE meat samples contaminated with high numbers of listeriae, combined incubation, fixation, hybridization, and cytometric analysis steps were accomplished in as little as 2 h. Without optimization, we have been able to detect approximately 105 cells/mL of stationary phase L. monocytogenes against a background of 107 cells/mL nontarget bacteria in spiked hot dog wash. Approaches for improving the sensitivity of detection include “gating out” or ignoring signals from smaller food particles, fat micelles, or unlabeled (nontarget) microflora and removal of larger food particles via filtration. The use of a cytometer optimized for microbial detection should further improve the sensitivity of this assay, as some such systems are able to detect fewer than 100 well-labeled target cells per milliliter.

BIOSENSORS Biosensors are compact analytical devices capable of translating the binding or recognition of a target analyte into an output signal that is proportional to the concentration of analyte in the sample [133]. The three major components of a biosensor are a recognition element or bioaffinity agent that is bound to the surface of the biosensor, a means of generating an electrical signal from the binding of the target to this recognition element (a transducer), and an output device [75]. The types of recognition

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FIGURE 8.3 Detection of L. monocytogenes using a surface plasmon resonance-based biosensor. Output from a flow-through surface plasmon resonance (SPR) biosensor is shown. The presence of L. monocytogenes in suspension was detected through shifts in resonant wavelength upon binding of target cells to antilisterial antibodies immobilized on the sensor’s surface. The magnitude of each shift was proportional to the number of Listeria present. Although the sensitivity of this system was low (106 CFU/mL), it was comparable to ELISA using the same antibodies. Advantages over standard ELISA include label-free detection of target cells and the ability to follow the capture reaction in real time. Replacement of solutions, addition of various levels of L. monocytogenes, and changes in flow rate are indicated by arrows. Key: tw = 0.05% Tween in PBS; LM 106 = 106 CFU/mL L. monocytogenes in the same buffer; f20 = flow rate of 20 µL/min). (Reprinted from Koubová, V., B. Brynda, L. Karasová, J. Skvor, J. Homola, J. Dostálek, P. Tobiska, and J. Rosicky, 2001. Detection of foodborne pathogens using surface plasmon resonance biosensors. Sens. Actuators B Chem. 74: 100–105. With permission from Elsevier.)

elements that could be incorporated into biosensor design include nucleic acids (e.g., single-stranded DNA, peptide nucleic acids, DNA or RNA aptamers, etc.), antibodies or recombinant antibody fragments, enzymes, lectins, peptides, or even whole cells [60,71,133,154]. Methods for transducing a specific binding reaction into a useful signal include optical, acoustic, electrochemical, or thermal approaches [71,75,133]. For example, surface plasmon resonance (SPR), an optical approach, detects minute changes in refractive index at the surface of a functionalized waveguide upon binding of the target analyte (Figure 8.3). The waveguide can be functionalized with either antibodies or nucleic acids, and changes in refractive index are proportional to the amount of analyte that binds to these bioaffinity agents [129]. Acoustic sensors, such as quartz crystal microbalances (QCMs), operate on the principle that binding of an analyte to the surface of a functionalized crystal will increase its mass and modulate the frequency at which the crystal resonates [75,133]. Again, changes in resonant frequency are proportional to the amount of analyte bound. Attractive features of biosensors include their potential to provide rapid, label-free detection with minimal sample preparation, the capacity to detect multiple analytes, and the ability to follow binding reactions in real time. Drawbacks include questions about their long-term stability under “real-world” processing conditions, whether or not the bioaffinity agent can be successfully

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regenerated, and the small volumes that are typically sampled [75]. Additionally, the specificity or sensitivity of a biosensor is ultimately limited by the binding properties or quality of the bioaffinity reagent used [70,128]. Several antibody-based biosensors to detect whole L. monocytogenes cells have been described. Depending on the experimental setup and the antibodies used, reported detection limits (in buffer) vary from 500 CFU/mL to 4 × 108 CFU/mL, with total assay times ranging from minutes to hours. To date, there are few reports detailing the use of biosensors to detect L. monocytogenes directly in foods. However, biosensor-based detection of L. monocytogenes in enrichment cultures could potentially shorten the time-to-result compared with traditional media-based detection. Biosensor formats that have been investigated to detect L. monocytogenes include QCM [89,135], enzymelinked amperometric detection using screen-printed electrodes functionalized with L. monocytogenesspecific antibodies [7,26], evanescent wave fiber-optic systems [41,128], and SPR [70].

CHIP-BASED MICROANALYTICAL SYSTEMS Microscale bioanalytical systems (or biochips) are a result of the convergence of materials science and biology and may combine electrical, mechanical, chemical, and/or microfluidic approaches for analysis of biological materials. Steps such as cell manipulation, addition, mixing and washing of reagents, temperature cycling, and analyte detection can be done sequentially within the same device [39,58]. Although the amount of analytical material handled by a single biochip device is small, so are the amounts of potentially expensive reagents used. Multiple devices may be operated in parallel, and they are amenable to automation. Analytical methods that have been successfully translated to the microscale and could potentially be incorporated within chip-based analytical systems include flow cytometry and cell sorting [39], impedance spectroscopy [45,46], the polymerase chain reaction, and various isothermal methods for nucleic acid amplification [58]. Movement of biological particles (molecules or cells) on or through a biochip can be directed through application of an electrical field. Examples include use of a DC electrical field for electrophoretic movement of charged bioparticles or the use of an AC field for the dielectrophoretic manipulation of polarized (neutral or charged) bioparticles [57]. Dielectrophoresis is especially useful, as it can be used to move, trap, separate, or concentrate cells based on their dielectric properties, which may differ according to cell volume, structure, composition, or viability status [19,77]. An example of dielectrophoresis for on-chip separation of L. monocytogenes from red blood cells is given in Figure 8.4. Other chip-based approaches have been described to detect and characterize Listeria spp. [19,45,46,77]. Bacterial metabolism of a low-conductivity medium alters its electrolyte concentrations, allowing detection of bacterial growth over time by measuring changes in impedance. Gómez et al. [46] constructed a microfluidic biochip able to measure impedance of bacterial suspensions in a very small volume of medium (~5 nL) over a 100 Hz–1 Mhz range. The small volume of the test chamber increases the effective concentration of bacterial cells, so that presence of only a few metabolizing cells can lead to rapid changes in impedance within a short time [46]. After off-chip incubations of 2 h, L. innocua or L. monocytogenes could be detected to a level of ~100–200 cells. Among limitations of the present work is that there is no mechanism for ensuring that impedance signals are derived from the activity of target cells. Antibody-based capture of Listeria spp. or use of Listeria-selective conductance media may be potential options for addressing this need. Microfabricated devices have also been described for distinguishing live Listeria from heat-killed Listeria based on either electrophoretic mobility or dielectrophoretic properties [19,77]. In one study, dielectrophoretic separation of mixed populations of live and dead L. innocua in water was possible with a reported efficiency of 90% [77].

SPECTROSCOPIC METHODS Vibrational methods such as Fourier transform infrared (FT-IR) and Raman spectroscopies provide a means of obtaining unique spectral “fingerprints” of specific cell types and can discriminate

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FIGURE 8.4 Dielectrophoretic separation of Listeria monocytogenes from red blood cells. A mixture of L. monocytogenes (LM) and red blood cells (RBC) was introduced onto a microelectronic array of platinum electrodes fabricated on the surface of a silicon wafer (A). A 10-KHz AC voltage was applied in a “checkerboard” mode, with adjacent electrodes along either vertical or horizontal lines receiving signals of the opposite polarity. In response to the applied field, LM cells accumulated on the circular electrodes and RBC accumulated at positions between the electrodes (B). Stronger dielectrophoretic forces acting on LM cells allowed the selective removal of RBC from the system by washing (C and D). The captured LM cells could then be released from the array by turning off the electric field. Alternatively, captured cells may be subjected to on-chip identification via electric field–mediated immunoassay or nucleic acid–based methods. (Example kindly provided by Y. Huang and D. Hodko, Nanogen, San Diego, California.)

between bacteria at the genus, species, or strain level [86,113]. These spectral signatures are a function of the intrinsic biochemical composition (lipids, proteins, carbohydrates, and nucleic acids) of each cell type and do not depend on the use of labels or dyes [86]. Whole-cell FT-IR and Raman spectroscopies are nondestructive techniques. Destructive methods such as matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) may also be used [86,92,113,141]. Typically, FT-IR analyses of bacteria are done by drying liquid cultures or resuspended colonies onto an IR-transparent substrate, such as a zinc selenide crystal [55,74,92,113]. Alternatively, disposable optical polymer films may be used. These provide a reproducible, archivable substrate—attributes that would be beneficial if FT-IR spectroscopy were to be used for routine identification of pathogenic bacteria [92]. During spectral collection, multiple scans (hundreds to thousands) may be averaged to enhance signal-to-noise ratios [86,92]. Total collection times vary according to experimental setup, but range from less than 1 min to 45 min [86]. Several multivariate statistical techniques can be used for analysis of the resulting complex spectra, including principal component

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analysis (PCA) and hierarchical component analysis (HCA) [86,92,113]. In addition to whole cells, preparations of cellular components, such as fatty acid methyl esters, may also be analyzed with spectroscopic techniques [147]. Advantages of spectroscopic methods are that they are fast, accurate, require minimal sample preparation, and can be coupled with objective means for data analysis [55,74,113]. Drawbacks are that cells must be isolated in pure culture before analysis and that high cultural densities (~108–109 CFU/mL) are often needed to obtain reproducible spectra [86,113]. However, recent advances include methods for analysis of mixed cultures using MALDI-MS [141] and the ability to collect spectra from microcolonies or individual cells using microspectroscopic methods [50,86]. A number of studies have focused on the use of spectroscopy to identify Listeria or L. monocytogenes. Holt et al. [55] used FT-IR spectrometry to discriminate between all six species of Listeria, with an additional distinction made between L. grayi and L. grayi subsp. murrayi. Figure 8.5 shows the graphical output of transformed spectral data for the Listeria strains examined in this work. Lefier et al. [74] examined effects on spectra of different cell growth conditions and sample preparation procedures and found the method to be robust and relatively unaffected by changes in these variables.

10

L. grayi L. ivanovii

Canonical Variate 1

5

L. grayi subsp. murrayi

L. innocua 0

L. monocytogenes

L. welshimeri –5

L. seeligeri

–10 –10

–5

0

5

10

Canonical Variate 2

FIGURE 8.5 Differentiation of Listeria spp. using Fourier transform infrared (FT-IR) spectroscopy. Listeria spp. grown in liquid culture were pelleted, washed, and resuspended in distilled water. Portions of these suspensions were dried (50°C, 45 min) on the surface of a ZnSe optical plate and infrared spectra were collected. Spectral data were transformed for canonical variate (CV) analysis. A projection of two of the resulting canonical variates is shown. All Listeria species (including L. grayi subsp. murrayi) could be distinguished from each other using this method. Although spectral clusters for L. monocytogenes and L. seeligeri are superimposed in this two-dimensional projection, the two species could be clearly differentiated using additional canonical variates (not shown). (Reprinted from Holt, C., D. Hirst, A. Sutherland, and F. MacDonald. 1995. Discrimination of species in the genus Listeria by Fourier transform infrared spectroscopy and canonical variate analysis. Appl. Environ. Microbiol. 61: 377–378. With permission from the American Society for Microbiology.)

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These authors also found that spectral analysis could be used to differentiate between two serotype groups of L. monocytogenes—one containing serotypes 4 and 4b and the other containing serotypes 1, 1/2b, and 1/2c. Using near-infrared Fourier transform (FT-NIR) spectroscopy, RodriguezSaona et al. [113] were able to clearly differentiate L. innocua from other bacteria, including Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa. Lin et al. [78] used FT-IR spectroscopy to differentiate between Listeria spp. and also found differences in spectra collected from “healthy” cells and those subjected to injury via sonication. Grow et al. [50] described a chip-based system for capture and on-chip spectral fingerprinting of microorganisms using surface enhanced Raman scattering (SERS) microscopy. Capture agents investigated for generic Listeria or L. monocytogenes included polyclonal antibodies, various lectins, and fibronectin. Once bound to the surface of the chip, Listeria and other target cells were identified using wholeorganism Raman spectra.

CONCLUSIONS To maintain a safe and wholesome food supply, the food industry needs access to rapid, reliable, and sensitive methods of detecting bacteria, including Listeria spp. and L. monocytogenes. Other desirable attributes include simplicity, cost-effectiveness, quantitative capacity, and online or real-time capabilities. Although no single method can meet these ideal demands, rapid microbial detection is a constantly evolving field characterized by continuous technological advances. Future developments will undoubtedly bring the state of the art closer to these ideals. This chapter has reviewed several analytical methods reported for detection of Listeria. These include both established methods (PCR, antibody-based methods) and more experimental technologies (biosensors, chip-based systems, spectroscopy). Rapid methods are valuable tools for routine screening of foods and environmental samples, but will not entirely replace standard culture in the near term. At present, negative results by rapid methods are considered definitive, but a positive result is viewed as presumptive and must be confirmed through culture [99]. Changes in this outlook will require both advances in rapid detection technology and regulatory acceptance of alternative methods. Apart from the detection step itself, sample preparation and adequate sampling procedures remain key challenges in development of effective rapid detection techniques [94]. The sheer diversity of food matrices and the fact that Listeria may be present at very low levels are particularly daunting challenges [32,94,114]. As a result, most rapid detection technologies still require a cultural enrichment step, although recent advances in immunological separation techniques have sought to circumvent this need [144]. Data regarding the performance of rapid detection methods are most often collected on laboratory-grown cells cultured under optimal conditions. Listeria present in foods or in the food-processing environment may be subjected to a variety of stressful or injurious conditions, including heating, freezing, exposure to sanitizers, and high acid or salt concentrations [32,40]. Rapid methods capable of discriminating between living, dead, or sublethally injured cells are needed. Finally, advances in technologies for microbial inactivation such as use of high pressure, pulsed electric fields, pulsed light, ohmic heating, or ozonolysis are not well characterized in terms of their physiological effects on bacterial cells. Further studies should address how use of such alternative processing methods may impact the ability of rapid methods to detect Listeria spp. in foods.

ACKNOWLEDGMENTS This work was supported in part by a grant from the North American Branch of the International Life Sciences Institute (ILSI N.A.). The opinions expressed herein are those of the authors and do not necessarily represent the views of ILSI. Additional support was provided by grants from sponsors of the Food Research Institute, and by the College of Agricultural and Life Sciences, University of Wisconsin–Madison.

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136. Vázquez-Boland, J.A., M. Kuhn, P. Berche, T. Chakraborty, G. Domínguez-Bernal, W. Goebel, B. González-Zorn, J. Wehland, and J. Kreft, 2001. Listeria pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev. 14: 584–640. 137. Vaz-Velho, M., G. Duarte, and P. Gibbs, 2000. Evaluation of mini-VIDAS rapid test for detection of Listeria monocytogenes from production lines of fresh to cold-smoked fish. J. Microbiol. Methods 40: 147–1551. 138. Volokhov, D., A. Rasooly, K. Chumakov, and V. Chizhikov, 2002. Identification of Listeria species by microarray-based assay. J. Clin. Microbiol. 40: 4720–4728. 139. Wagner, M., M. Schmid, S. Juretschko, K.-H. Trebesius, A. Bubert, W. Goebel, and K.-H. Schleifer, 1998. In situ detection of a virulence factor mRNA and 16S rRNA in Listeria monocytogenes. FEMS Microbiol. Lett. 160: 159–168. 140. Wagner, M., A. Lehner, D. Klein, and A. Bubert, 2000. Single-strand conformation polymorphisms in the hly gene and polymerase chain reaction analysis of a repeat region in the iap gene to identify and type Listeria monocytogenes. J. Food Prot. 63: 332–336. 141. Wahl, K.L., S.C. Wunschel, K.H. Jarman, N.B. Valentine, C.E. Petersen, M.T. Kingsley, K.A. Zartolas, and A.J. Saenz, 2002. Analysis of microbial mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Chem. 74: 6191–6199. 142. Wang, R.-F., W.-W. Cao, and M.G. Johnson, 1991. Development of a 16S rRNA-based oligomer probe specific for Listeria monocytogenes. Appl. Environ. Microbiol. 57: 3666–3670. 143. Wang, R.-F., W.-W. Cao, and M.G. Johnson, 1992. 16S rRNA-based probes and polymerase chain reaction method to detect Listeria monocytogenes cells added to foods. Appl. Environ. Microbiol. 58: 2827–2831. 144. Weimer, B.C., M.K. Walsh, C. Beer, R. Koka, and X. Wang, 2001. Solid-phase capture of proteins, spores, and bacteria. Appl. Environ. Microbiol. 67: 1300–1307. 145. Wells, J.M. and M.H.J. Bennik, 2003. Genomics of food-borne bacterial pathogens. Nutr. Res. Rev. 16: 21–35. 146. Wesley, I.V., K.M. Harmon, J.S. Dickson, and A.R. Schwartz, 2002. Application of a multiplex polymerase chain reaction assay for the simultaneous confirmation of Listeria monocytogenes and other Listeria species in turkey sample surveillance. J. Food Prot. 65: 780–785. 147. Whittaker, P., M.M. Mossoba, S. Al-Khaldi, F.S. Fry, V.C. Dunkel, B.D. Tall, and M.P. Yurawecz, 2003. Identification of foodborne bacteria by infrared spectroscopy using cellular fatty acid methyl esters. J. Microbiol. Methods 55: 709–716. 148. Wiedmann, M., J. Czajka, F. Barany, and C.A. Batt, 1992. Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction. Appl. Environ. Microbiol. 58: 3443–3447. 149. Wiedmann, M., J.L. Bruce, C. Keating, A.E. Johnson, P.L. McDonough, and C.A. Batt, 1997. Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential. Infect. Immun. 65: 2707–2716. 150. Wiedmann, M., 2002. Detection and characterization of Listeria monocytogenes. J. AOAC Int. 85: 494. 151. Wilson, R.L., A.R. Tvinnereim, B.D. Jones, and J.T. Harty, 2001. Identification of Listeria monocytogenes in vivo-induced genes by fluorescence-activated cell sorting. Infect. Immun. 69: 5016–5024. 152. Winters, D.K., T.P. Maloney, and M.G. Johnson, 1999. Rapid detection of Listeria monocytogenes by a PCR assay specific for an aminopeptidase. Mol. Cell. Probes 13: 127–131. 153. Winters, D.K., 2002. Polymerase chain reaction for detection of Listeria monocytogenes. Methods Mol. Biol. 179: 245–258. 154. Wittman, C., K. Riedel, and R.D. Schmid, 1997. Microbial and enzyme sensors for environmental monitoring. In Handbook of Biosensors and Electronic Noses, Medicine, Food, and the Environment, Ed., E. Kress-Rogers. CRC Press, Boca Raton, FL. pp. 299–347. 155. Zhang, C., M. Zhang, J. Ju, J. Nietfeldt, J. Wise, P.M. Terry, M. Olson, S.D. Kachman, M. Wiedmann, M. Samadpour, and A.K. Benson, 2003. Genome diversification in phylogenetic lineages I and II of Listeria monocytogenes: identification of segments unique to lineage II populations, J. Bacteriol. 185: 5573–5584.

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Subtyping Listeria 9 monocytogenes Lewis M. Graves, Bala Swaminathan, and Susan B. Hunter CONTENTS Introduction ....................................................................................................................................283 Conventional Methods ...................................................................................................................284 Serotyping.............................................................................................................................284 Bacteriophage Typing (Phage Typing).................................................................................285 Bacteriocin Typing ...............................................................................................................286 Antimicrobial Susceptibility Testing....................................................................................286 Molecular Methods ........................................................................................................................287 Multilocus Enzyme Electrophoresis ....................................................................................287 Chromosomal DNA Restriction Endonuclease Analysis (REA).........................................287 Restriction Fragment Length Polymorphism Analysis: Ribotyping and Other Methods............................................................................................288 DNA Macrorestriction Analysis by PFGE...........................................................................291 Random Amplification of Polymorphic DNA (RAPD).......................................................292 Repetitive Element-Based Subtyping...................................................................................294 Amplified Fragment Length Polymorphism ........................................................................294 DNA-Sequence-Based Subtyping Strategies .......................................................................295 Comparison of Methods Used to Subtype L. monocytogenes ......................................................297 References ......................................................................................................................................299

INTRODUCTION Most bacteria at the species level have sufficient phenotypic and genotypic diversity to allow for identification of different subtypes. Therefore, phenotyping and genotyping systems—singly or in combination—provide useful subtyping schemes for pathogenic bacteria. The various subtyping systems reviewed in this chapter provide different degrees of discrimination among Listeria monocytogenes isolates. Distinguishing individual strains or groups of strains using these systems in epidemiologic studies has allowed researchers to obtain information on relationships between isolates, identify disease outbreaks, identify source of infections in outbreaks and sporadic disease settings, and determine modes of transmission for the organism. We present a broad overview

Note: Use of trade names is for identification only and does not imply endorsement by the Public Health Service or by the U.S. Department of Health and Human Services. The manuscript for this chapter was submitted to the editors on December 2, 2003. The information provided in this chapter and the references cited herein are current as of December 2003.

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of the subtyping methods that have been applied to L. monocytogenes and where appropriate, discuss briefly the strengths and weaknesses of each. Since publication of the second edition, the genome of Listeria monocytogenes serotype 1/2a (EGD strain) has been sequenced [45]; the genome sequence of L. monocytogenes serotype 4b has been completed, and BLAST searches against the genome sequence can be made at the Web site of The Institute of Genomic Research (available at http://www.tigr.org). Consequently, development of more sophisticated molecular techniques are forthcoming or are awaiting evaluation and validation. However, in this chapter we will continue to separate the most commonly used typing methods for L. monocytogenes into two major categories: conventional methods and molecular methods. At present, each of these methods has some utility; however, with extraordinary developments in nucleic acid technology and wide availability of these technologies, some conventional methods may not be used in the future. L. monocytogenes is among the first of the pathogenic bacteria for which a concerted and internationally coordinated attempt has been made to critically evaluate various available subtyping methods and to standardize the more useful methods. In 1996, J. Bille and J. Rocourt organized the World Health Organization (WHO) Multicentre Listeria monocytogenes Subtyping Study. The results of Phase I of this study were published in a special issue of the International Journal of Food Microbiology [39] and will be referenced throughout this chapter.

CONVENTIONAL METHODS SEROTYPING Serotyping has been one of the classic tools for epidemiologic and sporadic case studies of L. monocytogenes [41,58], and many scientists consider it the “gold standard” for typing L. monocytogenes. Strains of L. monocytogenes differ in antigenic determinants expressed on the cell surface. Such antigenic variations are produced by many different surface structures, including lipoteichoic acids, membrane proteins, and extracellular organelles (e.g., flagella and fimbriae). These differences can be identified by serologic typing (serotyping). Strains of Listeria species are divided into serotypes based on somatic (O) and flagellar (H) antigens [101]. Flagellar antigens as well as somatic antigens must be identified to type strains of serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, and 3c. The remaining serotypes all have the same flagellar antigens (A, B, and C). Serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, and 3c can be identified with two O antisera (one with antibodies to factor I and the other with antibodies to both factors I and II) and three H antisera (one with antibodies to factors A and B, one reacting with C, and one reacting with D). With antisera for O factors V and VI, VII and IX, VIII, X, XI, and XV, strains of serotypes 4a, 4b, 4c, 4d, 5, 6a, and 6b can be typed [116]. Serotype 4bX is a variant of serotype 4b and was implicated in an outbreak of listeriosis in the United Kingdom that was traced to contaminated pâté [73]. Most (95%) human infections are caused by strains of L. monocytogenes belonging to serotypes 1/2a, 1/2b, and 4b. Therefore, serotyping alone is of limited value in epidemiologic investigations. In the WHO Multicentre L. monocytogenes Subtyping Study, Schönberg et al. [99] found that all of 80 strains tested by serotyping were typeable. However, there was complete agreement between the six participating laboratories on the serotype of only 49 (61%) of the tested strains (21 of serotype 1/2a and 28 of serotype 4b). The intralaboratory reproducibility, assessed on 11 duplicate strains, ranged from 82 to 100%, with a median value of 91%. Interlaboratory reproducibility varied from 64 to 95%; no laboratory correctly identified the two serotype 4bX strains in the set. Schönberg et al. [99] concluded that there is a critical need for good-quality antisera prepared by using standardized strains. Also, they emphasized the need to completely and efficiently absorb these antisera to produce good-quality factor sera.

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During the 14th International Symposium on Problems of Listeriosis in Mannheim, Germany, Lehmann and Schönberg [62] reported the results of the WHO Phase III Serotyping Study of L. monocytogenes. Six strains of L. monocytogenes (serotype 1/2a, 3a, 3c, 4b, 4c, and 4d) were used representing all of the important O- and H-factors. Seven laboratories from various countries were selected for serotyping these strains using two methods: their in-house method and a suggested simple procedure that required commercially produced Listeria Antisera (Denka Seiken, Tokyo). Of seven laboratories, five sent in results to be analyzed. Four of the five laboratories using their in-house method identified the expected serotype of the six strains and one identified the serotype for five strains. Using the suggested simple method, four out of five laboratories were able to identify the expected serotype of the six strains; one identified the serotype for four strains. They concluded that it is possible to use the simple method for the identification of O- and H- antigenic factors of L. monocytogenes and that the six strains used in the evaluation are suitable as reference strains for serotyping L. monocytogenes. Serotyping has poor discriminating power when compared to other subtyping methods. Isolates from foods and the environment are frequently nontypeable with standard typing antisera. Nevertheless, serotyping provides valuable information for rapid screening of groups of strains isolated during suspected outbreaks. Serotype information allows elimination of isolates that are not part of an outbreak and facilitates the efficient application of other more sensitive but time-consuming subtyping methods.

BACTERIOPHAGE TYPING (PHAGE TYPING) Numerous lytic bacteriophages have been identified for Listeria spp. [64]. Listeria monocytogenes isolates can be characterized by their patterns of resistance or susceptibility to a standard set of phages as demonstrated by Rocourt et al. [90]. Until the recent advent of molecular subtyping, phage typing was often used in conjunction with serotyping for epidemiologic investigations because of its high discriminating power [7,71,90,98]. The WHO Multicentre L. monocytogenes Subtyping Study on phage typing was done using an international phage set in five laboratories and unique phage sets in two laboratories [70]. When typed using the international phage set, 20–51% of the isolates were found to be nontypeable. Nontypeability was a greater problem among strains of serogroups l/2 and 3 (28–72%) than for serogroup 4 (11–22%) when these isolates were typed using the international phage set. The two laboratories that used unique phage sets had fewer problems with nontypeable strains. One of these laboratories was able to type all strains of serogroup 4 and 81% of strains of serogroups 1/2 and 3. The reproducibility of phage typing among the participating laboratories was 79% when laboratorians used criteria for interpretation previously proposed for the international phage set [72]. Based on findings of this WHO Multicentre Study, McLauchlin et al. [72] recommended that researchers review phages in the international set and consider additions to increase typeability of strains. Lemaitre et al. [63] have proposed a method that facilitates detection of induced phages; this procedure may be useful for identifying additional typing phages. McLauchlin et al. [70] suggested that better interlaboratory reproducibility may be achieved by standardization of phage suspensions, propagation strains, and methodology. Marquet-van der Mee et al. [68] used a panel of 395 L. monocytogenes isolates to evaluate seven experimental phages for inclusion in the international phage set for epidemiological typing of L. monocytogenes. Results of their evaluation showed that inclusion of five of the experimental phages contributed greatly to the overall typeability and discriminating power of the system, especially for strains within serogroup 1/2. Using centrally propagated phages—as does the Central Public Health Laboratory, London—for phage typing of Salmonella serotypes may be helpful. Despite its high discriminating power and easy applicability to large numbers of strains, phage typing is available only at selected national and international reference laboratories because this

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method necessitates maintaining stocks of biologically active phages and control strains. Although the procedure is technically not very demanding, it suffers from considerable experimental as well as biologic variability. The percentage of nontypeable strains may vary with the standard phage set used. Nevertheless, phage typing remains the only practical method that can be rapidly applied to type strains in massive outbreaks. Rocourt et al. [91] phage-typed more than 16,000 isolates in 1 year during the investigation of an outbreak in France in 1993 in which “pork tongue in jelly” was implicated as the vehicle of infection.

BACTERIOCIN TYPING Bacteriocins (monocins) were first isolated from L. monocytogenes by Sword and Pickett [111] and Hamon and Peron [55]. Monocins are resistant to trypsin, sensitive to heating at 56°C for 30 min and stable at 4°C. In monocin typing, an isolate is assessed for susceptibility to a set of bactericidal peptides produced by selected strains [80,121]. Curtis and Mitchell [31] studied monocin interactions of 97 strains of L. monocytogenes using an improved production method and standardization of the monocins against the type strain of Listeria ivanovii. Only serotype 4 strains acted as indicators. A typing system using 8 producer and 11 indicator strains showed poor discrimination. Bacteriocin typing has limitations similar to those of phage typing. Bannerman et al. [5] typed 100 strains of L. monocytogenes from sporadic cases and epidemic outbreaks by a combination of monocin typing and phage receptor/reverse phage receptor methods. The combination monocin-phage receptor subtyping method had a discrimination index of 0.99 for 87 epidemiologically unrelated strains, which was the highest of seven subtyping methods evaluated. The authors suggested that the combination monocin-phage reversal method was simple enough to be done in a nonspecialized laboratory, highly discriminatory, and reproducible, but they cautioned that the method and the indicator test strains must be rigorously standardized.

ANTIMICROBIAL SUSCEPTIBILITY TESTING Antimicrobial susceptibility testing has relatively limited utility for typing Listeria species simply because for many years L. monocytogenes susceptibility patterns have all but remained constant. However, resistance plasmids conferring resistance to chloramphenicol, macrolides, and tetracyclines have been found in L. monocytogenes [54,84]. Resistance to other antibiotics such as streptomycin, erythromycin, kanamycin, sulfamethoxazole, and rifampin have been observed in Listeria species [30,37,66,104]. In a minireview, Charpentier and Courvalin [28] give an overview of the mechanisms for recent emergence of antibiotic resistance in L. monocytogenes and Listeria spp. by acquisition of three types of mobile genetic elements: self-transferable plasmids, mobilizable plasmids, and conjugative transposons [2,29,30,54]. They conclude by suggesting that in all probability the current favorable situation of quasiuniform susceptibility of L. monocytogenes and Listeria spp. to the antibiotics used in clinical practice will deteriorate under the selective pressure exerted by mis- or overuse of the drugs. Recently, Godreuil et al. [46] screened 488 L. monocytogenes isolates from human cases of listeriosis for antibiotic resistance; they found that five isolates were resistant to fluoroquinolones. Fluoroquinolone resistance was attributed to active efflux of the drug. The authors’ observations indicate that increasing use of fluoroquinolones can select resistant mutants in nontarget species. Because antimicrobial susceptibility testing may be used as a method of typing bacteria according to antibiotic susceptibilities, with the emergence of resistance in L. monocytogenes strains antimicrobial susceptibility testing may provide an additional tool in epidemiologic investigations.

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MOLECULAR METHODS MULTILOCUS ENZYME ELECTROPHORESIS The characterization of prokaryotes and eukaryotes by multilocus enzyme electrophoresis (MEE) is based on differences in electrophoretic mobility of their metabolic enzymes. The electrophoretic mobility differences of these enzymes are a result of charge differences caused by amino acid substitutions in the polypeptide sequence; these charge differences, in turn, reflect changes in the nucleotide sequence of the DNA encoding the polypeptide [102]. In MEE, cell extracts containing the soluble metabolic enzymes are electrophoresed in nondenaturing starch gels. After electrophoresis is completed, the gel is sliced, and each slice is treated with a specific chromogenic substrate for a specific enzyme (e.g., aldolase) to render the enzyme band visible. Mobility variants of each enzyme are considered to be different electromorphs and are subjectively designated by different numbers. Combinations of a set of electromorphs (usually 10–20) constitute an electrophoretic type (ET). Thus, each ET represents a multilocus genotype. Some isolates may present nulls (absence of activity for specific enzymes); this complicates the analysis of MEE data. In the early 1990s, MEE was used in the United States and Europe for epidemiologic investigations of listeriosis outbreaks [6,49,77] and to determine the extent of involvement of contaminated foods in sporadic listeriosis [83]. Also, MEE has been useful for taxonomic and genetic characterization studies of L. monocytogenes [11,12,82]. Boerlin et al. [12] used MEE to analyze the genus Listeria and to estimate the genetic relatedness among Listeria species. The MEE data allowed not only identification of different genotypes within a population, but also provided an estimation of the genetic relatedness between strains. Although MEE is a very powerful tool for population genetic, taxonomic, and evolutionary studies, it is only moderately discriminatory for use as a subtyping tool in epidemiologic investigations. Caugant et al. [24] coordinated evaluation of MEE for the WHO Multicentre L. monocytogenes Subtyping Study. Seven laboratories participated in the study, assaying a total of 24 enzymes. Reproducibility and discriminating power of the method varied greatly among the seven laboratories. Five of the seven laboratories reported null alleles. In some instances, the nulls could be attributed to less-than-optimal activity of the enzyme in the cytoplasmic extracts applied to gels, whereas in other instances it was clearly the characteristics of the strains. Caugant et al. [24] concluded that to ascertain the immediate epidemiologic relationships of L. monocytogenes strains, the laboratorian will need, in some instances, to supplement MEE with other methods that provide further discrimination. Similar conclusions were reached by Norrung and Gerner-Smidt [78], who reported an overall discrimination index (DI) of 0.83 for MEE. When results of MEE were combined with those of restriction endonuclease analysis, the DI increased to 0.92. Further, MEE is a labor-intensive method that requires techniques and equipment that are available in relatively few laboratories. For these reasons, this method presently has relatively limited application in epidemiologic studies.

CHROMOSOMAL DNA RESTRICTION ENDONUCLEASE ANALYSIS (REA) Several authors have demonstrated the usefulness of chromosomal DNA restriction endonuclease analysis (REA) using frequently cutting restriction endonucleases for typing L. monocytogenes [38,44,76,119]. The number and sizes of restriction fragments generated by digesting a given piece of DNA are influenced by both the recognition sequence of the enzyme and composition of the DNA. Thus, investigators expect enzymes with a four-base recognition sequence to yield more and smaller restriction fragments than enzymes with six-base recognition sequences. However, an enzyme whose recognition sequence is composed of only guanine (G) and cytosine (C) will cut DNA with a low G + C content less frequently and consequently will generate fewer and larger restriction fragments than an enzyme that recognizes sequences of adenine and thymine. Selection

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of an appropriate restriction endonuclease is based on both the recognition sequence of a restriction endonuclease and the G+C content of the test organism [42,81]. Because of the high specificity of restriction endonucleases, complete digestion of a given DNA with a specific enzyme provides a reproducible array of fragments. Within a size range from 30 to 1 kb, the fragment can be separated by agarose gel electrophoresis to obtain DNA “fingerprint” patterns. For L. monocytogenes, the advantages of REA are that it is universally applicable, sensitive (because the entire genome is evaluated), cost effective, and relatively easy to perform. GernerSmidt et al. [44] evaluated the REA method for the WHO Multicentre L. monocytogenes Subtyping Study. They reported that of the restriction endonucleases tested, HaeIII, HhaI and CfoI were the most useful enzymes for REA typing of L. monocytogenes. The 80 test strains were divided into 27 REA types with HaeIII, 24 with HhaI, and 25 with CfoI. DNA fragment patterns obtained with HhaI and CfoI were nearly identical to each other and were the easiest to interpret by visual examination; EcoRI divided the strain set into only 14 REA types and the patterns were difficult to compare. The major limitation of REA is the difficulty of comparing the complex profiles, which consist of hundreds of bands that may be unresolved and overlapping. Patterns generated by some restriction enzymes may not be stable. Investigators need to clearly establish the differentiation criteria (the minimum number of band differences that are indicative of differences between the isolates) for each enzyme before they can reliably evaluate the discrimination [44]. Consequently, REA is not the method of choice for large-scale comparisons and for building a dynamic database of DNA patterns for comparative evaluation of new patterns.

RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS: RIBOTYPING AND OTHER METHODS Ribotyping is one type of Southern hybridization analysis in which strains are characterized for the restriction fragment length polymorphism (RFLP) associated with the ribosomal operons. Southern blot analyses detect only the particular restriction fragments associated with specific chromosomal loci, thereby significantly reducing the number of DNA fragments to be analyzed [106]. Ribotyping involves transferring chromosomal DNA restriction fragments that have been electrophoretically separated on a gel matrix onto a nitrocellulose or nylon membrane. After immobilizing the DNA fragments, laboratorians probe the membrane with an appropriately labeled 16 + 23S ribosomal RNA (rRNA) or rDNA probe [51,108]. Because the genes coding for rRNA are highly conserved, E. coli rRNA [108] or a cloned ribosomal operon (rrnB) of E. coli [17] can be used to probe L. monocytogenes. In general, ribotype patterns appear to be stable and reproducible after in vitro and in vivo passage of strains [17]. Therefore, this method may be best suited for long-term epidemiologic or phylogenetic studies. Figure 9.1 shows a gel containing genomic DNA restriction fragments of L. monocytogenes. Figure 9.2 shows a typical nylon membrane containing ribotype patterns obtained when the strains are Southern-blotted and probed with a cloned E. coli rrnB operon (plasmid pKK3535) labeled with digoxigenin. Several investigators have used ribotyping for subtyping L. monocytogenes [4,18,32,49, 50,59,75,78]. Most investigators have used EcoRI for ribotyping of L. monocytogenes. Baloga and Harlander [4] compared HaeIII and HindIII with EcoRI and concluded that EcoRI was the most discriminating enzyme for subtyping L. monocytogenes. Nocera et al. [75] reported that 69 of 96 serotype 4b isolates clustered in two closely related EcoRI ribotypes; they found bacteriophage typing to be more discriminating than ribotyping. Norrung and Gerner-Smidt [78] also found ribotyping to be less discriminating than bacteriophage typing, REA, and MEE for subtyping 99 clinical, food, and slaughterhouse isolates of L. monocytogenes.

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FIGURE 9.1 Genomic fingerprints of EcoR1-digested DNA of L. monocytogenes strains isolated from human and food sources. Lanes 2 and 14, human isolates (serotype 1/2a); lanes 3–13 and 15–20, food isolates (serotype 1/2a); lane 21, human isolate (serotype 4b); lanes 22 and 23, food isolates (serotype 4b); lanes 1 and 24, molecular size marker.

Swaminathan et al. [110] evaluated ribotyping and another probe derived from repeat sequences of L. monocytogenes DNA for the WHO Multicentre L. monocytogenes Subtyping Study. Six laboratories did ribotyping using EcoRI enzyme to restrict the L. monocytogenes DNA and rRNA or DNA as the probe for Southern hybridization. A seventh laboratory used NciI to restrict the DNA, and two probes, one randomly cloned and the other containing repeat sequences cloned from L. monocytogenes DNA. Their results showed that the overall Simpson’s index of diversity (DI) for five of the six laboratories that ribotyped most or all study strains ranged from 0.83 to 0.88. A DI value of 0.91 was obtained for the combination of two probes used by one laboratory. Also, DI values for strains that were serotypes 1/2a or 1/2c were greater than DI values for strains of serotypes 1/2b, 3b, 4b, or 4bX for ribotyping as well as for the randomly cloned probes used by one laboratory. Swaminathan et al. [110] concluded that although ribotyping satisfies two requirements for a good subtyping method, namely typeability and reproducibility, its discriminating ability for serotype 4b strains may not be adequate for epidemiologic investigations. They recommended that ribotyping be supplemented by other methods such as pulsed-field gel electrophoresis (PFGE) for determining the molecular epidemiology of L. monocytogenes serotype 4b.

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FIGURE 9.2 Ribotype profiles obtained by Southern blot analysis of genomic fingerprints of EcoR1-digested DNA of L. monocytogenes from Figure 9.1.

Probes other than rRNA or rDNA have been used to type L. monocytogenes. Saunders et al. [97] selected cloned DNA fragments of L. monocytogenes from a bacteriophage lambda gene library to type 64 isolates of serogroup 1/2 using restriction enzyme NciI and found good discrimination among epidemiologically unrelated isolates. Ridley [89] evaluated the same probe’s ability to type 862 isolates representing serogroups 1/2, 3, and 4. Although the utility of the method for subtyping serogroup l/2 isolates was demonstrated in the evaluation, Ridley observed that the method did not adequately discriminate among serogroup 4 isolates. The cloned probe evaluated by Ridley [88] and another probe derived from repeat sequences of L. monocytogenes DNA were used in the WHO Multicentre L. monocytogenes Subtyping Study because of their abilities to subtype L. monocytogenes [110]. Similar to ribotyping, using the two cloned probes did not provide adequate discrimination between epidemiologically unrelated serotype 4b isolates. Also, these probes did not discriminate between a serotype 4d isolate and other serotype 4b isolates associated with a listeriosis outbreak. However, the repeat sequence probe discriminated between serotype 4b isolates and serotype 4bX isolates [110]. The RiboPrinter is an automated ribotyping system that generates, analyzes, and stores riboprint patterns of bacteria. The first version of the RiboPrinter was configured to generate ribotype patterns using only EcoRI and was used to generate a database of patterns for 1346 isolates of L. monocytogenes [18,57]. Ryser et al. [93] used the RiboPrinter to demonstrate that different ribotypes of L. monocytogenes were favored by different selective enrichment protocols. Wiedmann et al. [120] characterized 133 isolates of L. monocytogenes by automated ribotyping using the

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RiboPrinter and examined them for polymorphisms in virulence-associated genes. They concluded that the isolates could be separated into three distinct phylogenetic lineages; human isolates were found in lineages one and two but lineage three was composed of exclusively animal isolates. Recently, Aarnisalo et al. [1] subtyped 486 L. monocytogenes isolates originating from 17 Finnish food-processing plants using the RiboPrinter and compared the results to those of DNA macrorestriction analysis. Although the automated RiboPrinter base typing was not as discriminatory as PFGE typing, the author suggested that automated ribotyping would be appropriate for screening large numbers of isolates from food processing plant environments. Gendel et al. [43] studied the temporal and spatial distribution patterns of L. monocytogenes strains from smoked salmon by ribotyping 72 isolates recovered over a 3-year period. No geographical or temporal stratification of strains was observed; 12.6% of the food samples yielded multiple ribotypes of L. monocytogenes. Polymerase chain reaction-ribotyping (PCR-ribotyping) is a method that uses oligonucleotide primers designed to be complementary to conserved regions of the 5S, 16S, and 23S regions of the rRNA genes. These primers are amplified with a purified or crude preparation of template DNA by PCR. The resulting PCR products may be digested with a restriction endonuclease of choice or added to an agarose gel, electrophoresed, and visualized by ethidium bromide staining. The potential of PCR-ribotyping for discriminating among and within various species of Listeria, as well as among strains of L. monocytogenes, has been explored [106]. Sontakke and Farber [105] analyzed 49 strains of L. monocytogenes and 12 isolates of other Listeria species. They subjected genomic DNA isolated from bacteria to PCR amplification using the region of DNA encoding 16S and 5S rRNA. They found that PCR-ribotyping distinguished between L. monocytogenes serotypes 1/2a and 1/2b with no overlap in composite profiles. The sensitivity of this method for differentiating serotype 1/2a and 1/2b isolates appears to be as good as that of other molecular methods. However, the PCR-ribotyping method was less discriminatory for serotype 4b strains [40]. Sontakke and Farber [106] concluded that PCR-ribotyping could be considered as an alternate molecular subtyping technique. However, they recommended combining the PCR-ribotyping method with another highly discriminatory molecular subtyping method for serotype 4b isolates for confirmation or association between similar isolates.

DNA MACRORESTRICTION ANALYSIS

BY

PFGE

DNA macrorestriction analysis by PFGE has revolutionized precise separation of DNA fragments greater than 40 kb. Schwartz and Cantor [100] developed PFGE, a variation of agarose gel electrophoresis in which the orientation of the electric field across the gel is changed periodically (“pulsed”) rather than kept constant as it is in conventional agarose gel electrophoresis used for the REA. This technology separates large fragments of unsheared microbial chromosomal DNA obtained by embedding intact bacteria in agarose gel plugs, enzymatically lysing the cell wall and digesting the cellular proteins. The intact DNA is digested with an infrequently cutting restriction endonuclease. Subsequent RFLP analysis allows differentiation of clonal isolates from unrelated ones. PFGE analysis has been used for epidemiological subtyping of L. monocytogenes by several investigators [15,16,19,23,56]. Brosch et al. [15] first demonstrated the usefulness of PFGE for subtyping L. monocytogenes by applying the method to type serotype 4b strains. Using ApaI, SmaI, and NotI, they showed that PFGE can distinguish between closely related strains that are indistinguishable by other typing methods. The ability of PFGE to subtype L. monocytogenes serotypes l/2 and 3 was subsequently demonstrated [20]. The applicability of PFGE for outbreak investigations was demonstrated by Buchrieser et al. [19], who typed 75 L. monocytogenes strains isolated during six major and eight smaller listeriosis outbreaks. PFGE divided these strains into 20 subtypes. Strains within each major epidemic (Switzerland [1983–1987], California [1985], and Denmark

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[1985–1987]) demonstrated indistinguishable patterns, whereas strains responsible for other outbreaks were characterized by specific combinations of patterns. Variations within PFGE patterns occurred more frequently within epidemiologically unrelated isolates. PFGE was used to demonstrate the link between contaminated chocolate milk and febrile gastroenteritis among a group of people who attended a cattle show in Illinois [33,85]. Destro et al. [34] found that RAPD and PFGE were the most useful methods for tracing dissemination of L. monocytogenes in a shrimp processing plant. Brosch et al. [14] evaluated the PFGE method for the WHO Multicentre L. monocytogenes Subtyping Study. Four laboratories participated in evaluating PFGE by analyzing 80 coded strains of L. monocytogenes. Two restriction endonucleases (Apa1 and SmaI) were used by all laboratories; one laboratory used an additional restriction endonuclease (Asc1). Agreement among the four laboratories ranged from 79 to 90%. Sixty-nine percent of the strains were placed in exactly the same genomic group by all four laboratories; most of the epidemiologically related strains were correctly identified by all four laboratories. This study validated the previous claims that PFGE is a highly discriminating and reproducible method for subtyping L. monocytogenes and is particularly useful for subtyping serotype 4b isolates, which are not typed satisfactorily by most other typing methods [14]. Currently, PFGE is widely used for molecular subtyping of L. monocytogenes for listeriosis cluster detection, epidemiologic investigations, and monitoring food production facilities. The method has been standardized for subtyping L. monocytogenes and is used internationally [1,3,48,65,95,109,115]. In the United States, the Centers for Disease Control and Prevention has established a network (PulseNet) of public health and food regulatory laboratories that routinely subtype foodborne pathogenic bacteria to rapidly detect foodborne disease clusters that may have a common source. PulseNet laboratories use highly standardized protocols for subtyping bacteria by PFGE and are able to quickly compare PFGE patterns of foodborne pathogens from different locations within the country via the Internet. Routine and timely subtyping of L. monocytogenes by participating PulseNet laboratories has significantly enhanced investigators’ ability to recognize and investigate outbreaks of listeriosis [9,25–27]. PFGE could possibly be considered the current “platinum standard” for molecular subtyping. Figure 9.3 shows the patterns obtained when genomic DNA of L. monocytogenes was digested with restriction endonucleases (RE) ApaI and AscI using the PulseNet’s L. monocytogenes standardized protocol. The major disadvantages of PFGE are the time required to complete the procedure (1 day), the requirement for large quantities of expensive restriction endonucleases, and the need for relatively expensive, specialized equipment for electrophoresis.

RANDOM AMPLIFICATION

OF

POLYMORPHIC DNA (RAPD)

Arbitrarily primed polymerase chain reaction (AP-PCR) and random amplified polymorphic DNA (RAPD) analysis are polymerase-chain-reaction-based methods in which researchers allow a single arbitrarily selected primer to anneal to nearly complementary sequences on the target DNA by doing the annealing step at a very low temperature (37°C). Typically, the primer anneals to several locations on the target and amplifies an array of DNA fragments of different sizes, yielding a DNA pattern suitable for typing. RAPD uses primers of 10-bp length, whereas AP-PCR uses longer primers [117,122]. RAPD was first applied to subtyping of L. monocytogenes by Mazurier et al. [70]. They used a 10-mer primer (HLWL 74) to analyze 104 L. monocytogenes isolates that included representative strains from six outbreaks. All but one of the outbreak-associated isolates were classified by RAPD in complete agreement with phage typing. Mazurier et al. [69] suggested that RAPD offers an attractive alternative to phage typing. Lawrence et al. [61] used a different 10-mer primer to type 91 isolates from raw milk, food, veterinary, environmental, and clinical sources. They obtained 33 different patterns. Farber and

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FIGURE 9.3 Pulsed-field gel electrophoresis separation of AscI (lanes 2–5) and ApaI (lanes 7–9) macrorestriction fragments of L. monocytogenes genomic DNA test isolates for certifying PulseNet participating laboratories. Lanes 1, 6, and 10, XbaI-digest of Salmonella ser. Braenderup standard reference strain (CDC. no. H 9812).

Addison [40] applied RAPD to type 52 L. monocytogenes isolates representing 11 serotypes and concluded that the method offered much promise as a subtyping method for L. monocytogenes. Niederhauser et al. [74] used a 19-mer primer to subtype 57 L. monocytogenes isolates and reported that the method allowed them to trace back L. monocytogenes contamination in several food outlets to a food-processing plant. Boerlin et al. [10] did an extensive evaluation of RAPD by typing 100 L. monocytogenes isolates that had been characterized by serotyping, phage typing, MEE analysis, REA, and ribotyping. They found RAPD to be a highly discriminating method for subtyping. O’Donoghue et al. [79] found RAPD to be useful for typing serogroup 1/2; also they found that the method distinguished the serotype 4bX strains involved in a pâté-associated outbreak from other serotype 4b isolates. Wernars et al. [118] evaluated RAPD for the WHO Multicentre L. monocytogenes Subtyping Study. Six laboratories participated in the study. Using three different 10-mer primers, the six participating laboratories obtained a median reproducibility of the RAPD results of 86.5% (range 0–100%). Any failure in reproducibility was mainly attributable to results obtained with one particular primer. Wernars et al. [118] concluded that RAPD analysis is a rapid and relatively simple technique for epidemiologic typing of L. monocytogenes isolates and that reproducible useful results can be obtained. Despite the simplicity and high discriminating ability of RAPD, much more work is needed to make RAPD typing a standard technique for general and widespread use. Its primary drawback is the inconsistent reproducibility of patterns. Although some investigators claim that the method is reproducible, there is a need to identify all steps in the procedure that are critical for obtaining consistently reproducible results. Because RAPD conditions are less stringent than those of other

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typing methods to facilitate initiation of the polymerization reaction at sites having one or more sequence mismatches, the polymerization is initiated with various efficiencies. The final quantities of DNA produced may vary widely among the different fragments amplified from a given isolate. Such variation is inherent in RAPD analysis and introduces two specific problems. First, comparison and interpretation of patterns with differences in intensity become quite difficult. Second, because some of the products may represent relatively inefficient reactions, the actual fragments obtained from a single isolate may vary in different amplification reactions. It is, therefore, very important that a well-standardized protocol be followed in RAPD analysis and used consistently for reliable results.

REPETITIVE ELEMENT-BASED SUBTYPING Repetitive element-based (REB) typing is a PCR typing method that incorporates use of primers based on short extragenic repetitive sequences [113] or generic rRNA intergenic spacer oligonucleotides [47]. Sequences are typically present at many sites around the bacterial chromosome such that when two sequences are located near enough to each other the DNA fragment between those sites is effectively amplified. Because the number and location of the repetitive sequences are quite variable, the number and size of the interrepeat fragments generated can similarly vary from strain to strain. Ericsson et al. [36] used an REB analysis to investigate 133 strains of L. monocytogenes serotype 4b. A segment of 2,916 bp containing parts of the two genes inlA and inlB in L. monocytogenes was amplified by the PCR technique. The PCR product obtained was digested with the restriction enzyme Alu1. The fragments generated provided two distinct groups—one containing 37 types and the other, 96 types. These results indicate that REB analysis may be a useful tool for subtyping L. monocytogenes serotype 4b strains.

AMPLIFIED FRAGMENT LENGTH POLYMORPHISM Amplified fragment length polymorphism (AFLP) is a DNA fingerprinting technique based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. Vos et al. [114] developed AFLP; the technique involves restriction of the DNA and ligation of oligonucleotide adapters, selective amplification of sets of restriction fragments, and gel analysis of the amplified fragments. Investigators can use the technique to generate fingerprints from DNA of any origin and from any complexity without prior sequence knowledge. The method allows the specific coamplification of a high number of restriction fragments. The number of fragments that can be analyzed simultaneously is dependent on the resolution of the detection system. The optimal number of fragments (50–100) are amplified and detected on denaturing polyacrylamide gels. Ripabelli et al. [89] used AFLP to analyze a set of 33 L. monocytogenes isolates collected from patients and from foods implicated in outbreaks, from human sporadic cases, or from foods. Of four selective primers used, one generated 20 different-sized DNA fragments with the AFLP technique. The 33 cultures segregated into 14 different patterns, each comprising 7–12 different fragments. They concluded that AFLP analysis reconfirmed the observation that L. monocytogenes comprises two major genetic groups. In a recent study Guerra et al. [52] evaluated 84 EcoRI digests of L. monocytogenes DNA using AFLP. The results were compared with those obtained with serotyping, phage-typing, and cadmium/arsenic resistance typing. They found that the results of the AFLP technique were reproducible and that 14 different banding patterns comprising between five and eight DNA fragments were produced. There were associations with AFLP results and those from phage-typing and cadmium/arsenic resistance typing, although each method showed some independence. The evaluation suggests that the AFLP method may be useful for epidemiologic studies.

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The advantages of AFLP are high reproducibility, high PCR multiplex ratio, the fact that this method is not hampered by genome complexity, the possibility of generating a virtually infinite number of markers, and the fact that no prior sequence information is required. Disadvantages of AFLP include the requirement for an automated DNA sequencer and the complexity of the protocols. Also, the epidemiologic relevance of AFLP has not been firmly established.

DNA-SEQUENCE-BASED SUBTYPING STRATEGIES Much work has been done on DNA-sequence-based subtyping and several approaches have been suggested; however, much work remains to be done. Some potential targets have been identified. Listeria monocytogenes is an attractive candidate for implementation of DNA-sequence-based subtyping for the following reasons. There is an excellent set of well-characterized, epidemiologically related and unrelated strains of L. monocytogenes that have been subtyped by all available phenotypic, protein-based, and DNA-RFLP-based subtyping methods. These strains will be invaluable for developing a database of DNA sequences for L. monocytogenes. Also, the extensive characterization of virulence-associated genes of L. monocytogenes that has been accomplished over the past 15 years provides valuable information on virulence-associated genes of L. monocytogenes and the sequence heterogeneities in these genes [35,86,87,97,103,123]. The virulenceassociated genes of L. monocytogenes that have now been sequenced include iap (gene encodes an invasion-associated protein), inlA (a family of genes that are involved in the internalization of the organism into the host cell), hlyA gene (encodes a β-hemolysin), the plcA gene (encodes a phosphatidyl inositol-specific phospholipase C, which is involved in lysis of the membrane during cell-to-cell spread of L. monocytogenes), the mpl gene (encodes a metalloprotease), the actA gene (encodes factors involved in actin polymerization), and the lma operon (associated with inducing delayed-type hypersensitivity reactions in L. monocytogenes-immune mice). In addition, the flaA gene that encodes the flagellin protein, the flaR gene that appears to modulate DNA topology, and the genes encoding 16S and 23S ribosomal RNA in L. monocytogenes have been sequenced. Some virulence-associated genes such as hlyA are highly conserved and may not be suitable targets for strain identification. Other virulence-associated genes such as the inlA operon and the genes encoding cell surface structures such as cell membrane and flagella may be more polymorphic and, hence, are likely to be more useful for discrimination of strains. Rasmussen et al. [87] sequenced internal fragments of the flaA, iap, hly, and 23S rDNA genes of isolates of L. monocytogenes from clinical, food, and environmental sources. These isolates represented different serotypes. A 150-bp region of the hly gene was sequenced in 75 strains. A total of 27 strains were sequenced for the other genes. Although the DNA sequence data for hly, iap, and flaA were useful for identifying three lineages, the genetic diversity within the sequencing targets was insufficient to provide adequate sensitivity for subtyping. However, because DNA insertions, deletions, and rearrangements are very frequently encountered, the targeting of a single gene for subtyping may provide misleading answers. Also, targeting a single gene, even a hypervariable one, may not provide adequate discrimination for epidemiologic subtyping, and will not necessarily provide reliable estimates of genetic relatedness between strains. Multilocus sequence typing (MLST) is a novel typing method in which alleles for multiple housekeeping enzyme loci are assigned directly by nucleotide sequencing. This method differs from MEE, in which alleles are inferred and assigned indirectly from the electrophoretic mobilities of gene products [67,107]. Complete or partial nucleotide sequences are determined for several bacterial genes or chromosomal regions, thereby providing unambiguous and discrete data. MLST provides excellent assessment of genetic relatedness between strains; therefore, it is very useful for the study of genetic structure, evolution, and population biology of living organisms including pathogenic bacteria.

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Salcedo et al. [94] described an MLST-based subtyping method for L. monocytogenes that uses the sequence diversity data of seven housekeeping genes (abcZ, bglA, cat, dapE, dat, ldh, and lhkA). Two additional loci (pgm and sod) that were examined in preliminary tests were not considered further because of low sequence diversity. Salcedo et al. [94] evaluated the MLST method against 62 strains of L. monocytogenes that had been previously characterized by PFGE. Although quite a bit of congruence was found among groupings obtained using sequence analysis of housekeeping genes and those obtained using PFGE, PFGE was much more discriminating than MLST (29 MLST sequence types [ST] versus 46 PFGE types). Five STs included multiple PFGE types (ST4:2; ST 11; ST14: 4; ST6:6 and ST2:7). In contrast, only one PFGE type (pattern 31) was shared between two STs (ST6 and ST8). Cai et al. [21] used a different approach to selecting multiple genomic targets on L. monocytogenes. They selected two housekeeping genes (prs and recA), one stress response gene (sigB), two virulence genes (actA and inlA), and two intergenic regions (plcA-hly and hly-mpl) and sequenced their complete open reading frames in 15 well-characterized isolates. The virulence genes and the two intergenic regions provided higher discrimination than did the housekeeping and stress-response-related genes; actA exhibited the highest sequence variability with 14.3% of the nucleotides being polymorphic, whereas the phosphoribosyl synthetase gene showed the lowest sequence variability (4.9%). Cai et al. [21] also used the complete sequence information for these genes and regions to define the most discriminatory 600-bp fragments within them that could be used for rapid sequencing-based subtyping. This approach needs to be further evaluated and validated using a larger set of epidemiologically and microbiologically wellcharacterized strains. Rudi et al. [92] used another variation of MLST employing a DNA-array-based approach in which only informative genetic changes in multiple genetic loci were investigated by sequencespecific labeling of oligonucleotide probes. They identified 29 such informative regions in the following virulence-associated genes of L. monocytogenes: hlyA, iap, flaA, actA, and inlA. They used a set of 32 L. monocytogenes strains to type by multilocus array hybridization and compared the results with AFLP. Although the strains were tightly clustered by multilocus array hybridization, in many instances they also clustered by AFLP. However, the two techniques were less congruent for distantly related strains. It is difficult to assess the utility of multilocus array hybridization for subtyping L. monocytogenes from their data [92] because AFLP is not extensively used for typing L. monocytogenes in public health laboratories and its utility in the context of public health surveillance and outbreak investigations has not been demonstrated. Borucki et al. [13] constructed a mixed-genome microarray using a genomic library based on 10 strains of L. monocytogenes. They evaluated the microarray using 24 isolates that were from humans, ovine/bovine brain, bulk milk, and the environment. The four major serotypes of L. monocytogenes were represented. The isolates were separated into two major clusters. This result was in agreement with serotype-based clustering and previously described phylogenetic lineages. Later, the investigators interrogated the mixed-genome microarray with an expanded set of 50 L. monocytogenes isolates [22]. They concluded that the major serotypes could be discerned by using the data from only four probes in the mixed-genome microarray. Further, they were able to distinguish between isolates that clustered within a serotype. The potential of this approach for molecular epidemiologic investigations of listeriosis remains to be demonstrated. Multilocus variable number tandem repeats analysis (MLVA) is a powerful subtyping technique for bacterial pathogens; it has been successfully used for subtyping even those bacterial pathogens that do not show much diversity by other methods. In this method, tandem repeats on the bacterial genome are determined by scanning the whole-genome sequence with specialized software [53,60] developed specifically for this purpose. Gene sequences that encompass these

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repeat sequences are targeted for amplification by PCR and are sized by separation on gel-based DNA sequencing systems. Multiple repeat sequences on the genome are evaluated for diversity in number of repeats in each target using a small set of carefully selected strains of the pathogen to identify the highly polymorphic loci. Multiple polymorphic loci that allow amplification from different strains are selected for further evaluation with a larger set of previously characterized strains. Although there are no reports of the application of MLVA to L. monocytogenes, we anticipate publication of such reports in the near future given the fact that whole-genome sequences for L. monocytogenes serotypes 1/2a and 4b, and L. innocua are now available. A third approach is to assess the diversity among strains of L. monocytogenes by assessing single-nucleotide polymorphisms (SNP) at multiple locations on the genome. An attempt to develop SNP-based subtyping was made by Unnerstad et al. [112], who used pyrosequencing to catalog variations in positions 1575 and 1578 of the inlB gene of L. monocytogenes. Using this approach, they were able to categorize 106 L. monocytogenes strains into four groups: serogroups 1/2a and 1/2c were in one group, 1/2b and 3b were clustered in another group, and serotype 4b strains were separated into two groups. By extending this approach to additional genomic targets, investigators may be able to develop a sensitive subtyping method.

COMPARISON OF METHODS USED TO SUBTYPE L. MONOCYTOGENES Several studies report comparisons of various methods for subtyping L. monocytogenes. Researchers compared different methods using different sets of isolates. For example, Baloga and Harlander [4] used genomic DNA fingerprints, ribotyping, serotyping, and MEE to study 28 strains of L. monocytogenes. DNA fingerprinting was more discriminating than ribotyping in this study. Norrung and Gerner-Smidt [78] compared MEE, ribotyping, REA, and phage typing. In this study, phage typing was the most discriminatory with a DI of 0.88. REA, MEE, and ribotying had DIs of 0.87, 0.83, and 0.79, respectively. Norrung and Gerner-Smidt [78] also saw differences in discrimination depending on O serotype. For serotype 1, REA gave the best discrimination, and for serotype 4, phage typing gave the best discrimination. Nocera et al. [75] also characterized Listeria strains from an outbreak using ribotyping and four other typing methods (serotyping, phage typing, MEE, and REA). They studied 134 isolates, of which 96 were serotype 4b. Within 4b isolates, phage typing gave the highest DI, followed by REA, MEE, and ribotyping. They also showed that combining methods could give a higher DI. Graves et al. [49] compared ribotyping and MEE for 305 isolates of L. monocytogenes. Overall, they found MEE to be more discriminating with this set of isolates than ribotyping. They concluded that these methods did not provide adequate discrimination for serotype 1/2b and 4b. At the beginning of this chapter, we discussed the WHO Multicentre Listeria monocytogenes Subtyping Study. Phase I of this study used a set of 80 coded L. monocytogenes strains that included 11 sets of duplicates. Bille and Rocourt [8] published an overview of the study. Because several different methods were compared using a well-defined set of isolates, this study is very useful in comparing the utility of the various subtyping methods. Table 9.1 shows a comparison of the methods used in the study. On the basis of these results, serotyping, phage typing, REA, PFGE, and RAPD were selected for standardization in Phase II. Currently, antisera are commercially available for serotyping, ribotyping can be done using automated technology, and PFGE methodology is standardized. These methods provide a selection of standardized subtyping that can be employed in listeriosis cluster detection and epidemiologic investigation using multicenter comparison of data. Novel DNA-sequence-based subtyping methods (MLST, multilocus array hybridization, MLVA, and SNP) must be evaluated and validated.

ND 98 ND 84

0–100, median 86.5

27–91 88–97 80–100 ND

0–100 for 3 primers

100

100

100

100

100

MEE

REA

Ribotyping

PFGE

RAPD

0.75–0.95 for 3 primers

0.95–0.96

0.83–0.88

0.93–0.98

0.83–0.93

ND

0.68

Discriminatory Power (DI)

Highly discriminating and useful for epidemiologic investigations. One primer gave serious reproducibility problems. Standardize method and address problems with reproducibility.

Highly discriminating and useful for epidemiologic investigations; standardize method.

Not sufficiently discriminating to be used alone for epidemiologic investigations.

Develop standardized nomenclature for types.

Not sufficiently discriminating to be used alone for epidemiologic investigations.

Centrally propagate phages and standardize phage suspensions, propagation strains, and methodology.

For standardization, reagents should be produced and distributed by one laboratory. Serotyping is not very discriminatory, but is a useful prerequisite to other methods, especially in outbreak investigations.

Recommendations

119

14

110

44

24

72

99

Reference

298

Note: DI = Simpson’s index of diversity; ND = not done; NA= not available; * = the remaining isolates did not give a strong reaction with any of the phages in the international phage set.

79

NA

49–80*

83

Interlaboratory Reproducibility (%)

Phage typing

82–100

Intralaboratory Reproducibility (%)

100

Percentage Typeability

Serotyping

Method

TABLE 9.1 Characteristics of Phenotypic and Molecular Subtyping Methods Used in the WHO Multicentre L. monocytogenes Subtyping Study

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Foodborne Listeriosis Dawn M. Norton and Christopher R. Braden

CONTENTS Identification of Listeria monocytogenes as a Foodborne Pathogen ............................................308 Role of Molecular Subtyping in Listeriosis Outbreak Detection and Investigation ....................309 Dairy Products................................................................................................................................310 Soft, Unripened “Mexican-Style” Cheese, Los Angeles County ........................................310 Vacherin Mont d’Or, Western Switzerland ..........................................................................312 Brie de Meaux, France.........................................................................................................313 Blue-Mold or Hard Cheese, Denmark .................................................................................314 Raw-Milk Soft or Semihard Cheese, Quebec, Canada .......................................................314 Illicitly Produced or Distributed Raw-Milk Fresh, Unripened Cheese...............................315 Fluid Milk.............................................................................................................................317 Other Dairy Products............................................................................................................318 Meat and Poultry Products ............................................................................................................320 Ready-to-Eat Meat and Poultry Products in the United States: Frankfurters and Deli Meat ..................................................................................................321 Other Processed Meats .........................................................................................................327 Reduction of Meat and Poultry Product–Associated Invasive Listeriosis ..........................333 Seafood Products............................................................................................................................333 Smoked Mussels...................................................................................................................334 Cold-Smoked Fish Products.................................................................................................334 Vegetables.......................................................................................................................................335 L. monocytogenes–Associated Febrile Gastroenteritis ..................................................................336 Potential Association with a Mild Clinical Syndrome: Shrimp, 1989, United States ................................................................................................337 Rice Salad, 1993, Northern Italy .........................................................................................338 Chocolate Milk, 1994, United States...................................................................................338 Corn and Tuna Salad, 1997, Northern Italy ........................................................................340 Cold-Smoked Rainbow Trout, Finland ................................................................................340 Ready-to-Eat Meat and Poultry Products, New Zealand and United States ......................341 Fresh, Raw-Milk Cheese, Sweden .......................................................................................342 Cheese, Japan .......................................................................................................................343 Control of L. monocytogenes–Associated Febrile Gastroenteritis ......................................343 Surveillance of Listeriosis in the United States ............................................................................344 Foodborne Outbreak Surveillance........................................................................................344 Surveillance of Sporadic Listeriosis ....................................................................................344 Improved Estimates of the Burden of Listeriosis in the United States ..............................348 References ......................................................................................................................................349

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Although listeriosis is a well-documented veterinary infectious disease, it was historically thought to occur only rarely in humans and primarily as a result of contact with ill animals or animal reservoirs (35). Indeed, food has been recognized as a primary mode of transmission for human disease only within the last 25 years. Hypotheses regarding the potential transmission of Listeria monocytogenes to humans through food can be found as early as 1926, when Murray et al. described a disease in rabbits caused by a bacterium subsequently identified as L. monocytogenes [115]. Results of feeding trials indicated successful infection via the oral route, leading the authors to suggest that the gastrointestinal tract may serve as a route of infection. Strong evidence for foodborne transmission to humans was first reported by Potel in the 1950s [123]. He described a marked increase in stillbirths in Halle, Germany, with up to 100 instances documented at a single clinic from ~1945 to 1952. Investigation efforts led to isolation of the same L. monocytogenes serotype from the milk of a cow with atypical mastitis and stillborn twins of a mother who had consumed the cow’s raw milk. Today, non-foodborne transmission of L. monocytogenes to humans is considered to occur only rarely. Clusters of late-onset neonatal listeriosis have been identified in newborn nurseries, suggesting that nosocomial transmission is possible [116,134]. It is also possible that late-onset neonatal listeriosis in infants born healthy and at full term may have been acquired during passage through the birth canal, though the route of infection in this circumstance is not well understood. Nonetheless, the proportion of listeriosis cases due to foodborne transmission has been recently estimated to be 99% [110]. The epidemiologic and laboratory-based investigation of foodborne listeriosis outbreaks has contributed significantly to our understanding of this disease. This chapter will thus place significant emphasis on outbreaks. However, invasive listeriosis outbreaks are often very difficult to identify and investigate. A discussion of challenges unique to their detection and investigation will facilitate a better understanding of investigative findings and conclusions. Many outbreaks occur among geographically dispersed populations over long time periods and affect relatively few exposed individuals, making their recognition difficult and often delayed. Surveillance for listeriosis and identification of cases potentially associated with outbreaks is also limited, for example, by the lack of routine testing to diagnose listeriosis among cases of spontaneous abortion. Long periods between exposure and diagnosis hamper the ability to identify the vehicle of infection. In the 1985 outbreak of listeriosis due to Mexican-style soft cheese in Los Angeles, reviewed in detail in this chapter, the median incubation period was 31 days, with a range of 11 to 70 days [97]. Investigators typically assess food exposures for patients in listeriosis outbreaks for the entire four to six weeks prior to the patient’s diagnosis, and patients often have difficulty recalling their specific food exposures. The narrow range of susceptible persons makes it difficult to identify and enroll appropriate control persons in case– control studies. Although approximately 20% of listeriosis cases may occur in healthy adults, most cases occur among pregnant women, neonates, the elderly, or immunocompromised adults [135]. Limited selection of comparable control persons potentially exposed to the food vehicle but not infected by L. monocytogenes or, specifically, the outbreak-C associated subtype may compromise the study. Successful identification of the food vehicle in an outbreak is further confounded by microbiological aspects of food contamination. L. monocytogenes is common in the natural environment (see Chapter 2), and multiple subtypes may contaminate foods. Subtyping L. monocytogenes isolates from patients and food is a critical tool for linking foods to human infections. However, the finding of multiple subtypes in food vehicles and among patients, some of which may not match, can add a layer of complexity to identification of cases and potential food vehicles. Last, food vehicles responsible for listeriosis are often those with prolonged shelf lives, and growth of L. monocytogenes can occur at refrigerator temperatures. Thus, the increased opportunities for cross-contamination of multiple foods during food production and storage can make identification of the original source vehicle in outbreaks difficult. Despite these obstacles in surveillance, outbreak detection, and investigations, we have learned much about the foods associated with listeriosis. In this chapter, we will review the current knowledge gained from listeriosis outbreaks and surveillance. Table 10.1 summarizes the reported invasive listeriosis outbreaks and the associated food vehicles.

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TABLE 10.1 Foodborne Invasive Listeriosis Outbreaks with Ten or More Cases Year 1945–1952 1954 1956 1960–1961 1966 1969 1975–1976 1977–1978 1978–1979 1979 1979–1980 1980 1981 1981 1981 1981–1982 1983 1983 1983 1983–1987 1985 1985–1987 1986 1986–1987 1986–1987 1987 1987 1987–1989 1989 1989–1990 1990 1992 1993 1995 1997 1998–1999 1999 1999 1999–2000 1999–2000 2000 2000 2002 2002 a b c

Location

Suspect/Implicated Vehicle

Halle, East Germany

Raw milk, sour milk, cream, cottage cheese Jena, East Germany Unknown Soviet Union Pork, mouse Bremen, West Germany Unknown Halle, East Germany Unknown Auckland, New Zealand Unknown Anjou, France Unknown Johannesburg, South Africa Unknown Western Australia Raw vegetables Massachusetts Raw vegetables Auckland, New Zealand Unknown Auckland, New Zealand Shellfish, raw fish suspected East Cambria, England Cream Slovakia Unknown Maritime Provinces, Canada Coleslaw Christchurch, New Zealand Unknown Houston, Texas Unknown Saxony, West Germany Unknown Massachusetts Pasteurized milk Vaud, Switzerland Vacherin Mont d’Or cheese Los Angeles Mexican-style cheese Denmark Unknown Linz, Austria Raw milk, vegetables Los Angeles Raw eggs Philadelphia Unknown, multiple vehicles likely Los Angeles Butter England Unknown England, Wales, North Ireland Pâté New York Shrimp Denmark Blue-mold or hard cheese Western Australia Processed meats or pâté France Pork tongue in aspic (jelly) France Rillettes France Brie de Meaux cheese France Pont l’Évêque cheese United States, multistate (n = 24) Processed meats Finland Butter, pasteurized Connecticut, Maryland, NewYork Pâté France Rillettes France Pork tongue in aspic United States, multistate Delicatessen turkey meat (n = 11) North Carolina Homemade Mexican-style cheese United States, multistate Delicatessen turkey meat (n = 9) Quebec, Canada Raw milk cheese

Number of cases reflects laboratory-confirmed and epidemiologically linked cases. 63 cases from the epidemic subtype. Goulet, 1998. Personal communication to E.T. Ryser.

No. Cases a

Reference

~100

[123]

26 19 81 279 13 162 14 12 20 10 22 11 49 41 18 10 25 49 122 142b 35 20 33 36 11 23 366 10 26 11 279 38 33 14 108 25 11 10 32 30

[146] [79] [62] [121] [27] [45] [88] [141] [82] [124] [96] [106] [124] [132] [60] [43] [117] [63] [41] [97] [131] [2, 145] [136] [137] [103] [107] [108] [125] [91] [94, 148] [89, 130] [74] [72]

13 54

[99] [70]

17

[9]

c

[109] [98] [11] [52] [52] [119]

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IDENTIFICATION OF LISTERIA MONOCYTOGENES AS A FOODBORNE PATHOGEN In 1981, the investigation of a large epidemic of adult and perinatal listeriosis in the Maritime Provinces of Canada provided overwhelming evidence for foodborne transmission of L. monocytogenes to humans [132]. First detected from the occurrence of perinatal listeriosis in 1.3% of births at a Nova Scotia hospital over a 3-month period, this outbreak of serotype 4b infections resulted in 7 adult and 34 perinatal cases (including 5 fetal deaths and 4 stillbirths, Figure 10.1). The casefatality rate among live-born infants was 27%, and 2 of 6 nonpregnant adults who developed meningitis died. The magnitude of the outbreak provided a unique opportunity to investigate risk factors for infection. Investigators conducted two case–control studies, in which prior exposures of case-patients are compared to an appropriate control population of persons without the disease; differences in exposure between the groups indicates an association between exposure and the disease [53]. For this study, information on potential exposures was collected from case-patients and a healthy control population. The first study primarily explored hypotheses of transmission by exposure to ill persons, animals, and environmental sources based on the body of knowledge at that time, but did not identify potential risk factors. However, collection of a comprehensive food history for the 3 months before illness and a second case–control study implicated coleslaw as the most likely vehicle of infection. Case-patients were 2.3 times more likely to have consumed coleslaw than controls (P = 0.04). The vehicle was laboratory-confirmed when coleslaw obtained from a patient’s refrigerator yielded L. monocytogenes serotype 4b, the same serotype associated with the human infections. This serotype was also isolated from unopened packages of coleslaw prepared by the same manufacturer. The isolates were further characterized by molecular means in subsequent studies [21,40,49,77,150], and were found to share molecular profiles. In an effort to find the source of contamination, investigators traced ingredients back to their suppliers. Carrots and cabbage were obtained from several regional farms. One also raised sheep and routinely fertilized with raw and composted sheep manure. Environmental samples did not yield L. monocytogenes. However, reports of listeriosis among the sheep and long-term storage of cabbage in a cold-storage shed, conditions that would allow growth of this organism in the cabbage, support the hypothesis that contaminated cabbage from this farm was the source of L. monocytogenes. On the other hand, because this organism

16 14

Adult cases Perinatal cases

12 Cases

10 8 6 4 2 0

J F MAM J J A S O N D J F MAM J J A S O N D J F MAM J J A S O N D 1979 1980 1981

FIGURE 10.1 Nonpregnant adult and perinatal listeriosis cases — Maritime Provinces, Canada, 1979–1981. (Adapted by E.T. Ryser from Schlech, W.F.I., P.M. Lavigne, R.A. Bortolussi, A.C. Allen, E.V. Haldane, A.J. Wort, A.W. Hightower, S.E. Johnson, S.H. King, E.S. Nicholls, and C.V. Broome 1983. Epidemic listeriosis— evidence for transmission by food. N Engl J Med 308: 203–206.)

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is often found in food processing environments (see Chapter 17), product contamination during or after preparation is also possible. In addition to bringing listeriosis to international attention, this outbreak definitively documented foodborne transmission of L. monocytogenes to humans. Since the recognition of food as the primary mode of transmission of L. monocytogenes to humans, subsequent investigations of many outbreaks and sporadic listeriosis cases have implicated a wide variety of food types. A common characteristic of many implicated foods is that they are eaten without cooking by the consumer; that is, they are “ready to eat.” Many of these foods are cured, pasteurized or cooked in the production process, indicating that postprocessing contamination from the production environment is an important route of food contamination. These products also have a prolonged shelf life compared to fresh foods. As L. monocytogenes grows well at refrigeration temperatures (see Chapter 6), even cold-storage of products allows conditions favorable for growth of this organism to levels posing a high risk for human infection and disease, especially in susceptible individuals. In the following sections, we review epidemiological and laboratory investigations implicating different types of foods. We will first discuss invasive listeriosis, and then turn the focus to febrile gastroenteritis. As molecular subtyping has become a critical component of epidemic investigations, we first discuss its important role and provide an example of a molecular surveillance system in place in the United States and several other countries—PulseNet.

ROLE OF MOLECULAR SUBTYPING IN LISTERIOSIS OUTBREAK DETECTION AND INVESTIGATION Historically, foodborne disease outbreaks were of local scale, often linked to a single restaurant or social event and attributed to improper food handling. Today, outbreaks often involve food products that are centrally produced and widely distributed to many states and, occasionally, other countries. Cases of illness are often geographically diffuse, making it more difficult to detect, investigate, and control the outbreak. Subtyping methods have proven to be a valuable tool during investigations of foodborne disease outbreaks, beginning with the use of methods including serotyping, plasmid profiling, and phage typing over 20 years ago [84,85]. A number of highly discriminatory subtyping methods have since been developed (see Chapter 9 for a detailed discussion). However, their widespread adoption and the ability to compare results from different laboratories have been hindered by issues including cost, reproducibility, and time to completion [142]. Pulsed-field gel electrophoresis (PFGE) (detailed in Chapter 9) is a highly discriminatory and reproducible DNA-based subtyping method for several foodborne pathogens, including L. monocytogenes. In 1993, PFGE was applied for characterization of clinical and food isolates during the investigation of an Escherichia coli O157:H7 outbreak in the western United States. Subtyping facilitated investigation of the extent of the outbreak and confirmed the link between patient clinical isolates and hamburger patties served by a regional restaurant chain, thus demonstrating the utility of PFGE in outbreak investigations [25]. Over the next several years, the Centers for Disease Control and Prevention (CDC) standardized the methodology for PFGE and collaborated with the Association of Public Health Laboratories to develop PulseNet, a national public health laboratory network for molecular surveillance of foodborne disease and rapid outbreak response. Participating laboratories are certified to perform PFGE analysis and rapidly compare the resulting patterns. Beginning in 1996 with ten laboratories, PulseNet has grown to include all state public health laboratories in the United States, several large county public health laboratories, the U.S. Department of Agriculture’s (USDA) Food Safety and Inspection Service (FSIS), and the U.S. Food and Drug Administration’s (FDA) Center for Food Safety and Applied Nutrition (CFSAN) and Center for Veterinary Medicine (CVM). PulseNet routinely monitors five foodborne bacterial pathogens, including L. monocytogenes. Canadian provincial public health laboratories have also joined together to form PulseNet Canada, and share PFGE results with PulseNet USA. Other international PulseNet networks are forming in Europe, Latin America, the Pacific Rim, and China.

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Number of outbreaks

6

PulseNet begins subtyping clinical Listeria isolates

5 4 3 2 1 0 ’78

’80

’82

’84

’86

’88

’90

’92

’94

’96

’98

’00

’02

Year

FIGURE 10.2 Outbreaks of listeriosis detected in the United States by public health officials, 1978–2003. Arrow denotes initiation of subtyping and routine surveillance by the National Molecular Subtyping Network (PulseNet).

The severity of listeriosis and potential for geographically diffuse and prolonged outbreaks underscored the need for continued vigilance for listeriosis clusters and the ability to rapidly compare isolate subtypes. PulseNet scientists developed a standardized protocol enabling isolate analysis within 30 h [76], and routine surveillance was initiated in 1998. PulseNet surveillance has revolutionized the ability to detect listeriosis outbreaks. Figure 10.2 shows the number of known outbreaks of listeriosis in the United States by year, from 1978 through 2003. From 1978 to 1997, five outbreaks were detected and reported by health officials. Following initiation of subtyping and surveillance of Listeria by PulseNet in 1998, the number of outbreaks detected increased dramatically. A total of five outbreaks were detected in 1999 alone. This increase can be largely attributed to enhanced surveillance, as opposed to an increase in listeriosis, as surveillance data have shown an overall national decrease in listeriosis in recent years (discussed later in this chapter).

DAIRY PRODUCTS SOFT, UNRIPENED “MEXICAN-STYLE” CHEESE, LOS ANGELES COUNTY In 1985, a large epidemic occurred in Los Angeles County, which brought listeriosis to the forefront of public health and regulatory concern [97]. First recognized as an increase in perinatal listeriosis, 142 cases of listeriosis were eventually confirmed in Los Angeles County over an 8-month period. A striking feature was the predominance of perinatal infections (65%), of which 87% occurred among Hispanic women. In contrast, only 29% of cases in nonpregnant adults occurred among Hispanic persons. The case-fatality rate among 87 perinatal or early fetal infections was 63%, and the overall case-fatality rate was 34%. Predisposing factors or conditions among nonpregnant adult patients placed 98% in a higher risk category for infection due to immunosuppression, and included cancer, chronic illness such as diabetes, acquired immune deficiency syndrome (AIDS), taking steroids, and being over 65 years of age. A total of 5 L. monocytogenes serotypes were among the 105 available clinical isolates, of which 82% were serotype 4b. Among the serotype 4b isolates, 73% were the same phage type and were defined as epidemic-associated cases (Figure 10.3). Of the remaining 42 non-epidemic-associated cases, 73% occurred predominantly among nonpregnant adults, and likely represented sporadic cases. Two case–control studies among perinatal listeriosis patients resulted in the implication of a specific brand of soft, unripened Mexican-style cheese as the most likely vehicle of infection. Results of the first study, which explored dietary, occupational, health, and personal behaviors

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14 Epidemic Nonepidemic

12

Cases

10 8 6 4 2 0 Jan

Feb

Mar

Apr

May

Jun

Jul

Aug

Week of Positive Culture

FIGURE 10.3 Cases of listeriosis according to phage type—Los Angeles County, 1985. Arrow designates the time of product recall. (Adapted by E.T. Ryser from Linnan, M.J., L. Mascola, X. Dong Lou, V. Goulet, S. May, C. Salminen, D.W. Hird, M.L. Yonekura, P.S. Hayes, R. Weaver, A. Audurier, B.D. Plikaytis, S. Fannin, L. A. Kleks, and C.V. Broome 1988. Epidemic listeriosis associated with Mexican-style cheese. N Engl J Med 319: 823–828.)

found a statistical association between illness and consumption of Mexican-style cheese (odds ratio [OR] = 5.5, 95% confidence interval [CI] = 1.2–24.8]. Case-patients were also significantly more likely than controls to have consumed the root vegetable jicama; however, this food item is a less likely vehicle as it was consumed by only 41% of cases. In a second case–control study, the investigators focused upon brands of Mexican-style cheese common to the Los Angeles County area; case-patients were significantly more likely than controls to have consumed Brand A cheese (OR = 8.5, 95% CI = 2.4–26.2). Isolation of L. monocytogenes serotype 4b from Brand A cheese collected from patients’ homes and from unopened packages purchased at supermarkets prompted a Food and Drug Administration (FDA) recall of this product, which was subsequently expanded to encompass approximately 500,000 lb of products distributed by the manufacturer to 26 states and the U.S. Protectorates of Guam, American Samoa, and the Marshall Islands [10]. These cheese isolates were confirmed to be of the same phage type that characterized most cases caused by serotype 4b, thus providing laboratory confirmation of the vehicle of infection for epidemicassociated cases. That epidemic-associated cases continued to occur following the product recall can be explained by the long incubation period for listeriosis, and reports that some stores continued to sell the product up to a week after the announcement. Further investigation at the Brand A production facility identified raw milk as the likely source of Brand A cheese contamination. Although the milk was reportedly pasteurized on-site, FDA investigators documented deficiencies in the pasteurization process, including the delivery of quantities of raw milk that would overwhelm the capacity of the pasteurizer by 10% and the presence of high levels of alkaline phosphatase in samples of cheese produced over a 6-month period. These findings indicated that either the milk was inadequately pasteurized or that raw milk was added to pasteurized milk. Postprocessing contamination of the cheese by L. monocytogenes residing in the facility environment was also possible, as investigators isolated the epidemic phage type of L. monocytogenes from the environment of the Brand A production facility. However, recovery of the epidemic phage type from Mexican-style sour cream and cottage cheese produced at a different

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production facility, which received raw milk from the same supplier as used for Brand A cheese, supports the hypothesis that raw milk was the source of L. monocytogenes in Brand A cheese. Reanalysis of data from the aforementioned studies revealed that case-patients were more likely than controls to have consumed the Mexican-style sour cream product (OR = 4.0, 95% CI = 1.1–14.2) [97]. The potential association between consumption of cottage cheese and illness was not explored, however, as the epidemiological investigation focused exclusively on perinatal cases and foods that the patients routinely consumed. Occurrence of the majority of cases in pregnant women of a single ethnic group who sought care at a single facility in Los Angeles County enabled investigators to detect this outbreak, launch a successful investigation, and intervene such that further transmission was prevented. Without this focused cluster of cases to herald the outbreak, it may not have been detected. Such clustering is less common today as more outbreaks are geographically diffuse and are related to centrally produced, widely distributed and commonly consumed products. This underscores the importance for enhanced surveillance, in place in the United States and other countries via notifiable disease reporting systems and standardized, routine subtyping of L. monocytogenes isolates. This epidemic also provided early indication of significant ethnic disparities in the incidence of listeriosis. Last, as mentioned earlier, this epidemic brought L. monocytogenes to the forefront of public health and regulatory attention. In 1986, CDC initiated enhanced surveillance for listeriosis in several U.S. sites and launched epidemiological studies to facilitate a better understanding of dietary risk factors for listeriosis (see Chapter 6 for a detailed discussion). FDA began a program to monitor dairy products for L. monocytogenes, which was later expanded to include other ready-to-eat foods. In 1989, the FDA established a zero-tolerance policy (i.e., no detectable levels of viable organisms permitted) for L. monocytogenes in FDA-regulated ready-to-eat foods [138].

VACHERIN MONT D’OR, WESTERN SWITZERLAND An outbreak of L. monocytogenes serotype 4b, electrophoretic type (ET) I infections occurred in western Switzerland over a 5-year period from 1983 to 1987, and was associated with consumption of a soft cheese produced with pasteurized milk [12,30,31,33,36,41,102]. This outbreak, the first known to have occurred in Switzerland, was also recognized due to an increase in listeriosis cases. Over a 15-month period, 25 cases were diagnosed at a single medical facility (14 in nonpregnant adults and 11 perinatal infections), compared to a mean of 3 cases per year at that facility from 1974 to 1982 [102]. Fifteen additional cases were identified in neighboring facilities during the same time period. The cases were of uniform geographic distribution by patient residence and peaked during winter months. A similar increase in cases during winter months was observed in the following years. Investigators suspected a common source, but results of two case–control studies addressing a variety of food, occupational, and household exposures were inconclusive [12,30]. In addition, attempts to isolate L. monocytogenes from several hundred food items did not identify a potential source. In an independent effort following the 1985 Los Angeles County outbreak in the United States, Swiss health officials conducted studies to determine the prevalence of L. monocytogenes in cheese and other dairy products. These efforts resulted in isolation of L. monocytogenes from regionally produced Vacherin Mont d’Or soft cheese, including the two predominant phage types found in patients [36]. In 1987, a third case–control implicated this cheese as the most likely vehicle of transmission, with 84% of 37 case-patients having consumed Vacherin Mont d’Or cheese, compared to 39% of 51 controls (OR = 8.0, 95% CI = 2.8–22.6, P < 0.05) [33,41]. Further, investigators isolated the subtypes representing most clinical isolates from a patient’s open package of cheese. A total of 122 cases of listeriosis occurred in the Swiss canton of Vaud from 1983 to 1987; 65 (53%) occurred in pregnant women and neonates and 57 (47%) occurred in nonpregnant adults [41]. The overall case-fatality rate was 28% (32% among 57 nonpregnant adults). Among 120 clinical isolates available for characterization, 93% were serotype 4b. Among these, 85% were one

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of two phage types also recovered from the cheese, later confirmed to match by a variety of other subtyping methods [32,34,40,122]. PFGE analysis using two restriction enzymes later distinguished a total of four subtypes each among the human epidemic and cheese isolates [34]. Three subtypes matched between the two groups, lending further support to epidemiological findings. These analyses highlight the importance of a combined laboratory-based and epidemiological approach to outbreak investigations, and the utility of highly discriminatory subtyping methods such as PFGE. This outbreak had notable clinical features. First, underlying conditions associated with immunosuppression were present in 42% of nonpregnant adults, a proportion much lower than that typical of invasive listeriosis outbreaks [41]. Second, this outbreak was characterized by an unusually high proportion of cases with central nervous system (CNS) involvement (79% of cases among nonpregnant adults), with 39% of patients developing meningoencephalitis. Evaluation of cases among nonpregnant adults found an independent association between increasing age and presentation with meningoencephalitis and an increased risk of death. Neurological sequelae were observed among 30% of surviving patients upon evaluation after 5 months to 3 years. Interestingly, a higher proportion of CNS listeriosis cases were caused by the two epidemic-associated subtypes (although not statistically significant). This finding suggests a role for subtype-specific virulence properties. Upon implication of Vacherin Mont d’Or cheese, Swiss officials initiated an international recall and halted production of this cheese. The cheese production process is instructive with regard to epidemiological features of this outbreak, potential routes of contamination, and the effect of a rigorous cleaning and sanitation program. This regional cheese has been produced during winter months both in western Switzerland and in the neighboring area in France (Vacherin Du HautDoubs/Mont d’Or) for up to two centuries [104]; this correlates well with the observed seasonal increase in epidemic cases. Although produced from raw milk in France, the Swiss began producing this cheese from pasteurized milk in 1983 in an effort to better control production and ripening. The cheese associated with this outbreak was reportedly made by forty producers [31,33]. Following coagulation of the milk, the curd was dipped into wooden hoops and allowed to drain for 1 to 2 days. The cheese was then allowed to ripen in one of 12 caves for 3 weeks prior to packaging for sale, where ripening continued. It was not uncommon for cheese to be transferred among different cellars during ripening and distribution or for hoops to be returned to different producers and reused without disinfection, facilitating dissemination of potential contaminants. Indeed, follow-up studies indicated that half of the cellars were contaminated with one or both epidemic subtypes of L. monocytogenes. Implementation of a rigorous cleaning and sanitation program, along with placement of more easily cleaned equipment, was highly effective in ending the outbreak. Only two cases of listeriosis were reported in western Switzerland during the months of January to September 1988 [31].

BRIE

DE

MEAUX, FRANCE

The French National Reference Centre for Listeriosis, which has conducted routine surveillance for L. monocytogenes in humans and in food since 1987, facilitated detection of a geographically diffuse listeriosis outbreak associated with the soft, raw milk cheese Brie de Meaux in 1995. This was the first outbreak in France to be associated with a raw milk cheese [72,127]. The outbreak was first identified as a cluster of 6 cases caused by an unusual phage type, which had represented only 33 cases in France since 1987; this was distinct from subtypes identified in other European and North American outbreaks. Twenty cases were identified over approximately 2 months. Four of 11 perinatal infections resulted in fetal death. Nine cases were among nonpregnant adults; all recovered except for one patient, who remained in a coma at the time of the report. Routine food surveillance data provided evidence for a potential source of the infections, as the epidemic subtype had also been isolated from four surveillance samples of Brie de Meaux. A case–control study, along with Ministry of Agriculture investigations, implicated a specific production batch of this cheese. Health officials initiated a recall and enforced disinfection and

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control measures at the production facilities. Targeted health education messages regarding prevention were also disseminated. In total, 33 cases caused by the epidemic phage type were identified during 1995 [127].

BLUE-MOLD

OR

HARD CHEESE, DENMARK

During a case–control study to identify risk factors for sporadic listeriosis in Denmark, laboratorybased surveillance efforts identified an outbreak that occurred over a 21-month period, from 1989 to 1990 [91]. A total of 26 outbreak-related cases were identified, caused by a serotype 4b phage type (the epidemic subtype) matching that implicated in foodborne listeriosis outbreaks due to Vacherin Mont D’Or cheese in Switzerland from 1983 to 1987 and pork tongue in aspic in France in 1992 (both discussed elsewhere in this chapter). Meningitis was more common (65%) among persons infected with the epidemic subtype than in sporadic cases (40%) that occurred over the same time period; perinatal cases occurred among 12% of epidemic cases. Among nonpregnant adults, 57% had underlying conditions associated with immune suppression, including leukemia, lymphoma, and renal transplant. The overall case-fatality rate was 26%. Subanalyses of data collected for the larger case–control study, in which data for patients infected with the epidemic subtype were compared to data for listeriosis patients infected with non-epidemic subtypes and control persons matched by hospitalization date and department, age, and gender implicated a specific brand of blue-mold cheese as the most likely vehicle of infection (OR = 4.6, 95% CI = 1.0–21, P = 0.03). The epidemic subtype was isolated from seven brand types of a different, popular hard cheese type during routine dairy product screening conducted during the outbreak period; however, it was consumed by over 90% of both listeriosis patients and controls and, therefore, could not be epidemiologically linked to the outbreak. This epidemic is unique in that it was identified and investigated during an effort to identify dietary risk factors for listeriosis in a nonoutbreak setting; case-patients were interviewed upon report of the diagnosis using a questionnaire developed for that purpose. A limitation of the outbreak investigation component was that a hypothesis was not established before interview of associated case-patients, resulting in a greater chance of missing similar exposures among them. On the other hand, there are also advantages to be gained by administration of a standardized food consumption questionnaire upon diagnosis, before an outbreak is recognized. As discussed earlier, the severity of invasive listeriosis and delays of up to several months due to the incubation period and time until outbreak recognition contribute to poor food consumption recall among patients. The longer time period also results in delay of interventions to prevent additional listeriosis cases. In an effort to improve the ability to respond quickly to listeriosis outbreaks, particularly those that are geographically diffuse, public health officials in the United States are currently piloting a standardized, hypothesis generation questionnaire that is administered to all listeriosis patients upon disease reporting to public health agencies. This allows rapid comparison of food exposures between patients infected with a specific subtype and patients infected with other subtypes, expedites the launch of focused epidemiological studies, and results in more rapid intervention measures. A similar approach was successfully applied during the investigation of an outbreak associated with rillettes in France [52]. Used in conjunction with routine molecular subtyping of human L. monocytogenes isolates, this strategy may help to substantially increase the timeliness of outbreak investigations. In turn, appropriate intervention measures can be implemented sooner, reducing the number of outbreak-associated cases and outbreak duration.

RAW-MILK SOFT

OR

SEMIHARD CHEESE, QUEBEC, CANADA

The first listeriosis outbreak to be documented in Quebec, Canada, was linked to consumption of a raw-milk soft or semihard cheese that had undergone <60 days aging [9]. The outbreak was recognized due to an increase in cases caused by the same PFGE subtype across several areas from

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April to November 2002, with a peak in the number of cases in September and October. Among 17 cases, 5 perinatal infections resulted in 3 infected neonates and 2 premature births. Twelve cases occurred in nonpregnant adults, and no deaths were reported. Although interviews of patients with listeriosis did not provide hypotheses for potential sources of infection, an investigation of unassociated reports of gastroenteritis following consumption of cheese from a specific producer led to isolation of the outbreak-associated L. monocytogenes from 56 samples among 16 batches of cheese. Sixty-one percent of 13 listeriosis patients re-interviewed later recalled consuming the cheese prior to illness. A recall conducted in October 2002 was an effective intervention, as only 1 additional case was identified thereafter. Not all of the cases with isolates matching the outbreak-associated PFGE subtype could be explained by cheese consumption, as production did not begin until May 2002. However, the September/October peak in the number of cases, isolation of the same subtype of L. monocytogenes from the cheese, and cessation of the outbreak following the recall provide convincing evidence that this cheese was the source of infection for most of cases. L. monocytogenes was not isolated from the processing environment or bulk milk from the producer’s herd during the investigation. The producer was said to have begun operating in May 2002, following renovations, which may have facilitated the introduction of L. monocytogenes into the production environment. L. monocytogenes was isolated from soil samples outside the facility, but the isolates were not available for characterization. Contamination of the processing area due to renovation and construction is highly possible, given the organism’s ubiquity in the environment [135]. Indeed, a similar hypothesis resulted from the investigation of a contaminated hot dog–associated outbreak that occurred following production facility renovations [109].

ILLICITLY PRODUCED

OR

DISTRIBUTED RAW-MILK FRESH, UNRIPENED CHEESE

Several outbreaks highlight a unique risk factor for listeriosis among members of the Hispanic community, namely, consumption of fresh, unripened cheese (often referred to as queso fresco, queso blanco, Hispanic-style, or Mexican-style soft cheese) made from unpasteurized milk. The cheeses, produced locally by unlicensed manufacturers or in people’s homes and internationally (e.g., Mexico), reach community members in the United States in a variety of ways, including distribution by family and acquaintances or sale at flea markets, by unlicensed street vendors, from people’s vehicles, from door to door, and in small markets. In addition to production from unpasteurized milk, often under conditions of suboptimal sanitation, this cheese style has a relatively high moisture content and pH (>5.6) [127] and is consumed without additional aging. Thus, if contaminated, there are no barriers inherent to the food matrix to prevent growth of L. monocytogenes. Early indication of health risks associated with consumption of this style of cheese occurred in 1997, when three outbreaks of multidrug-resistant Salmonella Typhimurium DT104 infections among Hispanic persons in Northern California and Washington were linked to consumption of Mexican-style soft, raw-milk cheeses [48,147]. In one outbreak, the cheese was purchased predominately from small Hispanic markets [147]. In the other two, unlicensed vendors or family members produced the cheese with raw milk purchased from local dairies. The laboratory investigations supported implication of contaminated raw milk as the source of contamination. The intrastate sale of raw milk is legal in California and Washington; however, the sale of milk to persons who intend to resell the milk or milk products without a license to do so is illegal. These outbreaks provided evidence that this practice was still occurring, emphasizing the importance of a continued focus on enforcement of existing regulations. Both states launched educational campaigns among producers and members of the Hispanic community regarding the risks of fresh, unpasteurized-milk cheese consumption. In 2000, an outbreak of listeriosis linked to illicitly produced Mexican-style soft cheese occurred among Mexican immigrants in North Carolina [99]. First detected as a small cluster of perinatal cases caused by serotype 4b, this outbreak resulted in 13 cases, including 11 perinatal infections and 5 stillbirths. Results of a case–control study implicated Mexican-style soft cheese as the most likely

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vehicle of transmission (matched OR = 17.8, 95% CI = 1.9–69.6). Patients reportedly purchased cheese in unlabeled packages from small Hispanic markets, street vendors, and by door-to-door sales. Two cheese makers, identified by a patient and store owner, reported purchase of raw milk from a local dairy; one person available for further interview reported not pasteurizing the milk during preparation. Personnel from one of three area dairies visited by investigators confirmed selling raw milk to unlicensed persons, despite a warning from the state’s Dairy and Food Protection Branch that the sale of raw milk is illegal in North Carolina and can pose a health risk. L. monocytogenes serotype 4b was isolated from unlabeled fresh, Mexican-style soft cheese obtained from the home of one patient and from two area Hispanic grocery stores, and from bulk tank raw milk samples from a local, manufacturing-grade dairy. Clinical, cheese, and raw-milk isolates were indistinguishable by serotyping (4b), ribotyping, and two-enzyme PFGE analysis. The PFGE pattern was rare, representing only seven isolates from three states among more than 2,000 human clinical isolates in the PulseNet database, further supporting the epidemiological link between consumption of the illicitly produced cheese and listeriosis cases. As a result of this outbreak, listeriosis was made a reportable disease in North Carolina. The source of L. monocytogenes in the bulk tank milk was not identified; milk samples from each of 49 cows were repeatedly culture-negative. Following initiation of a program emphasizing proper teat preparation before milking and thorough cleaning and sanitation of equipment and the facility, however, bulk milk samples no longer yielded L. monocytogenes. In 2003, another outbreak of listeriosis linked to Mexican-style soft, unripened cheese occurred in southern Texas, again characterized by perinatal infections [81]. Among six cases, one infection resulted in fetal death and one in the death of a neonate. Five of six women reported consuming Mexican-style soft cheese (queso fresco) during the month before illness. Further investigation by Texas health officials indicated that the queso fresco was made in Mexico from unpasteurized milk and sold illegally in the state at flea markets and by unlicensed street venders [18]. In a local health advisory, health officials warned against consumption of the illegally imported cheese and educated consumers about how to identify properly manufactured and labeled cheese made from pasteurized milk and sold in reputable stores and markets. Fresh, soft cheese was classified as posing a moderate risk for listeriosis to consumers in the 2003 USDA–FSIS/FDA joint risk assessment [16] (available at http://www.foodsafety.gov/ ~dms/lmr2-toc.html). Placement in the moderate-risk category was attributed predominately to reduced contamination rates, facilitated by improved control measures such as use of pasteurized milk. However, officials acknowledge that this classification may not be representative of the risk among members of the Hispanic community. Data used in the risk assessment were derived in part from several studies of contamination of soft, fresh cheeses, and not specifically related to cheeses of this style made from unpasteurized milk. Soft, unripened cheese made from unpasteurized milk is a traditional part of the Hispanic diet and presents a unique challenge for reducing the risk for listeriosis in this community. Indeed, in a qualitative study investigating knowledge, attitudes, and practices regarding consumption of unpasteurized milk products among 76 members of the Hispanic community in the state of Georgia, most believed that unpasteurized milk products were “healthier” than those made from pasteurized milk [87]. None had heard of listeriosis, other infections, or pregnancy complications associated with consumption of unpasteurized milk or milk products. Most (including pregnant women) reported regular consumption of homemade cheese. Studies addressing knowledge, attitudes, and practices regarding consumption of fresh, soft cheese made from unpasteurized milk, along with outbreak data, indicate that the cheese is often produced or sold illicitly by unlicensed vendors, rather than by licensed commercial establishments. It is therefore difficult to obtain estimates for the prevalence of L. monocytogenes in these cheeses, and regulations regarding their production and sale are difficult to enforce. In addition to continued regulatory enforcement efforts, a key strategy for prevention of listeriosis and other infections associated with consumption of fresh cheese made from unpasteurized milk in this community may lie in health education. Following the outbreak of Salmonella

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Typhimurium DT104 infections in Yakima County, WA, health officials collaborated with University of Washington Cooperative Extension to launch the Abuela Project among Hispanic community members [28]. Educators worked with abuelas (grandmothers), who are highly respected and traditionally make the cheese, to develop an organoleptically acceptable recipe and produce the cheese using pasteurized milk and improved safety measures. The abuelas, in turn, trained 15 additional community members. In total, more than 225 persons were trained; positive behavior changes were maintained for at least 6 months, and the incidence of Salmonella Typhimurium DT104 infections in Yakima County decreased dramatically. This project may serve as a model component of culturally appropriate educational intervention programs aimed at reducing the incidence of foodborne listeriosis in Hispanic communities [5].

FLUID MILK Few outbreaks of invasive listeriosis have been associated with consumption of contaminated fluid bovine milk. Aside from the raw milk–associated epidemic in Halle, Germany (late 1940s–1957), discussed earlier, we are aware of only one other documented invasive listeriosis outbreak potentially associated with raw-milk consumption. Austrian officials reported an outbreak in 1986 that resulted in 24 perinatal infections and 4 cases in nonpregnant adults [2]. Case histories indicated that all 4 nonpregnant adults, along with 5 of 20 pregnant women, had consumed raw milk. Consumption of vegetables produced using biologic agriculture by 11 patients was suggested as a second potential risk factor. In 1983, health officials linked an outbreak of serotype 4b infections in Massachusetts to consumption of a specific brand of 2% and whole pasteurized milk [63]. A dramatic increase in listeriosis occurred from June to August, with an incidence 4 to 5 times that for previous summers in the state. Of 49 cases, 42 (86%) occurred among nonpregnant adults, all with underlying conditions causing immunosuppression. Seven infections were perinatal. The overall case-fatality rate was 29%, with 2 cases resulting in fetal death. One case-patient acquired the infection in a hospital. Forty of 49 isolates were available for further characterization; 32 were serotype 4b. The first of two casecontrol studies, in which case-patients and controls were matched for age, gender, and neighborhood of residence, showed that case-patients were more likely than controls to have shopped at a specific grocery chain (Chain A). Comparison of a list of items purchased by 5 patients during the month before illness, obtained during store visits with the patients, helped to narrow the focus to dairy products. Further interviews regarding dairy product consumption showed that case-patients were significantly more likely to have consumed Chain A whole or 2% milk (OR = 9.3, P < 0.01). Pasteurized milk was an unexpected vehicle, and investigators were concerned that the association may have resulted from comparison of patients to healthy neighbors less susceptible to L. monocytogenes infection. Results of a second study, in which patients’ food consumption exposures were compared to those for persons with similar underlying health conditions, confirmed the findings of the first; patients were significantly more likely to have consumed Chain A whole or 2% milk than controls (OR = 11.5, 95% CI = 2.7–48.8, P < 0.001). Additional epidemiologic and laboratory studies supported these findings. Evaluation of the volume of Chain A milk consumed per day among patients and controls suggested a dose–response effect, and consumption of skim milk (which would decrease the chance of exposure to whole or 2% milk) was significantly protective against illness (OR = 0.25, 95% CI = 0.08–0.75, P < 0.02). Listeriosis cases in a neighboring state may have also been associated with this outbreak; an investigation of 12 cases found that 3 patients had consumed milk from, among 2600 total stores in the state, 1 of only 2 stores that sold milk from the implicated facility (P < 0.0001). Last, further characterization of clinical isolates found that one phage type was isolated significantly more often from patients who had reported drinking Chain A whole or 2% milk than from those who did not (P < 0.001). The epidemic-associated phage type was later shown to be genetically similar to subtypes implicated in European listeriosis outbreaks associated with pâté in the United Kingdom (1987–1989) [108] and France (rillettes, 1993) [74,127].

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The route of milk contamination was not determined; both improper pasteurization and postpasteurization contamination seemed unlikely based on environmental investigation findings. Listeriosis among cows at supplier dairy farms was reported to have occurred during the human epidemic period. However, time/temperature logs were consistent with proper pasteurization, and no equipment defects that would have compromised the pasteurization process were noted. Facility hygiene practices were appropriate, and skim milk (not associated with the epidemic) and 2% and whole milk were processed with the same equipment; this argued against long-term postpasteurization contamination that would not also affect skim milk. Investigation following the epidemic period resulted in isolation of several L. monocytogenes serotypes (including 4b) from milk filters and from raw-milk samples (12% of 124) collected from the facility, the cooperative that supplied raw milk, and from farms. However, the epidemic-associated phage type was not recovered. These findings, together with reports describing high heat resistance of L. monocytogenes during pasteurization [26] and postulations of enhanced heat resistance among organisms that had invaded bovine leukocytes led investigators to hypothesize that some organisms may have survived proper pasteurization. This called into question the efficacy of FDA pasteurization guidelines with regard to inactivation of L. monocytogenes in milk. However, results of subsequent, carefully designed studies addressing this issue found recommended pasteurization procedures to be efficacious for inactivation of L. monocytogenes [42,54,55].

OTHER DAIRY PRODUCTS Two outbreaks of invasive listeriosis, one in Southern California and one in Finland, have been linked to consumption of contaminated butter. Enhanced surveillance in Los Angeles County, initiated following the large 1985 outbreak associated with Mexican-style soft cheese, facilitated detection of a cluster of 11 perinatal cases in 1987 [103]. A case–control study implicated butter as the most likely vehicle of transmission (OR = 4.0, 95% CI = 0.9–16.7). Results also indicated a potential association of salad (OR = 6.0, 95% CI = 0.5–66.7) and carrots (OR = 4.5, 95% CI = 0.8–24.3). An outbreak of 25 listeriosis cases caused by an unusual serotype, 3a, occurred primarily among patients of a tertiary care hospital in Finland during 1998–1999 [98,100]. Twenty patients were diagnosed with sepsis, four with meningitis, and one with an abscess; six (24%) patients died. Their clinical isolates were indistinguishable by PFGE (designated as the outbreak-associated subtype). Patients infected with the outbreak-associated subtype were more likely to suffer severe immunosuppression from underlying conditions including malignancy or to have been admitted to the tertiary care hospital than patients infected with other subtypes (P = 0.02 and P < 0.001, respectively), and were also more likely to have been admitted to a specific tertiary care facility. The same serotype had been isolated previously from a sample of butter produced at the Finnish dairy that served as the exclusive supplier to the facility; the isolates were subsequently shown to match the clinical isolates by PFGE analysis. Samples of butter collected from the hospital kitchen yielded the outbreak subtype, prompting national and local food authorities to expand sampling (the dairy also supplied other hospital kitchens and a wholesaler) and conduct an environmental investigation at the dairy. Deficiencies in pasteurization were not identified; however, the outbreak subtype was isolated from butter packages of several sizes collected from the dairy and wholesaler, as well as from processing and packaging equipment at the dairy. A case–control study among outbreak-associated cases at the tertiary care facility and control patients matched by age and underlying condition did not statistically implicate butter as the likely vehicle of transmission, perhaps because of the availability of only seven outbreak-associated patients for interview and the frequency of butter consumption among all patients. Although not statistically significant, investigators found that case-patients consumed over four times as much butter as control patients during their stay. Further, hospital units with cases received significantly more butter than units with no cases (1378 vs. 181 g/100 patient-days, P = 0.01). The median length of the

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hospital stay before collection of clinical specimens that yielded L. monocytogenes was significantly longer among case-patients than controls (31 vs. 10 days, P = 0.008). This outbreak provided a unique opportunity to estimate probable levels of exposure among at-risk tertiary care facility patients, as packages of the implicated butter were available at the time of outbreak detection [100]. Using quantitative contamination data (obtained by screening samples from the hospital kitchen) and daily consumption data (obtained from five case-patients and accounting for length of stay), the estimated continuous daily dose ranged from 101 to 103 CFU/day; the dose ranged from 104 to 105 CFU/day if the higher level (105) detected in a wholesale sample was used. Using maximum contamination levels, the single dose (i.e., number of organisms consumed at one meal) causing illness was estimated to be 7.7 × 104. The data supported two hypotheses, each having a different impact on dose–response predictions from these data. For the first hypothesis, consumption of a single, high-level dose resulted in listeriosis. Based on sample screening results, occasional contamination (and therefore consumption in a single meal) of a package of butter with a disease-producing dose was possible. However, the same brand of butter, and likely the same dose of L. monocytogenes, was consumed in other units and hospitals without incident. Dose–response curves for a single high dose further predicted a low probability of listeriosis in the population. For the second hypothesis, prolonged exposure to a lower level of L. monocytogenes in the butter resulted in disease as opposed to a single exposure. This was also possible, as the majority of samples were contaminated at <100 CFU/g and as case-patients were found to have significantly longer stays. This hypothesis underscores the potential risk for listeriosis posed by consumption of foods with lower-level contamination (<100 CFU/g) among susceptible populations. Although dose–response curves must be interpreted with caution, as the unusual serotype, vehicle, and highly susceptible populations are not generalizable to all exposure scenarios or atrisk populations, these data provide a valuable contribution to risk assessment in humans, and were used for exposure assessment (dose–response estimates for persons with immunosuppression) in the USDA–FSIS/FDA joint risk assessment [16]. An unusual invasive listeriosis outbreak involving several different serotypes and, probably, several independent vehicles occurred in the Philadelphia, PA metropolitan area from December 1986 to April 1987 (Figure 10.4) [137]. The incidence during this period was 2.0/100,000 area 16

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FIGURE 10.4 Listeriosis cases reported between January 1986 and April 1987—Philadelphia Standard Metropolitan Statistical Area, Pennsylvania. (Adapted from Schwartz, B., D. Hexter, C.V. Broome, A.W. Hightower, R.B. Hischhorn, J.D. Porter, P.S. Hayes, W.F. Bibb, B. Lorber, and D.G. Faris 1989. Investigation of an outbreak of listeriosis: new hypotheses for the etiology of epidemic Listeria monocytogenes infections. J Infect Dis 159: 680–685. With permission.)

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population, compared to 0.3/100,000 during the preceding 11 months. Among 36 cases identified during the epidemic period, 32 occurred in nonpregnant adults (75% had underlying conditions resulting in immuno suppression) and 4 were perinatal. The case-fatality rate was higher than that for most outbreaks; 44% of patients, including 2 neonates, died. Also uncharacteristic of other outbreaks of invasive listeriosis, 35% of patients reported having diarrhea during the week before their illness was diagnosed. A case–control study among patients (or surrogates) and neighborhood- and underlying condition-matched controls identified several potential dietary risk factors for illness. Casepatients were significantly more likely than controls to have shopped for food at a specific grocery chain (OR = 5.0, 95% CI = 1.5–16.4), to have consumed ice cream (94% of casepatients compared to 75% of controls, OR = 9.1, 95% CI = 1.6–51.4), and to have consumed salami (22% of case-patients compared to 5% of controls, OR = 6.3, 95% CI = 1.2–33.3). Casepatients were also more likely to have consumed Brie cheese, although this association did not reach statistical significance. However, specific brands of these foods were not epidemiologically associated with illness. Molecular characterization identified 4 serotypes and 11 electrophoretic types (ET) among clinical isolates, of which ET 1 and ET 4 were most prevalent. ET 1 was isolated from three of four patients who had consumed a specific brand of ice cream (P = 0.04), but no other subtype was significantly associated with specific food types. An ET 4 isolate was recovered from Brie obtained from a patient’s refrigerator; this ET matched that of the patient’s clinical isolate. Samples of ice cream, cheese and other dairy products, vegetables, and meat products, some of which were obtained from patients’ homes, did not yield L. monocytogenes. The time and geographic clustering of cases in this area support the hypothesis that an outbreak occurred. The diversity of clinical subtypes and case–control study results, however, argue against a common source. This outbreak is illustrative of the utility of molecular characterization for interpretation of epidemiological data. It is possible, albeit an unusual scenario, that more than one cluster occurred simultaneously, as each food was a plausible vehicle (see Chapters 11 to 13 for discussion of the prevalence of L. monocytogenes in these foods). Also, more than one subtype of L. monocytogenes may be present in a contaminated food item [41,109,128], potentially contributing to the diversity of subtypes associated with an outbreak. Case–control study results showed use of antidiarrheal medication and family members who were ill during the month before illness among significantly more case-patients. These data, along with the presence of gastrointestinal symptoms among ~30% of patients led investigators to hypothesize that gastrointestinal carriage may have resulted from consumption of a contaminated item, with transition to invasive disease facilitated by coinfection by another enteric pathogen such as an enteric virus circulating in the community.

MEAT AND POULTRY PRODUCTS The first laboratory-confirmed association of meat/poultry products with invasive listeriosis occurred in 1988, when a case was linked to consumption of contaminated turkey franks (13). Coincidentally, results from the first case–control study to identify dietary risk factors for sporadic listeriosis were published; persons who had consumed undercooked chicken and uncooked hot dogs were more likely to develop listeriosis [136]. The patient, hospitalized with sepsis caused by L. monocytogenes serotype 1/2a, reported daily consumption of turkey franks that were heated in a microwave oven. L. monocytogenes serotype 1/2a was isolated from opened packages of turkey franks obtained from the patient’s refrigerator (MPN >1100/g) and from unopened packages of the same brand obtained from a local store (MPN < 3/g) (149). The patient and food isolates were of the same electrophoretic type. Based on these data, the USDA initiated a product recall. The following day, USDA–FSIS and CDC began an environmental investigation at the processing facility to identify potential routes of contamination and strategies to prevent product contamination [149]. L. monocytogenes was isolated from finished product samples representing six of seven

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lots tested, with isolates from five lots matching the case isolate by serotyping and multilocus enzyme electrophoresis (MEE). Production line sampling revealed an increased contamination frequency of samples after mechanical removal (peeling) of the cellulose casing, and a swab sample taken from the conveyer belt onto which the peeled franks dropped yielded the same subtype. Another L. monocytogenes serotype, 1/2b, was isolated from product and environmental samples in the room in which the cooked franks were cooled prior to peeling. The environmental investigation results provided strong evidence that the source of contamination was the processing environment, and that contamination occurred at the peeling step—after the cook step, which would inactivate L. monocytogenes. Isolation of this subtype from the plant environment over 4 months after the case occurred suggested its persistence in the processing area. The presence and persistence of L. monocytogenes in processing areas has proven to be a significant challenge to regulators and the food industry in the United States, as the contamination of ready-to-eat meat and poultry products after preparation of the finished product but before packaging led to three large, multistate listeriosis outbreaks over the following 12 years. This investigation provided early evidence that prevention of even low-level finished product contamination is critical to prevention of listeriosis, especially since L. monocytogenes grows well during storage of processed poultry products [69] and appeared to have grown by several logs during home refrigeration of the frankfurters implicated here. In 1987, USDA–FSIS responded to increased knowledge of the association of L. monocytogenes with deli meats and other processed foods by initiating a monitoring program for ready-toeat meat and poultry products produced in federally inspected establishments (4). In 1989, USDA–FSIS established a zero tolerance policy for L. monocytogenes and other pathogens in readyto-eat meat and poultry products [3].

READY-TO-EAT MEAT AND POULTRY PRODUCTS IN THE UNITED STATES: FRANKFURTERS AND DELI MEAT The largest listeriosis outbreak in the United States occurred during 1998–1999 [109]. In November, 1998, PFGE data submitted to PulseNet of serotype 4b isolates from contemporaneous listeriosis clusters in Connecticut, New York, Ohio, and Tennessee revealed that they shared an unusual pattern. Concerned about the potential for a multistate outbreak, CDC and collaborating state public health officials initiated an epidemiologic and laboratory-based investigation. Investigators defined a case as infection by L. monocytogenes with one of three closely related PFGE patterns (indistinguishable by AscI restriction and differing by no more than 2 in 26 bands on ApaI restriction [75]). A total of 447 clinical isolates from cases with illness onset from January 1998 to July 1999 were characterized by PFGE. Among them, 108 (Figure 10.5) from 24 states (Figure 10.6) met the case criteria, with most cases occurring between August 1998 and January 1999. Thirteen cases resulted from perinatal infection. Among 43 nonpregnant patients (infected with both outbreakand nonoutbreak-associated subtypes) for whom information regarding underlying conditions was available, all but 2 were either >65 years of age or had a condition that would result in immunosuppression. Hypertension, diabetes, and malignancy were most common, and there were no notable differences between the outbreak- and nonoutbreak-associated groups. The median age among nonpregnant adults infected with an outbreak-associated subtype was 70 years. Results from two case–control studies implicated frankfurters as the most likely vehicle of infection. In the second study and for the first time in a listeriosis investigation, patients with contemporaneous L. monocytogenes infection caused by a nonoutbreak-associated PFGE subtype served as the control population. Although a rather unconventional approach, this method allows for efficient identification of a comparable control group. Selection of control persons has historically been a challenge as the at-risk population for listeriosis is not representative of the general population. In the first case–control study, case patients were more likely to have consumed cooked frankfurters during the month preceding illness (89% of 18 case patients vs. 32% of 19 controls, OR = 17.3, 95% CI = 2.4–160.0, P < 0.0004). The second case–control study, which included

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FIGURE 10.5 Epidemic curve showing listeriosis cases caused by outbreak-associated subtypes, United States, January 1998–April 1999. Light arrow indicates removal of refrigeration unit from processing facility A production area. Dark arrow indicates date of recall of frankfurters and deli meats. (Adapted from Mead, P., E. F. Dunne, L. Graves, M. Weidmann, M. Patrick, S. Hunter, E. Salehi, F. Mostashari, A. Craig, P. Mshar, T. Bannerman, B. Sauders, P. Hayes, W. Dewitt, P. Sparling, P. M. Griffin, D. Morse, L. Slutsker, and B. Swaminathan 2006. Nationwide outbreak of listeriosis due to contaminated meat. Epidemiol Infect 134(4): 744–751. With permission.)

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FIGURE 10.6 States with listeriosis cases in an outbreak associated with contaminated ready-to-eat meat products — United States, 1998–1999. (Reprinted from Mead, P., E. F. Dunne, L. Graves, M. Weidmann, M. Patrick, S. Hunter, E. Salehi, F. Mostashari, A. Craig, P. Mshar, T. Bannerman, B. Sauders, P. Hayes, W. Dewitt, P. Sparling, P. M. Griffin, D. Morse, L. Slutsker, and B. Swaminathan 2006. Nationwide outbreak of listeriosis due to contaminated meat. Epidemiol Infect 134(4): 744–751. With permission.)

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questions about deli meat products, also implicated frankfurters as the most likely vehicle of transmission (OR = 6.0, 95% CI = 1.2 – 38.6, P = 0.01). Case patients were also more likely to have consumed turkey cold cuts sliced at a deli; however, the association was not statistically significant. Eleven of 14 case patients from the first study for whom purchase information was available reported consumption of products made by a specific company, and one available package bore the processor establishment code (processing facility A). The company, however, did not issue a recall at that time. Among case patients in both studies, 13 of 44 (30%) recalled consumption of brands produced at processing facility A, and an additional 9 recalled consumption of frankfurters or deli meats from retailers who received products from this facility. At the same time, a review of isolates obtained by academic researchers identified two deli meat isolates that matched the outbreak-associated subtype by PFGE and automated ribotyping (38,39). The isolates were from two brands of deli meat consumed by members of one family, of which three members developed febrile gastroenteritis. A specific diagnosis was not made; however, convalescent serum from one ill person indicated high titers of α-listeriolysin O antibodies. This is consistent with acute illness caused by L. monocytogenes. Processing facility A also produced one brand of this deli meat. An investigation at processing facility A revealed that construction had occurred in July 1998, shortly before the outbreak began, with the replacement of a refrigeration unit in the room where the frankfurters were collected on conveyer belts after their casings were removed. In-plant records indicated that the frequency of isolation of psychrotrophic microorganisms (organisms that can grow between 0°C and 7°C and produce visible colonies within 7 to 10 days [90]), which can be indicative of the presence of L. monocytogenes, sharply increased among samples collected from retail frankfurter processing lines during the months following construction. L. monocytogenes had likely colonized areas of the refrigeration unit, and the environmental disruption resulted in contamination of processing equipment and finished product. Whereas L. monocytogenes was not recovered from the processing environment at the time of the investigation, the outbreak strain was isolated from opened and unopened packages of frankfurters and deli meats. Following isolation of this subtype from an opened package of frankfurters, the company voluntarily recalled specific production lots of various brands of frankfurters and deli meats and issued a public notification [14]. As shown in Figure 10.5, the outbreak abruptly ended after the recall. This outbreak had several informative features. The rapid decrease in cases that followed identification and recall of the product suggested that the incubation period for invasive listeriosis may be shorter than historical estimates of 3 to 4 weeks, data which were derived primarily from studies involving perinatal infection. In this outbreak, the onset of cases in nonpregnant adults ended within 5 days of the recall. One person had illness onset within 48 hours of a single exposure. Thus, it is important to consider even recent exposures during investigations of invasive listeriosis. Another interesting feature was the isolation of a second serotype (1/2a) from deli meat at a much higher level (3,000 CFU/g) than the outbreak-associated subtype, serotype 4b (<0.3 CFU/g). This second subtype was not epidemiologically linked to any outbreak-associated cases and was not represented among any of the sporadic case isolates characterized for this outbreak. These data are supportive of the growing body of evidence for differences in pathogenic potential among different subtypes of L. monocytogenes [95,122,151]. However, such interpretations must be made with caution. As reported by Graves et al. [75], quantitative data for the outbreak-associated subtype were obtained from analysis of opened packages of frankfurters collected from patient refrigerators more than 3 weeks after illness onset, while unopened packages of deli meat obtained from the processor yielded serotype 1/2a. It is possible that the outbreak-associated subtype had entered the stationary or death phase by the time of sample collection; thus, the data may not be representative of the dose consumed by patients. In a recent study, the serotype 4b subtypes associated with this outbreak (designated EC II) were genetically compared with other serotype 4b isolates [59]. Analyses revealed genetic fragments that were conserved in the ECII subtypes but not conserved among other 4b isolates. The serotype 4b specific region in which these fragments were located lies adjacent to inlA, which codes for a virulence factor that has a critical role in invasion of several classes of mammalian cells. Whereas any association with host–pathogen

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interaction characteristics of this subtype remains to be determined, the divergent region provides potential targets for DNA-based tools designed for specific detection and monitoring of this subtype. In 2000, the processor linked to the 1988 listeriosis case discussed earlier (turkey franks [113]) was the source of contaminated turkey deli meat implicated in an outbreak involving 30 cases in 11 states [119]. The outbreak was first recognized as a cluster of nine serotype 1/2a infections that occurred over a 2-month period in New York City. The isolates matched by PFGE analysis and ribotyping, prompting health officials to notify CDC and share the PFGE pattern with other public health laboratories via the PulseNet Webboard. Comparison to patterns in the national PulseNet database revealed matches in New York State, Michigan, and Georgia. CDC initiated a multistate investigation; investigators posted a message on the PulseNet message board asking state laboratories to perform real-time PFGE analysis on all clinical L. monocytogenes isolates and requested case history information from epidemiologists at state health departments. Thirty cases in 11 states with matching PFGE patterns and ribotypes were identified, with culture dates ranging from May to December 2000; 90% occurred after mid-July (Figure 10.7). A case–control study, with patients contemporaneously infected with a nonoutbreak-associated subtype of L. monocytogenes serving as the control population, identified consumption of turkey deli meat from a delicatessen (76% of 17 case patients vs. 21% of 24 controls, OR = 12.4, 95% CI = 2.3–72.5) and lettuce (94% of 18 case-patients vs. 57% of 23 controls, OR = 13.1, 95% CI = 1.4–600.0). When the data were analyzed controlling for consumption of turkey deli meat, lettuce did not remain significantly associated with illness. However, turkey deli meat consumption remained significantly associated with illness when controlling for consumption of lettuce. Purchase information available from nine patients

10 9

Number of Cases

8 7 6 5 4 3 2 1

5/ 14 –5 /2 0 5/ 28 –6 /3 6/ 11 –6 /1 7 6/ 25 –7 /1 7/ 9– 7/ 15 7/ 23 –7 /2 9 8/ 6– 8/ 12 8/ 20 –8 /2 6 9/ 3– 9/ 9 9/ 17 –9 /2 3 10 /1 –1 0/ 10 7 /1 5– 10 /2 10 1 /2 9– 1 1/ 11 4 /1 2– 11 /1 11 8 /2 6– 12 12 /2 /1 0– 12 /1 6

0

Date of Culture (2000) FIGURE 10.7 Epidemic curve showing listeriosis cases caused by the outbreak-associated subtype, United States, May–December 2000. Arrow indicates date of recall of processed turkey and chicken meats, December 14, 2000. (Adapted from Olsen, S.J., M. Patrick, S.B. Hunter, V. Reddy, L. Kornstein, W.R. MacKenzie, K. Lane, S. Bidol, G.A. Stoltman, D. M. Frye, I. Lee, S. Hurd, T. F. Jones, T. N. LaPorte, W. E. Dewitt, L. Graves, M. Wiedmann, D. Schoonmaker-Bopp, A.J. Huang, C. Vincent, A. Bugenhagen, J. Corby, E.R. Carloni, M.E. Holcomb, R.F. Woron, S.M. Zansky, G. Dowdle, F. Smith, S. Ahrabi-Fard, A. Rae Ong, N. Tucker, N.A. Hynes, and P. Mead 2005. Multistate outbreak of Listeria monocytogenes infection linked to delicatessen turkey meat. Clin Infect Dis 40: 962–967. With permission.)

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was used in a traceback investigation to determine the source of the turkey deli meat. Investigators visited the places of purchase to collect environmental swab samples and identify turkey deli meat brands and production establishment codes. Swabs from four stores yielded L. monocytogenes, and isolates from three stores matched the outbreak strain. Twenty-seven production facilities supplied turkey deli meat to stores where the nine patients reported purchasing the meat. However, one establishment (plant A) supplied turkey deli meat to stores named by at least six (potentially eight, based upon brand) patients, and a second (plant B) supplied to retailers named by five. Combined, products from these two establishments could explain up to eight of these nine cases. Further investigation revealed that plant B was a copacker for plant A, meaning that product could be shipped to plant A to be processed further and packaged under the plant A label. Based upon this information, USDA–FSIS investigated the two processing facilities. Product and environmental samples did not yield L. monocytogenes; however, plant B initiated a voluntary recall of 16 million pounds of processed turkey and chicken meat, based upon results of the epidemiologic investigation. This outbreak highlighted the critical role of molecular subtyping and efficient information sharing in detection and investigation of outbreaks in the age of centrally produced, widely distributed food products. With a proportionately small at-risk population and long incubation period, listeriosis outbreak investigations present a unique challenge to public health officials. These tools enabled investigators to better determine the scope, magnitude, and source of the outbreak, such that the appropriate intervention prevented further illness. In 2002, contaminated turkey deli meat was implicated as the source of yet another large, multistate outbreak in the United States [70]. This outbreak was detected as a cluster of PFGEmatched serotype 4b isolates among 22 cases reported by Pennsylvania health officials. CDC initiated a collaborative, multistate investigation, asking that health officials in northeastern states conduct active case finding, perform real-time PFGE analysis of all clinical isolates, and submit the patterns to PulseNet for rapid comparison. Among 188 cases identified in nine states from July to November 2002, 54 clinical isolates shared the outbreak-associated subtype. This PFGE pattern had only been identified 20 times previously in the national database, which contained approximately 2000 isolates from the previous 6 years. Among the 54 patients infected with the outbreakassociated subtype, 8 (15%) died and 3 women lost their fetuses (Figure 10.8). Among the casepatients were 8 pregnant women, 4 neonates, 30 patients with medical conditions associated with immunosuppression, and 4 patients aged >65 years with no underlying medical condition. A case–control study, in which controls were listeriosis patients in states where case-patients resided with contemporaneous infection by a nonoutbreak-associated subtype, found that case-patients were significantly more likely than controls to have consumed turkey deli meat more than one to two times during the 4 weeks preceding illness (55% of 38 case-patients vs. 29% of 53 controls, OR = 4.5, 95% CI = 1.3–17.1). Consumption of precooked turkey breast purchased at grocery stores or restaurants was statistically associated with infection by the outbreak-associated subtype (P = 0.008). Based upon this information, health and regulatory officials launched a traceback investigation. Casepatients were unable to recall specific brands; however, 80 purchase sites were identified by the first 29 patients interviewed. Among 57 purchase sites investigated, turkey deli meat products were supplied by more than 50 processors. USDA–FSIS officials systematically evaluated the 15 most frequently identified processors, addressing factors including in-plant Listeria sampling results, food safety violations, and construction projects. Results from four processors warranted further investigation. The outbreak-associated subtype was isolated from the processing environment of one processor (processor C, designations differ from original reference), from unopened packages of turkey deli meat from another (processor D), and from opened packages of deli meats produced at both processors which were obtained during purchaser investigations and from patients. Notably, in a similar scenario to that observed in the 1998–1999 outbreak linked to ready-to-eat meats, a construction project had occurred at processor C in the room where cooked turkey was handled. Coincidentally, the frequency of Listeria-positive environmental samples increased in this processing area. In response to investigation findings, which provided strong evidence for the association of processor C and D products

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12 Died

Number of Patients

10

Had fetal death

8 6 4 2 0 7/1

7/15

7/29

8/12

8/26

9/9

9/23

10/7

10/21

11/4

11/18

Week of Culture, 2002 FIGURE 10.8 Epidemic curve showing listeriosis cases caused by outbreak-associated subtypes, United States, July–November 2002. Dark arrow indicates approximate date of initial recall of turkey deli meat products (October 9, 2002). (Adapted from Gottlieb, S.L., C.E. Newbern, P.M. Griffin, L.M. Graves, R.M. Hoekstra, N.L. Baker, S.B. Hunter, K.G. Holt, F. Ramsey, M. Head, P. Levine, G. Johnson, D. SchoonmakerBopp, V. Reddy, L. Kornstein, M. Gerwel, J. Nsubuga, L. Edwards, S. Stonecipher, S. Hurd, D. Austin, M.A. Jefferson, S.D. Young, K. Hise, E.D. Chernak, J. Sobel, and the Listeriosis Outbreak Working Group 2006. Multistate outbreak of listeriosis linked to turkey deli meat and subsequent changes in United States regulatory policy. Clin Infect Dis 42: 29–36. With permission.)

to this outbreak, both processors voluntarily recalled a combined >30 million pounds of ready-to-eat poultry products. Investigation findings further informed recent USDA–FSIS policy changes, detailed in the closing discussion for this section.

OTHER PROCESSED MEATS Two outbreaks of invasive listeriosis have been linked to pâté [11,108]. The first occurred in England, Wales, and northern Ireland, when trends in the epidemiology of listeriosis in 1988 and 1989 suggested the occurrence of one or more common source epidemics [108]. As shown in Figure 10.9, the number of laboratory-confirmed cases in 1987–1989 was nearly double the number of cases in 1983–1986. Consistent seasonal peaks in late summer and early fall disappeared during this period, and the percentage of perinatal infections increased to 52% in 1989, compared to 38% in 1983–1987. Isolate characterization revealed two frequent subtypes: a specific serotype 4b phage type (phage type 6,7) and serotype 4bX accounted for 30 to 54% of the cases annually. In 1989, routine public health follow-up of a listeriosis case resulted in isolation of L. monocytogenes from pâté obtained from the patient’s refrigerator. This prompted a comprehensive survey of pâté products for this organism, and survey of listeriosis patients with regard to their food exposures during the 3 weeks before illness. Of 1,698 pâté samples screened in 1989, 9.5% were positive. Notably, samples produced by one manufacturer (manufacturer Y) were contaminated at a higher frequency (48% of 107 samples positive, 11% at >1000 CFU/g) than samples produced by other manufacturers (4% of 781 samples). Further, serotype 4b phage type 6,7 and serotype 4bX accounted for 96% of isolates from manufacturer Y’s pâté samples, compared to 19% of samples from other producers. Patients infected with the two predominant subtypes were significantly more likely to have consumed pâté during the 3 weeks before illness, compared to patients infected by other subtypes (87% of 15 vs. 35% of 17, χ2 test P = 0.0095).

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350 Total Cases Non-pregnancy Pregnancy

300

Number of Cases

250

200

150

100

50

0 1983 84 85 86 87 88 89 90 91 92 93 94 Year

FIGURE 10.9 Listeriosis in England, Wales, and Northern Ireland, 1983–1994. (Adapted by E.T. Ryser from McLauchlin, J. and L. Newton 1995. Human listeriosis in England, Wales and Northern Ireland: a changing pattern of infection. Proc. XIIth International Symposium on Problems of Listeriosis, Perth, Western Australia, October 2–6, pp. 177–181.)

The chance finding of L. monocytogenes in pâté obtained from a patient’s refrigerator also prompted a government health warning (May–June 1989) to at-risk consumers regarding pâté consumption. Following this warning and suspension of sales of pâté from manufacturer Y, the incidence of listeriosis declined dramatically (Figure 10.9). Further, a concurrent decline in infection by the two predominant subtypes was also observed. Last, in a 1990 survey of 626 pâté samples, the frequency of L. monocytogenes serotype 4b phage type 6,7 and 4b X-positive samples declined to 4%. In 1999, a small outbreak of invasive listeriosis in three U.S. states (New York, Connecticut, and Maryland) was attributed to consumption of pâté [11]. Eleven cases caused by an unusual subtype of serotype 4b were identified. Two patients had consumed the same brand of pâté, and a third had consumed an unknown brand of pâté. Based upon USDA–FSIS investigation findings, the company recalled approximately 400 pounds of pâté and other similar products produced from poultry meat [17]. The recall was subsequently expanded to encompass approximately 80,000 pounds of product [17]. One of the largest documented outbreaks of invasive listeriosis occurred in France in 1992 [72a,73,89,130] and was associated with consumption of pork tongue in aspic (jelly). Detection of this outbreak was facilitated by two National Reference Centers (NRC), components of a surveillance system for listeriosis established in 1987. Clinical isolate serotyping at the Nantes facility, followed by phage typing at the Pasteur Institute, revealed that 279 of 758 captured cases (37%) were caused by a specific subtype of serotype 4b (Figure 10.10), compared to only 6 to 27 cases during previous years [89]. Among the 279 outbreak-associated cases, 182 (65%) outbreak-associated cases occurred in nonpregnant adults, 5 occurred in children (2%), and 92 (33%) in pregnant women.

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120 Epidemic

Non-epidemic

Number of Cases

100 80 60 40 20 0

J

F

M

A

M

J

J

A

S

O

N

D

Month

FIGURE 10.10 Listeriosis in France, 1992. (Adapted by E.T. Ryser from Jacquet, C., B. Catimel, R. Brosch, C. Buchrieser, P. Dehaumont, V. Goulet, A. Lepoutre, P. Veit, and J. Rocourt 1995. Investigations related to the epidemic strain involved in the French listeriosis outbreak in 1992. Appl Environ Microbiol 61: 2242–2246.)

This outbreak resulted in 63 deaths in nonpregnancy-associated cases, 22 fetal deaths, and 7 neonate deaths, for an overall case fatality rate of 33% [72a]. Health officials launched epidemiologic and laboratory-based investigations to identify risk factors for infection and potential control measures [72a, 73]. The first case–control study, conducted among 144 case-patients and 288 matched controls, did not identify risk factors for infection. However, a second case-control study among women with perinatal infection found that casepatients were more likely than controls to have consumed pork tongue in aspic (60% of casepatients vs. 6.1% of controls, OR = 9.2) and, to a lesser extent, other delicatessen items. A third study implicated a specific brand of pork tongue in aspic (OR = 14.8). In the contemporaneous laboratory-based based investigation, the clinical isolates were further characterized by PFGE and ribotyping along with a subset of >14,000 nonhuman isolates submitted to the NRC and isolates from samples collected in conjunction with this investigation [89]. A total of 247 (89%) human clinical isolates shared the same 3-enzyme PFGE profile, designated as the epidemic subtype sensu stricto. Notably, this subtype is closely related to that implicated in several U.S. and European outbreaks described in this chapter (California, 1985; Switzerland, 1983–1987; Denmark, 1989–1990). The sensu stricto subtype of L. monocytogenes was recovered from 154 food items collected during various stages of production and distribution, including abattoirs, processing facilities, retailers, and patient refrigerators. Among these food items were pork tongue in aspic from delicatessens (n = 112), other meat products (n = 19), cheeses (n = 12), and other food items with lower frequency. This subtype was further isolated from opened and unopened packages of the brand of pork tongue in aspic cited by patients, from environmental samples collected at the facility that produced that brand, and from retail stands. Thus, results of the laboratory-based investigation strongly supported the epidemiologic investigation findings. In another report describing the investigation of six processors suspected to have produced contaminated product, L. monocytogenes was isolated from 35% of 270 samples collected throughout the processing area [130]. Among these, 33% were from cooked product contact surfaces, correlating well with environmental investigation findings from other ready-to-eat meat and poultry products. The epidemicassociated phage type was isolated from raw brine in a facility producing pork tongue in aspic. In follow-up investigations in two plants after cleaning and disinfection, L. monocytogenes was isolated

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from 17% of raw product surfaces and 7% of finished product contact surfaces, most of which remained visibly soiled or covered by a biofilm; these results highlight the difficulty of eliminating this organism from the food processing environment (see Chapter 19 for a detailed discussion). Two invasive listeriosis outbreaks in France were linked to consumption of contaminated rillettes, a ready-to-eat pâté-like product produced by cooking ham meat in grease [52,74]. In July 1993, the National Reference Center notified Ministry of Health officials of a cluster of 10 listeriosis cases caused by an unusual phage type [73]. This serotype 4b subtype had accounted for <1% of cases from 1987 to 1992, and was later found to be closely related to that implicated in outbreaks linked to pâté (England, Wales, Northern Ireland) [108] and pasteurized milk (Massachusetts) [89]. Upon identification of eight additional cases in the following weeks, an investigation was launched to assess the magnitude of the outbreak, identify risk factors for infection, and implement control measures. As shown in Figure 10.11, 38 cases caused by the epidemic-associated phage type were identified from June to October 1993. This figure highlights the importance of routine subtyping in timely outbreak recognition, as the overall incidence of listeriosis did not increase significantly during the epidemic period. Most patients resided in western France. This outbreak was characterized by a high percentage of perinatal cases (82%), with seven cases occurring in nonpregnant adults. Twelve women gave birth prematurely. The overall case fatality rate was 32%, and included nine fetal deaths and one death in a neonate. Similar to observations in the 1998–1999 frankfurterassociated outbreak, the incubation period appeared to be shorter among three nonpregnant patients (11 to 19 days) than in six pregnant women (median 33 days). In the case control study, a standardized questionnaire addressing consumption history of >100 food items was administered to case-patients and controls matched by gestational age or age and underlying condition as appropriate. Within 6 days, preliminary analyses implicated rillettes

16

Sporadic cases Epidemic cases (Materno-neonatal)

14

Epidemic cases (Non-pregnant)

12

Cases

10

8

6

4

2

0 17 24 31

May

7

14 21 28

June

5

12 19 26

July

2

9

August

16 23 30

6

13 20 27

September

4

11 18 25

October

1

8

November

FIGURE 10.11 Listeriosis in France, 1993. (From Goulet, V., J. Rocourt, I. Rebiere, C. Jacquet, C. Moyse, P. Dehaumont, G. Salvat, and P. Veit 1998. Listeriosis outbreak associated with the consumption of rillettes in France in 1993. J Infect Dis 177: 155–160. With permission.)

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purchased at a specific retail chain as the most likely vehicle of transmission (OR = 18.0, CI = 2.2–208.0). The retail chain was supplied with a specific brand (designated “Brand A”) of meat products, including rillettes, produced at a single plant. Based upon these results and isolation of the epidemic phage type from a raw product sample from the implicated establishment, the producer recalled the product and ceased production. Further analysis of case–control study data resulted in the implication of additional Brand A meat products, including pâtés and sausages. Two Brand A products remained significantly associated with illness in a multivariate statistical model, and included rillettes (OR = 10.9, CI = 2.1–54.4) and country pâté (OR = 5.0, CI = 1.0 –24.1). Each of 35 case-patients interviewed reported exposure to Brand A products; 30 had consumed rillettes, 4 had consumed other meat products, and 1 had a family member who had consumed rillettes, suggestive of the potential for crosscontamination of other items during storage. Further interview of case-patients and controls who had consumed rillettes revealed that case-patients were more likely than controls to have stored the rillettes longer (> 6 days, 48% of case patients vs. 28% of controls, P = 0.12) and to have eaten them over more meals (> 4 meals, 48% of case patients vs. 23% of controls, P = 0.05). Results of the environmental investigation correlated well with the epidemiologic investigation findings. The epidemic-associated subtype was isolated from 15 of 508 containers of Brand A rillettes sampled prior to their sell-by dates, 20 containers removed from retail shelves, 11 containers returned to retailers in response to the recall, and 2 containers sampled from patients’ refrigerators and from several other Brand A meat products. Sell-by dates indicated contamination of at least 14 batches. The majority of unopened containers contained L. monocytogenes concentrations of <100 CFU/g. Two opened containers returned prior to their sell-by dates were contaminated at >10,000 CFU/g, demonstrative of the growth potential during home storage. An intensive environmental investigation at the processing facility revealed several potential sources of contamination. Records indicated isolation of coliforms from the rillettes production line. Production of rillettes had increased during that time, and time of disinfection had been shortened to the apparent detriment of product safety. Further, the epidemic-associated subtype of L. monocytogenes was isolated from smoked pork breast, the hood over the frankfurter processing line, and, two months after the outbreak ended, from one of the filling and packaging machines for rillettes. These results support both raw materials and the processing environment as potential sources of product contamination. As a result of these findings, raw materials and cooked product areas were better separated, a Hazard Analysis Critical Control Point (HACCP) plan was developed and implemented, bacteriological monitoring throughout the processing chain was reinforced, and the processor initiated postpackaging pasteurization of rillettes. Analysis of data from hospital surveys and two primary surveillance systems for invasive listeriosis in France indicated a dramatic estimated 68% decline in the incidence of listeriosis from 1987 to 1997 [71]. Data from a network of general and regional hospitals (EPIBAC) indicated a decline from 12.3 bacteremia and 3.4 meningitis cases per million population to 3.5 and 0.9 cases per million, respectively, whereas data from the National Reference Center showed a decline from 6.3 to 4.1 cases of listeriosis per million population. Contemporaneous declines among retail-level food product samples evidenced the contribution of aggressive prevention efforts implemented during that decade, including enhanced surveillance, intensified microbiological monitoring of food products throughout the production and distribution chains, improved hygiene at the retail level (particularly at delicatessen counters), and targeted health education messages for high-risk populations. Due to the severity of disease and high case-fatality rate, invasive listeriosis remained a public health priority. In 1998, health officials mandated notification of all laboratory-confirmed listeriosis cases, along with standardized collection of food exposure history (complete with purchase information). Detection and investigation of the two coincidental outbreaks described below were greatly facilitated by these initiatives, in addition to the molecular surveillance system already in place, which also enabled their differentiation.

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Between October 1999 and February 2000, health officials identified 10 cases of invasive listeriosis throughout France caused by the same serotype 4b subtype; clinical isolates were of the same phage type, and matched by two-enzyme PFGE analysis [52]. Six cases occurred in nonpregnant adults with underlying conditions known to be associated with increased risk of listeriosis, three cases were perinatal, and one occurred in a previously healthy adult. Two adults with underlying conditions and one infant died. A food history review for the first five recognized cases revealed that all patients had reported consumption of rillettes during the 2 months prior to illness, with four of five having purchased the products from the same market chain. In a review of nonclinical isolates, 21 matched the epidemic-associated subtype; 7 of those had been isolated from rillettes produced by the manufacturer that supplied the market named by the patients. In-plant records indicated isolation of L. monocytogenes from finished product stock during the outbreak period, and isolates from rillettes were later shown to match the epidemic-associated subtype. The manufacturer recalled its products (rillettes, pork tongue in aspic) in January, and the outbreak ended several weeks later. In response to this outbreak, French manufacturers reduced the shelf life for rillettes to 28 days to help reduce the potential growth of inadvertent contaminants to high levels during storage. A second, simultaneous outbreak occurred from November 1999 to February 2000 [52]. This outbreak involved a total of 32 cases and was caused by a subtype of serotype 4b with a different PFGE pattern than that associated with the first outbreak. Nine perinatal cases, 11 cases in nonpregnant adults with underlying conditions, and 12 cases in previously healthy nonpregnant adults (all male, median age 52.5 years, all with central nervous system listeriosis) were distributed throughout France. This outbreak had an overall case fatality rate of 31% and included death in five adults with underlying conditions, four premature neonates, and one fetus. A case–control study, in which patients infected with nonoutbreak-associated L. monocytogenes subtypes served as controls, found that case-patients were significantly more likely to have consumed pork tongue in aspic (OR = 28.0, 95% CI = 3.2–1,222.1), along with several ready-to-eat items including cervelas sausage, cooked ham, and pâté de campagne. Having consumed at least one meat product purchased from a delicatessen counter was also significantly associated with illness. On multivariate analysis, consumption of pork tongue in aspic (OR = 75.5, 95% CI 4.7–1,216.0) and pâté de campagne (OR = 8.9, 95% CI = 1.7–46.1) remained significantly associated with illness. It is likely that more than one vehicle of transmission was associated with this outbreak, as consumption of pork tongue in aspic explained 14 of 29 cases (48%) and all other case patients reported consumption of pâté and/or ham. Patients reported a variety of purchase sites. A review of distribution records allowed identification of two processors that had supplied the majority of purchase sites named by patients. Available records, extensive product sampling, and in-plant environmental investigations did not, however, enable investigators to implicate a single brand of product or processor. At the time of this outbreak, L. monocytogenes was permissible in ready-to-eat meat products manipulated between the microbial inactivation and packaging steps if studies were available to show that the concentration of L. monocytogenes would not exceed 100 CFU/g at the end of the product shelf life. In response to this outbreak, the food safety agency advised a production level zero-tolerance policy for these products. In 2000, investigators used PCR-based detection methods and automated ribotyping to quickly link a cluster of three cases of invasive listeriosis (serotype 1/2) in Western Australia, one of which resulted in fetal death, to contaminated cooked chicken products [86]. The efficient turn-around time (8 hours for ribotyping) resulted in data that guided generation of hypotheses for illness and allowed the investigators to make efficient recommendations for intervention. The products were recalled, and isolation of high levels of L. monocytogenes from unopened packages of cooked chicken pieces prompted implementation of a more rigorous cleaning and sanitation program and additional heat treatment at the end of production. Other isolated listeriosis cases linked to consumption of meat or poultry products have been reported and include (1) isolation of >106 CFU/g of L. monocytogenes serogroup 4 from a homemade sausage consumed by a man prior to

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development of listeriosis caused by the same serogroup [44]; (2) in the context of a study to identify risk factors for listeriosis, isolation of the same electrophoretic enzyme type from two patients and retail packages of pork sausage and ground beef [133]; and (3) isolation of the same phage type from cooked-chilled chicken and from fetal tissue obtained from a patient who had consumed the chicken prior to development of listeriosis [93].

REDUCTION

OF

MEAT

AND

POULTRY PRODUCT–ASSOCIATED INVASIVE LISTERIOSIS

Findings from the investigation of outbreaks associated with meat and poultry products strongly support the USDA–FSIS/FDA joint risk assessment, in which analysis of per serving and per annum relative risk rankings placed deli meats and frankfurters (not reheated) in the highest risk category (very high risk) and pâté and meat spreads in the second highest category (high risk) [16]. The categorization of unreheated frankfurters and deli meats reflects several factors, including (1) a relatively high rate of contamination, (2) rapid growth of L. monocytogenes under refrigerated storage temperatures, (3) likely storage for extended periods, and (4) frequent consumption by members of the U.S. population. Supportive data indicated a relatively low annual consumption rate for pâté and meat spreads (<1 × 109 annual servings) and a moderate level of contamination at retail; however, the high growth rate during home storage and likely long home storage time (>6 to 10-day range) contributed to the high risk ranking. Thus, these products warranted particular attention with respect to reducing the incidence of listeriosis. Outbreak investigation findings, in conjunction with risk assessment results, have led to an aggressive USDA–FSIS regulatory policy. Following the 2002 turkey deli meat-associated outbreak in the United States, USDA–FSIS issued a final rule mandating that establishments producing ready-to-eat meat and poultry products have in their HACCP plans or Sanitation Standard Operating Procedures (SSOPs) controls to prevent product contamination by L. monocytogenes from the processing environment [8]. These controls must be scientifically validated, and are stratified in accordance with the number of control steps taken (i.e., a post-lethality step treatment and/or a growth inhibitor). Controls could include a postpackaging thermal step or the addition of growth inhibiting compounds. Irradiation would also be an effective measure; however, FDA has not yet approved it for use with these products. The final rule also states that processors relying solely upon sanitation for prevention of contamination would be subject to intensified testing by USDA–FSIS. Establishments would also be mandated to share in-plant control measure verification data. Last, this final rule clarified that isolation of L. monocytogenes from processing equipment would provide sufficient justification for product recall, regardless of whether finished product yielded this organism or not. In addition to efforts to reduce the frequency and level of L. monocytogenes contamination, another critical prevention strategy for reduction of listeriosis associated with ready-to-eat meat and poultry products lies in effective consumer education programs.

SEAFOOD PRODUCTS Several small outbreaks of listeriosis have been linked to seafood items, including imitation crab meat, smoked mussels, gravad, and cold-smoked fish. Two 1996 cases in Canada were suspected to have been caused by consumption of imitation crab meat produced from the Alaskan Pollock fish [61]. A healthy adult female was hospitalized with severe gastrointestinal symptoms including explosive diarrhea and projectile vomiting. L. monocytogenes serotype 1/2b was isolated from her blood and stool. Her husband experienced similar but less severe symptoms; L. monocytogenes serotype 1/2b was isolated from his stool. Both reported consuming imitation crab meat during the 18 hours before onset of illness. Samples of imitation crab meat and four other food items obtained from the patients’ refrigerator yielded L. monocytogenes isolates that matched the patient isolates by serotyping, PFGE, and randomly amplified polymorphic DNA (RAPD) analysis. The imitation crab meat, along with two other items, was contaminated at a high level (2 × 109 CFU/g). Retail samples

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of imitation crab meat also yielded matching isolates, although at low levels, suggestive of growth during subsequent home storage. Based upon these findings, along with information indicating that two well meal companions had not consumed this item, investigators concluded that the imitation crab meat was the most likely vehicle of infection. Although one case was invasive, the predominance of gastrointestinal symptoms following consumption of a high inoculum correlates well with reports of noninvasive L. monocytogenes-associated gastroenteritis [120]. This outbreak also highlights the importance of consumer education regarding safe food storage, as food sampling results demonstrated the likelihood of cross-contamination of several other food items.

SMOKED MUSSELS Investigators in New Zealand linked three cases that occurred from October to December 1992 to consumption of contaminated smoked mussels [37]. Initially, two perinatal cases caused by serotype 1/2a were detected. A food exposure history indicating consumption of the same brand of mussels prior to development of listeriosis prompted investigators to analyze all 1991 and 1992 serotype 1/2a clinical isolates by PFGE. Two additional patient isolates, along with L. monocytogenes isolates from an unopened package of mussels obtained from a patient’s refrigerator, were indistinguishable by PFGE. One patient with illness in October 1992 reported consumption of mussels, but the fourth patient, with illness in May 1991, did not. Isolates from three patients reporting mussel consumption and the mussel isolates were indistinguishable by phage typing, sensitivity to arsenic and cadmium, and restriction fragment length polymorphism (RFLP). The fourth patient’s isolate was untypable via phage typing. This is an oft-encountered caveat of this subtyping method, and likely means that the isolate was of a type not represented in the phage set [105]. These laboratory findings highlight the higher discriminatory power offered by characterization with more than one typing method when feasible. Confirmation of “Brand X” mussels as the vehicle of transmission prompted officials to screen additional Brand X products, along with samples collected from the processing environment. Of 27 isolates from Brand X products, 26 matched the human clinical isolates, along with 4 of 7 from environmental samples taken from the processing facility. Notably, this subtype had been isolated from the processing environment 3 years earlier. These data are supportive of long-term colonization of the processing environment, as has been observed in other studies focused upon the smoked seafood industry [23,24,58,64,83,118,126], which can then serve as an ongoing source of contamination. Another small outbreak with four cases of L. monocytogenes serotype 1/2b associated gastroenteritis, which occurred in Tasmania in 1991, was also microbiologically confirmed to have been caused by mussels produced in New Zealand [112,113]. A high concentration of L. monocytogenes (1.6 × 107 CFU/g) was isolated from both open and unopened packages of mussels. Traceback investigations identified three batches for which the shelf life had been overestimated by at least 3 months, prompting a recall. The isolates were indistinguishable by RFLP and phage typing (J. McLauchlin, unpublished data) but did not match those isolated from Brand X products or the respective processing environment.

COLD-SMOKED FISH PRODUCTS Despite frequent isolation of L. monocytogenes in cold-smoked fish products [29,57,92,118], few listeriosis outbreaks have been attributed to these food items. In a 1995 report from Australia, smoked salmon was implicated as the vehicle of transmission for two cases of perinatal listeriosis which resulted in fetal death [20]. Another outbreak, associated with cold-smoked rainbow trout and gravad (made by curing a raw fillet with sugar, salt, pepper, and dill), occurred in the province of Värmland, Sweden from August 1994 to June 1995 [58,144]. Of nine cases due to serotype 4b, three were perinatal infections and six cases were in elderly or immune-compromised nonpregnant adults.

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Two patients, including one neonate, died. All patients reported consumption of gravad or smoked rainbow trout or salmon, five of whom recalled definitely or possibly consuming the same brand. Four subtypes were identified among patient isolates using restriction enzyme analysis (REA) with three enzymes and phage typing (designated clonal types A–D); six isolates were clonal type B, seen only once previously in Sweden. Investigators launched an environmental investigation, in which samples from the producer processing area along with gravad and smoked fish collected from patient refrigerators, retailers, and the producer were screened. Samples from the processing area and from fish yielded L. monocytogenes. Molecular subtyping results linked environmental and food isolates to eight of nine cases. Type B was recovered from samples of gravad fish from the home of one patient and from the producer, along with residue found in the packing machine, providing laboratory confirmation of the vehicle and identifying the likely environmental source of product contamination. Similar to several other investigations, the same subtype had been isolated from a product from this producer 6 months earlier, indicating that this subtype was likely among the resident environmental microflora. In addition, L. monocytogenes types C and D were isolated from product sampled at the processing plant, explaining two additional cases. One sample yielded two subtypes, highlighting the utility of characterizing more than one L. monocytogenes colony from a given sample during investigations. In the 2003 USDA–FSIS/FDA joint risk assessment, smoked fish was categorized as a high risk food on a per serving basis [16]. The high risk classification resulted largely from several characteristics: (1) a high frequency of contamination (12.9%), as indicated by data compiled from 30 studies evaluating prevalence; (2) a high estimated contamination level at retail (>0.6% of servings containing 103–106 CFU); (3) a moderate growth rate during storage; and (4) a typical storage duration of 3 to 5 days. Given the risk ranking, the infrequency of association of these products with listeriosis outbreaks is intriguing. Differences in pathogenic potential among L. monocytogenes subtypes common to these products do not provide an explanation, as several studies have reported the isolation of human epidemic- and sporadic-case–associated subtypes from smoked fish and smoked fish processing environments [23,29,118,144]. Potential contributors to this phenomenon could include the relatively low frequency of consumption by a large segment of the population or an inherent feature of the food matrix or production process that affects the organisms’ ability to cause disease once they are consumed. It is also possible that outbreaks have occurred but have gone undetected. Control of L. monocytogenes in the cold-smoked fish-processing environment and products presents a unique and significant challenge to the industry. This organism is ubiquitous in the environment, thus easily introduced into production facilities. Further, persistence of L. monocytogenes in smoked fish processing facilities, thereby establishing an environmental reservoir that is difficult to eliminate, has been well documented [23,24,58,64,83,118,126]. The production process, which typically involves brining via rubbing with salt, cure injection, or soaking in 3 to 5% aqueous phase NaCl followed by smoking at 25 to 30°C [144], does not include a step that would inactivate L. monocytogenes or prevent growth during refrigerated storage. As a result of these factors, sporadic contamination with low levels of viable L. monocytogenes may be difficult to control. Strategies to minimize L. monocytogenes contamination should include a continued focus on rigorous in-plant cleaning and sanitation programs, verified and modified in accordance with data obtained from routine environmental screening, and exploration of measures effective against established microflora. Options to inhibit growth of sporadic contaminants include exploration into process modification to employ potential inhibitory properties of lactic acid bacteria, salt and smoke, post-packaging pasteurization (i.e., irradiation), and frozen storage [5].

VEGETABLES Compared to other food categories, few invasive listeriosis outbreaks have been linked to consumption of contaminated vegetables or vegetable products. The large coleslaw-associated outbreak that occurred in the Canadian Maritime Provinces, discussed earlier in this chapter, was among the first

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to provide convincing evidence for transmission of L. monocytogenes by food. Although reported after this outbreak, an outbreak likely associated with raw vegetables occurred among hospitalized patients in Massachusetts a few years earlier in 1979 [82]. Twenty-three patients hospitalized during September and October had invasive listeriosis. Of these cases, 20 (87%) were caused by serotype 4b (identified as the epidemic serotype), compared to 33% of cases over the previous 26 months. Fifteen of these 20 patients acquired their infection while hospitalized. All cases occurred among nonpregnant adults, half of whom had underlying conditions contributing to immune suppression. Five patients died, although two deaths were attributed to other causes. Epidemiologic studies among patients and controls matched by age, gender, and hospitalization date did not result in identification of specific dietary risk factors. Case-patients were more likely than controls to have consumed tuna fish, chicken salad, and hard cheese; the common factor was the inclusion of these items in salads that also contained lettuce, celery, and/or tomatoes. This investigation also revealed intriguing clinical findings. Outbreak-associated case patients were more likely than patients with sporadic listeriosis to have had gastrointestinal symptoms at the time of fever onset (85% of outbreak-associated case-patients, compared to 22% of patients with sporadic listeriosis), a phenomenon similar to that observed in the 1987 outbreak that occurred in Philadelphia [137]. They were also more likely to have received antacids or histamine-blocking drugs prior to the onset of listeriosis (60% compared to 17%). These results were corroborated by case–control study findings; 60% of case-patients took antacids or histamine blockers before onset of listeriosis, compared to 25% of controls, and were also more likely to have undergone gastrointestinal procedures. Decreased stomach acidity may have resulted in improved survival of L. monocytogenes during passage through the stomach, as has been observed for other enteric pathogens [66]. In addition, underlying gastrointestinal lesions, for which these medications may have been used, may have resulted in an increased risk for invasive disease. A small outbreak of serotype 4b infections that occurred among patients subsequently admitted to a hospital on the Texas–Mexico border was epidemiologically associated with frozen vegetables [140]. There were five cases (four perinatal, one in an immunocompromised nonpregnant adult) detected over a 5-week period, with two additional cases identified over the following 2 months. A case–control study implicated frozen broccoli and cauliflower, and L. monocytogenes serotype 4b was later isolated from opened and unopened packages of these frozen vegetables. Human clinical and food isolates were indistinguishable by PFGE subtyping. In the recent USDA–FSIS/FDA risk assessment, vegetables were classified in the moderate risk category, due largely to the high number of annual servings and moderate frequency of contamination by L. monocytogenes (about 2 to 5% of samples). However, vegetables remain infrequently associated with outbreaks of listeriosis. Potential differences in pathogenic potential among different subtypes of L. monocytogenes do not offer a conclusive explanation for this phenomenon, as both outbreak and sporadic-case–associated subtypes have been isolated from vegetables (see Chapter 16 for a detailed discussion). The overall low growth rate during storage [16], along with the relatively short shelf life of unpreserved vegetable products may contribute to the infrequent association with invasive disease, compared to other food categories. Continued emphasis on processing facility hygiene, along with careful handling of raw vegetables and ready-to-eat vegetable products by individuals in high-risk populations, will help to further reduce the risk of listeriosis from consumption of vegetables.

L. MONOCYTOGENES–ASSOCIATED FEBRILE GASTROENTERITIS A second, increasingly well-described disease state resulting from infection by L. monocytogenes is febrile gastroenteritis (for a recent review with a focus on clinical practice, see Ooi and Lorber [120]). As shown in Table 10.2, at least eight outbreaks of foodborne gastroenteritis have been attributed to this organism. These outbreaks have largely been recognized as point-source outbreaks among otherwise healthy members of a cohort and are characterized by consumption of a high-level

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TABLE 10.2 Listeria monocytogenes-Associated Febrile Gastroenteritis Outbreaks Year

Location

Serotype

No. Cases

Implicated Vehicle

Reference

1993 1994 1997 1998 2000 2001 2001 2001

Northern Italy Illinois Northern Italy Finland New Zealand Los Angeles Sweden Japan

1/2b 1/2b 4b 1/2a 1/2 1/2a 1/2a 1/2b

18 45 1,566 5 31 16 48 38

Rice salad Chocolate milk Cold corn and tuna salad Cold-smoked rainbow trout Ready-to-eat meat products Turkey delicatessen meat Fresh, raw-milk cheese Cheese

[129] [50] [22] [110a] [139] [65] [46,51] [101]

inoculum followed by onset of fever, gastrointestinal symptoms including watery diarrhea and nausea, headaches, and joint and muscle pain within 24 hours. Symptoms typically resolve within a few days. Here, we discuss several well-characterized outbreaks.

POTENTIAL ASSOCIATION WITH A MILD CLINICAL SYNDROME: SHRIMP, 1989, UNITED STATES The suggestion of milder illness in otherwise healthy adults was reported by Riedo et al. following an investigation that grew from follow-up of two perinatal cases of L. monocytogenes bacteremia [125]. After learning that the patients’ only common exposure was attendance at a party, public health officials launched a retrospective cohort investigation among 36 attendees to evaluate the potential for mild illness and determine risk factors for illness. In this type of study, food and/or other exposures are collected from all or a representative sample of persons in a well-defined population among whom disease occurred (e.g., attendees of an event after which illness occurred among some attendees), allowing the calculation of attack rates of illness in those who did or did not report a certain exposure [53]. The attack rates can then be compared to determine the food item or other exposure associated with the greatest increase in risk. For this investigation, based upon the body of knowledge regarding foodborne listeriosis at that time, a case of illness was defined as isolation of L. monocytogenes from blood or stool or illness with any two concurrent gastrointestinal or musculoskeletal symptoms in a party attendee within 6 weeks after the party. Illness in 10 patients met the case definition, 9 met the symptom criteria, and L. monocytogenes was isolated from 1 of 25 stool samples collected from attendees. Symptom onset and/or isolation of L. monocytogenes from a clinical specimen occurred throughout the 6 weeks after the event. The 3 clinical isolates (2 from the blood of the women with perinatal infection and the stool isolate) were serotype 4b and matched by multilocus enzyme electrophoresis. Among 24 food items served at the party, persons who consumed greater amounts of shrimp (boiled and chilled, served with a sauce) and, to a lesser extent, nonalcoholic beverages, Camembert cheese, and cauliflower were at significantly increased risk for illness (relative risk [RR] for shrimp consumption 7.0, 95% CI = 2.3–21.5). After controlling for consumption of other foods, only consumption of shrimp and cauliflower remained significantly associated with illness. Only three ill persons, however, recalled consumption of cauliflower. No leftover foods were available for screening, and foods purchased from suppliers approximately 6 weeks after the party did not yield L. monocytogenes. Although a matching L. monocytogenes subtype was isolated from a person with noninvasive illness, the longer incubation period (19 to 23 days for index cases) is not representative of other L. monocytogenes-associated outbreaks of foodborne gastroenteritis. It is possible that another organism was responsible for the mild gastrointestinal illness observed in the patients who subsequently

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developed invasive listeriosis and that the syndrome observed in others was not associated with food consumed at the party. However, this report highlighted the potential for a milder illness caused by L. monocytogenes in otherwise healthy adults and raised awareness of this hypothesis for consideration in subsequent investigations.

RICE SALAD, 1993, NORTHERN ITALY In 1993, an outbreak of febrile gastroenteritis occurred among 39 attendees of a supper [129]. Four attendees were hospitalized during the 24 hours following the event with high fever (average 39.6 °C), diarrhea, nausea, abdominal pain, arthromyalgia, headache, and sore throat. Several days later, blood cultures yielded L. monocytogenes serotype 1/2b, whereas stool cultures were negative for enteric pathogens including Salmonella and Shigella. Routine public health follow-up revealed that the outbreak occurred among nonpregnant, otherwise healthy persons. Based upon these unusual findings, health officials launched epidemiologic and laboratory investigations to better characterize the outbreak. Investigators defined a case as onset of fever plus diarrhea, nausea, vomiting, and/or arthromyalgia within 3 days of attending the supper. Among the 39 attendees, 18 met the case definition, for an attack rate of 46%. Ill persons ranged in age from 17 to 54 years; their median age was significantly higher than that of well attendees (36 years compared to 22 years, P < 0.001). Their clinical syndrome largely mirrored that of hospitalized attendees, with the exception of a flu-like syndrome (arthromyalgia, sore throat, headache) in four persons in the absence of gastrointestinal symptoms. Among patients with gastrointestinal symptoms, onset occurred a median of 18 hours following the supper. None of the stool specimens collected from all attendees approximately 1 month after the event yielded L. monocytogenes. Among convalescent serum tested from 18 ill attendees, however, 39% showed αsomatic antigen type 1, compared to 1 of 4 well persons. None had α-type 4b antibodies, and none of 11 community members who served as a comparison group had detectable antibody titers against either. Consumption of rice salad was significantly associated with illness. Eighteen of 20 persons (90%) who consumed rice salad developed illness, compared to none who did not consume that item (RR undefined, P < 0.001). A food preparation review revealed that, due to limited refrigeration space, the rice salad had been held at ambient temperature during the 24 hours before the supper. Samples of rice salad and other available food items did not yield pathogens including pathogenic Escherichia coli, Salmonella, Bacillus cereus, or Staphylococcus aureus. Although there was no rice salad left for further screening, samples of several other food items, along with environmental samples collected from the blender and freezer in the home of the cook, yielded L. monocytogenes. The clinical, food, and freezer isolates all matched by MEE and phage typing. Although it is possible that the gastrointestinal syndrome characterizing this outbreak may have been caused by an organism not detected in clinical specimens, which were collected approximately 1 month after the event, several factors support L. monocytogenes as the etiologic agent: (1) isolation of the same subtype from the blood of two patients, food, and environmental samples; (2) several ill persons with high titers of α-Listeria antibodies, compared to a low percentage among well attendees and nondetectable titers in a comparison population; and (3) a median incubation period longer than that typically observed for toxin-mediated illness, yet shorter than that typical for viral etiologies. This outbreak highlights the importance of proper food handling and storage in noncommercial settings, as L. monocytogenes was isolated at >103 CFU/g from two food items. Further, investigation findings are supportive of considering L. monocytogenes as a potential etiology in outbreaks of febrile gastroenteritis.

CHOCOLATE MILK, 1994, UNITED STATES The investigation of a 1994 outbreak linked to contaminated chocolate milk provided overwhelming evidence for the association of L. monocytogenes with febrile gastroenteritis [50]. Again recognized

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as a point-source outbreak, illness occurred among approximately 92 persons who attended a picnic at a Holstein cow show in Illinois. Of 82 persons interviewed, 45 (55%) met the case definition of onset of symptoms from two to four symptom complexes including (1) fever; (2) diarrhea, nausea, or vomiting; (3) myalgia or arthralgia; and (4) headache within a week of attending the picnic or consuming food from it. As observed in the previous outbreak, the median age of ill persons was higher than that of well persons (31 years vs. 24 years). None of the attendees reported underlying conditions; one pregnant woman at 40 weeks gestation had diarrhea the day after the picnic and delivered a healthy baby several days later. Common symptoms among ill persons included diarrhea, fatigue, fever, chills, headache, myalgia, and abdominal cramps. The median incubation period was 20 hours (range 9 to 32 hours), and diarrhea lasted a median of 42 hours (range 3 to 50 hours). Four patients were hospitalized as a result of their illness, and others reported loss of work time due to the severity of their symptoms. L. monocytogenes was isolated from 11 stool specimens. All positive specimens were from persons with illness meeting the case definition. Analysis of convalescent serum collected from 48 attendees and a control group with nonoutbreak-associated enteric infections showed a significantly higher median α-Listeriolysin O antibody titer (143 ELISA units) in persons with illness meeting the case definition than in controls (63 ELISA units, P < 0.001). Lower titers were also reported among attendees who were not ill and persons with mild illness. The strong association of higher α-Listeriolysin O antibody titers with illness highlights the diagnostic utility of this serologic assay. Among 60 persons who consumed chocolate milk, 45 (75%) became ill, compared to none of 22 persons who did not consume this item (RR undefined, 95% CI = 13.6 – ∞). Consumption of Swiss cheese was also significantly associated with illness; however, fewer attendees had consumed the cheese, and all ill persons who consumed cheese had also consumed chocolate milk. Culture of a carton of milk leftover from the picnic and a carton collected from the dairy yielded L. monocytogenes at 1.2 × 109 and 8.8 × 108 CFU/mL, respectively. An 8-ounce carton of milk would, therefore, potentially provide an inoculum of 2.9 × 1011 CFU. These and all but one clinical isolate were indistinguishable by MEE, ribotyping, and PFGE analysis. The PFGE pattern of one clinical isolate differed by one band. Inspection of the dairy and review of handling during transport to the picnic provided a clear explanation for the presence of high levels of L. monocytogenes in the chocolate milk. After flavor addition and milk pasteurization, the chocolate milk was held in a jacketed tank for 2 hours before being pumped to a filler line over a 7-hour period. The refrigerated jacket had been in disrepair, thus unrefrigerated, for 3 years. Inspection revealed a breach in the tank lining, which allowed milk to leak into the insulation jacket and pool there. When milk was pumped out, the pooled milk could leak back into the tank. Sanitizer spray nozzles were clogged, thus inhibiting the flow of sanitizer into the tank lining. A total of 180 cartons of chocolate milk for the picnic were in transport for over 2 hours without refrigeration. Upon delivery, they were held in a noncommercial refrigerator overnight. They were placed in an unrefrigerated cooler on the morning of the picnic and were available throughout the day. Based upon these data, it is likely that postpasteurization contamination of the milk occurred due to poor facility hygiene practices, with subsequent temperature abuse allowing rapid growth of L. monocytogenes. Records further indicated that chocolate milk had been distributed to three additional states. Consumption of chocolate milk from the implicated dairy was associated with similar illness in a family of five traveling through a neighboring state. A review of clinical isolates submitted to health officials in Illinois, Michigan, and Wisconsin resulted in identification of three additional cases of invasive listeriosis with some association with chocolate milk from the implicated dairy. Two patients had consumed chocolate milk from the implicated dairy, and the third had consumed chocolate milk purchased at a store that sold milk from the implicated dairy. Further, each clinical isolate was indistinguishable from the outbreak-associated subtype. Recommendations resulting from this investigation included diagnostic screening for L. monocytogenes in outbreaks with similar clinical features, enhanced monitoring of food products for pathogens including L. monocytogenes, and rigorous cleaning and sanitation programs.

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CORN

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AND

TUNA SALAD, 1997, NORTHERN ITALY

A massive outbreak of L. monocytogenes-associated febrile gastroenteritis occurred in northern Italy in 1997 [22]. On a single day, local health units in three different towns received unusually high reports of febrile illness and gastroenteritis among students and staff of two primary schools and students of a university. All had dined at cafeterias supplied by the same local caterer, which prepared approximately 8,000 meals daily. Health officials immediately launched epidemiologic and laboratory-based investigations to investigate the scope, magnitude, and potential source of illness. Investigators determined that a cohort of 2,930 persons had consumed cafeteria meals on the day before the illnesses began. Of 2,189 primary school students and teachers enrolled in a cohort study, 93 adults and 1,473 students reported having at least one flu-like or gastrointestinal symptom, for an overall attack rate of 72%. Headache (88% in adults, 86% in children), abdominal pain (72% in both groups), and fever (68% in adults, 86% in children) were most commonly reported, and the median incubation period was 24 hours. Fever and vomiting occurred with significantly more frequency in children, whereas diarrhea, arthralgia, and myalgia were significantly more frequent in adults. A total of 292 persons (19%) were hospitalized as a result of their illness for a median of 3 days, underscoring the severity of associated symptoms. Of 292 stool specimens screened by 9 hospital laboratories, 290 were negative for bacterial and viral enteric pathogens and two yielded different Salmonella serotypes. L. monocytogenes was isolated from the blood of one patient. Among 141 stool specimens subsequently screened for L. monocytogenes, 123 (87%) were positive. All isolates were serotype 4b, and were indistinguishable by PFGE and RAPD analysis. Persons who consumed a salad made from corn and tuna were over six times more likely to become ill than those who did not (84% vs. 14%, RR = 6.2, 95% CI = 4.8–8.0, P < 0.001). No other food items were significantly associated with illness. Investigators conducted an environmental investigation at the catering facility and tested food samples obtained there. Although unopened cans of corn and tuna did not yield microorganisms, a sample of the leftover corn and tuna salad yielded L. monocytogenes at >106 CFU/g. These, along with L. monocytogenes isolates from facility sink drains and a meal preparation surface, were indistinguishable from the clinical isolates by PFGE and RAPD analysis. In a food preparation review, caterers reported opening the cans of tuna and corn during early morning on the day of preparation and allowing them to drain at ambient temperature. They were then mixed (no additional ingredients were added), portioned, and transported to the schools for lunch service. A corn salad without tuna was prepared later that day for dinner service at the university that had reported gastrointestinal illness among students. Studies with inoculated corn showed the potential for growth of L. monocytogenes to high concentrations (>106 CFU/g) during storage at 25°C. Based upon the environmental and laboratory-based investigation results, salad ingredients or the prepared salad were likely cross-contaminated with L. monocytogenes at the catering facility. Storage at ambient temperatures for several hours prior to serving could then allow the contaminants to grow to high levels. This outbreak highlights the importance of rigorous cleaning and sanitation programs in food production areas and proper food handling practices in reducing illness caused by L. monocytogenes. The high hospitalization rate underscores the potential severity of febrile gastroenteritis.

COLD-SMOKED RAINBOW TROUT, FINLAND A small outbreak in Finland was linked to consumption of cold-smoked rainbow trout [110a]. Four adults and one child developed a gastrointestinal illness with nausea, abdominal cramps, and diarrhea within 27 hours of sharing a meal together. Fever was reported by three adults, and other symptoms included vomiting, headache, fatigue, and arthralgia. The child was hospitalized overnight as a result of the illness. Information from patient interviews identified cold-smoked rainbow

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trout as the most likely vehicle of infection. No leftover fish was available; however an unopened package from the same lot obtained from the retailer yielded 1.9 × 105 L. monocytogenes CFU/g. Retail inspection findings indicated that the storage temperature of the cold-smoked trout was higher than that recommended by the producer. Stool samples collected from the patients did not yield enteric pathogens. However, subsequent stool swabs obtained from two ill persons yielded L. monocytogenes. Clinical and smoked trout isolates were both serotype 1/2a, and were indistinguishable by two-enzyme PFGE analysis.

READY-TO-EAT MEAT AND POULTRY PRODUCTS, NEW ZEALAND AND UNITED STATES In 2000, investigators in New Zealand linked several independent foodborne illness reports to consumption of contaminated ready-to-eat meats produced by the same manufacturer [139]. The first two reports of febrile gastroenteritis on South Island were associated with corned beef. Two additional reports of a similar syndrome indicated consumption of products from the same manufacturer. Leftover corned beef samples yielded L. monocytogenes at concentrations of >2.5 × 105 CFU/g, prompting the manufacturer to issue a product recall. On that date and shortly afterward, two foodborne outbreaks were reported among 7 residents of South Island and among 21 of a cohort of 24 residents of North Island; both meals had included ready-to-eat products produced by the same manufacturer. In the latter case, the retailer had failed to remove the product from sale despite the product recall. The two reported outbreaks were characterized by different clinical syndromes; flu-like symptoms including fever, myalgia, and headache predominated in South Island cases, whereas the majority of persons associated with the North Island outbreak also experienced gastrointestinal symptoms including diarrhea, vomiting, and abdominal cramps. Most experienced symptom onset within 24 hours of meal consumption. L. monocytogenes serotype 1/2 was isolated from stool specimens collected from 2 of the 4 ill persons from the initial reports, 2 ill persons associated with the South Island outbreak, and from 20 of 21 specimens collected from 17 ill and 3 well persons associated with the North Island outbreak. Several of the manufacturer’s food items were available for screening. L. monocytogenes serotype 1/2 was isolated from leftover corned beef (as noted above) and ham (North Island outbreak, 1.8 × 107 CFU/g), luncheon ham (<100 CFU/g) obtained from retailers cited by patients, and unopened retail packages of bulk corned beef and roast pork (102 to 104 CFU/g from each of six samples). All clinical and food isolates were indistinguishable by two-enzyme PFGE analysis. A review of the manufacturing process revealed two potential sources for contamination: (1) crosscontamination of cooked product prior to repackaging for retail and (2) raw meat, due to the identified potential for an inadequate heat treatment. Investigators further found that the estimated shelf life for corned beef (90 days) was erroneous, and it was subsequently reduced to 6 weeks. Overall, the results of this investigation support the relatedness of these reports of febrile gastroenteritis and the likelihood of a specific subtype of L. monocytogenes serotype 1/2 as the etiology. Although many outbreaks of invasive listeriosis have been linked to contaminated readyto-eat meat products, this report provides evidence for an association with febrile gastroenteritis. This underscores the importance of efforts to reduce the frequency of L. monocytogenes contamination in these products, along with proper food handling practices at the retail and consumer levels. In 2001, an outbreak of febrile gastroenteritis occurred among approximately 60 persons who attended a birthday party in Los Angeles [65]. Food served at the party had come from a variety of sources, including a delicatessen, a bakery, a local farm, and a grocery store. Among 44 attendees interviewed, 16 (36%) had experienced at least one systemic symptom (fever, headache, body ache) and one gastrointestinal symptom (diarrhea, cramps, vomiting, nausea), thus meeting presumptive case criteria. Ill persons were a median of 15.5 years of age (range 7 to 66 years), and half were female. Similar to other outbreaks described here, predominant symptoms included fever, headache, diarrhea, and vomiting. Symptom onset occurred a median of 25 hours after the party.

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Stool specimens collected initially from 6 attendees were negative for enteric pathogens; however, L. monocytogenes serotype 1/2a was isolated from 6 of 8 additional specimens requested from ill persons with presumptive cases. The isolates were indistinguishable by PFGE analysis. Consuming a portion of 1 of 3 kinds of sandwiches served—a 6-foot-long half-turkey, halfvegetable sandwich with pepper-jack cheese—was significantly associated with illness (RR = 5.6, P < 0.002). In analyses addressing specific ingredients, consumption of turkey (RR undefined, P < 0.0001) and pepper-jack cheese (RR = 6.3, P = 0.0002) were significantly associated with illness. Consumption of pepper-jack cheese explained fewer cases, however, and only turkey remained significantly associated with illness when controlling for consumption of each food item. Leftover, home-refrigerated portions of the sandwich yielded the outbreak-associated subtype; a sample of turkey from the sandwich, obtained 14 days after the party, contained Listeria spp. at 1.6 × 109 CFU/g. Inspection of the delicatessen revealed multiple retail food code violations, including inadequate sanitation procedures and inadequate cold-holding temperatures in a walk-in refrigerator. Although traceback efforts allowed identification of a processor, it was determined that an in-plant investigation was unfeasible due to the lack of a specific lot number and the apparent limited nature of the outbreak. Nonetheless, based upon investigation findings, the likely source of this outbreak was contaminated turkey delicatessen meat.

FRESH, RAW-MILK CHEESE, SWEDEN During 2001, health officials in Sweden were alerted to an increase in gastrointestinal illness among residents of one county who had visited a summer farm prior to their illness [46,51]. Patient interviews rapidly identified consumption of on-farm–produced dairy products, including fresh, raw-milk cheeses and butter, as the likely source of illness. Health officials initiated active casefinding and launched an investigation to determine the etiology, specific source, and evidence for a dose–response effect between the amount of each food item consumed and development of illness. Among a composite cohort consisting of 42 persons in 2 groups who had visited the farm and 50 additional community members identified by active case-finding efforts, 48 persons had illness meeting the case definition. All had consumed a dairy product from the farm during the study period and had developed 2 or more symptoms consistent with febrile gastroenteritis (fever, diarrhea, vomiting, arthralgia, headache, or body pain) within the 2 weeks following consumption. Approximately half of all persons were female, and their median age was 52 years (range 2 to 85 years). Predominant symptoms included diarrhea (88%), fever (60%), and stomach cramps (54%), and onset occurred a median of 31 hours following product consumption. Ill persons reported a symptom duration of 1 to 15 days. One person who had been on immunosuppressive therapy was hospitalized upon development of a joint abscess due to L. monocytogenes. Stool samples collected from ill persons were negative for bacterial and viral pathogens, with the exception of the detection of genetic markers for verotoxigenic E. coli (VTEC) in 6 of 35 specimens and enterotoxigenic E. coli (ETEC) in 1 specimen. L. monocytogenes was isolated from 27 (84%) of 32 stool specimens collected from persons whose illness met the case definition, compared to 9 (82%) of 11 specimens from persons whose illness did not, indicating common exposure but varying clinical symptoms. Results of the epidemiologic study indicated that consumption of raw-milk cheeses produced from cow’s milk (RR = 2.23, 95% CI = 1.49–3.34) or cheese of an unknown type (RR = 2.23, 95% CI = 1.49–3.34) was significantly associated with illness. Although not statistically significant, the attack rate increased as consumption of dairy products increased, suggesting a potential dose–response effect. Samples of goat’s milk cheese, cow’s milk cheese, whey cheese, and butter yielded L. monocytogenes serotype 1/2a at concentrations of 101 to 107 CFU/g. However, it is difficult to estimate the dose consumed as some samples had been in home-storage for 1 to 2 weeks before collection. Further isolation of high concentrations of coagulase-positive Staphylococcus aureus and presumptive E. coli was indicative of compromised product quality. Notably, all clinical isolates from ill persons, isolates from persons who did not have illness meeting the case definition,

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and food L. monocytogenes isolates matched by restriction enzyme analysis (REA) and by PFGE. The potential contribution of pathogenic E. coli these illnesses is uncertain. Further investigation at the farm revealed significant food hygiene issues and a lack of process controls. L. monocytogenes was isolated from environmental samples including a wooden bench in the production area and brine used for cheese storage prior to sale [51]. Stool specimens from 5 of 19 lactating cows, along with milk samples from 10 of 14 goats and 1 fecal sample from a goat yielded L. monocytogenes. Samples of milk from one goat collected twice over the following month remained positive, leading to a diagnosis of subclinical mastitis. Isolates from the environmental specimens, milk from the mastitic goat, and a subset of additional fecal and milk specimens (number unspecified) matched the human clinical and food isolates by PFGE analysis. This outbreak of febrile gastroenteritis was likely caused by consumption of on-farm–produced dairy products contaminated with L. monocytogenes. The data support the milk of a goat with subclinical mastitis as a source, although isolation of the outbreak-associated subtype from the production environment demonstrated the likelihood of cross-contamination of other products. The data also supported carriage of the outbreak-associated subtype by other animals whose milk was used for production. The summer farm was subsequently closed to visitors. This outbreak contributes to the enormous body of evidence regarding the risks associated with consumption of raw milk and raw milk products, and emphasizes the importance of enhanced food safety programs for summer farms and other similar venues such that visitors can continue to enjoy these historical cultural traditions.

CHEESE, JAPAN Investigators recently reported the first identified outbreak caused by L. monocytogenes in Japan [101]. This gastroenteritis outbreak was identified and investigated after routine sampling resulted in isolation of high levels of L. monocytogenes serotype 1/2b from several soft and semi-hard cheeses produced at a single facility. Further investigation revealed extensive contamination of the processing environment. The same subtype, along with serotypes 4b and 1/2a, was also isolated from samples collected throughout the facility. Following isolation of L. monocytogenes from the cheese, investigators identified persons who had consumed it and collected fecal specimens for screening, along with health information. L. monocytogenes was isolated from 60% of fecal specimens, a proportion much higher than expected for fecal carriage [78]. Among persons from whom L. monocytogenes was isolated, 38 reported a gastrointestinal or “cold-like” illness within several days of cheese consumption, and 38% sought medical care. A subset of human clinical and cheese isolates was further characterized by PFGE, ribotyping, mouse bioassay, cellular virulence assays, and PCR-based subtyping. Human and cheese isolates showed >85% similarity by PFGE, and were genetically similar according to other characterization methods.

CONTROL

OF

L.

MONOCYTOGENES–ASSOCIATED

FEBRILE GASTROENTERITIS

Strategies outlined for the continued reduction of invasive listeriosis would serve this goal well. As described in the examples above, the sources and route of contamination often parallel scenarios observed for invasive listeriosis outbreaks, with growth to high levels facilitated by subsequent temperature abuse or long periods of storage. In addition to efforts to reduce the frequency and level of L. monocytogenes contamination at the farm, producer, distribution, and retail levels, prevention strategies should include educational programs focused on proper handling and storage of products during preparation and prior to consumption. Such messages should target the general population in addition to persons at higher risk for invasive listeriosis. L. monocytogenes-associated gastroenteritis has been well characterized only recently. Outbreaks are likely underrecognized because of a lack of awareness of this syndrome and because stool samples are infrequently screened for L. monocytogenes. Healthcare workers and public health

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officials should consider L. monocytogenes as a cause of febrile gastroenteritis, particularly in cases where routine screening fails to identify an enteric pathogen. The factors leading to febrile gastroenteritis as opposed to invasive disease are currently poorly understood. As with invasive listeriosis, a better understanding of factors resulting in development of this illness along with the disease process may contribute to improved control and prevention strategies.

SURVEILLANCE OF LISTERIOSIS IN THE UNITED STATES FOODBORNE OUTBREAK SURVEILLANCE In the United States, the CDC has collected reports of foodborne outbreaks since 1973. These reports comprise summary information concerning outbreaks including the number of illnesses, hospitalizations, and deaths, the etiologic agent for illnesses, implicated food vehicles, and other outbreak-associated factors. Officials in all states currently report foodborne outbreaks in this surveillance system, now called the electronic Foodborne Outbreak Reporting System (eFORS). This system was recently enhanced by efforts to ensure complete and accurate reports, including verification of the number and content of reports with state officials and the implementation of Internet-based data entry into a national database. The eFORS system receives between 1,200 and 1,500 outbreak reports annually. Among 6,647 foodborne outbreaks reported to eFORS from 1998 to 2002, 2,168 had an identified etiology; 11 were due to L. monocytogenes, including a total of 256 cases and 38 (14.8%) deaths (CDC unpublished data). These deaths accounted for 48% of all deaths among outbreakassociated cases reported. The number of cases in listeriosis outbreaks ranged from 2 to 101. Four outbreaks occurred in multiple states. Four outbreak reports indicated delicatessen meats as the implicated vehicle (two sliced turkey, one multiple delicatessen meats, and one unspecified delicatessen meat), three implicated hot dogs, one implicated deli sandwiches, one implicated pâté, one implicated queso fresco (cheese), and one implicated potato salad. Foodborne outbreak surveillance provides an opportunity to construct statistical models to assess the burden of illnesses due specific food categories (i.e., poultry). These models incorporate data from case surveillance to estimate the total number of illnesses associated with various foods, as outbreak-related cases comprise only a fraction of the total burden. Investigators in the United Kingdom have published one such model [1]. From 1996 to 2000, an estimated 221 cases and 78 deaths were attributed to listeriosis. Though the authors provided estimates of the total estimated number of illness attributed to food categories due to all etiologies, they did not provide estimates due to listeriosis specifically. Several groups in the United States and other countries are conducting similar analyses; these efforts will undoubtedly enhance our knowledge of the burden of listeriosis due to specific food categories.

SURVEILLANCE

OF

SPORADIC LISTERIOSIS

Although we have learned much about the epidemiology of listeriosis through outbreak investigation, timely, effective surveillance of listeriosis is critical to the overall goals of control and prevention of this disease. Reliable surveillance data are critical in estimating the burden of listeriosis, determining disease trends, guiding development of targeted intervention strategies, providing a framework for their assessment, and developing risk assessment models used to guide regulatory actions. Furthermore, determining the expected incidence of sporadic listeriosis in a population through surveillance is critical for efficient outbreak detection and response, as both local and diffuse outbreaks are often detected by an increase of cases over the baseline rate. Prior to the mid-1980s, when the Los Angeles County outbreak solidified the public health significance of foodborne listeriosis, the incidence of sporadic human disease in the United States was poorly understood. Listeriosis was monitored exclusively by passive surveillance, which relies

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upon voluntary reporting by physicians and clinical laboratories to state health officials, who then voluntarily report to CDC [114], and by analysis of hospital discharge data [47]. The low sensitivity of these methods likely greatly underestimated its true incidence in the population. The 1985 outbreak underscored the importance of improved surveillance for listeriosis in the United States. An active surveillance program, for which public health officials routinely contacted personnel at all clinical laboratories and acute-care hospitals in five states and Los Angeles County to ascertain listeriosis cases, was initiated in 1986 in an effort to more precisely estimate the incidence of laboratory-confirmed disease [68,133]. The resulting estimates are detailed in Chapter 4. In an effort to improve nationwide reporting in the United States, listeriosis was made a nationally notifiable disease (one for which routine and timely reporting is considered necessary for prevention and control measures) in 2000. Although disease reporting is mandated only at the state level, and laws and regulations regarding which diseases and conditions must be reported to public health officials vary slightly from state to state, most currently require reporting of listeriosis. CDC provides uniform case criteria to increase specificity and data comparability [7], and reports the data weekly in its Morbidity and Mortality Weekly Report. Comprehensive annual summaries of nationally notifiable diseases, including listeriosis, present data stratified by important risk markers including geographic distribution, gender, age group, and race/ethnicity. The incidence and trends in listeriosis, as estimated by this surveillance program, along with international listeriosis estimates, are detailed in Chapter 4. Last, as discussed earlier in this chapter and in Chapter 9, participating PulseNet laboratories routinely subtype L. monocytogenes isolates by PFGE to facilitate rapid detection of clusters of listeriosis that may have a common source. In an effort to address emerging infectious diseases in the United States, CDC launched the Emerging Infections Program (EIP) in collaboration with selected state health departments, local health departments, academic institutions, and additional partner agencies in 1994. The Foodborne Diseases Active Surveillance Network (FoodNet), a collaboration among CDC, the USDA, FDA, and the EIP sites, is the principle foodborne disease component of the EIP [111]. FoodNet’s primary goals include (1) determining more precisely the burden of foodborne diseases in the United States, (2) monitoring trends in foodborne diseases, (3) determining the proportion of foodborne disease attributable to specific foods, and (4) developing and assessing interventions to reduce the burden of foodborne illness. In 1996, FoodNet began active, laboratory-based surveillance of selected foodborne diseases including listeriosis. As described for the active surveillance program initiated in 1986, which was continued and expanded as a component of the EIP, public health officials in FoodNet sites contact laboratory directors frequently to ascertain new cases of laboratory-confirmed foodborne disease. As a result, the burden of specific foodborne diseases in the United States can be more precisely estimated over time. Surveillance began in 5 geographically diverse sites with a catchment population of 14.3 million people. By 2003, FoodNet had expanded considerably to include 10 sites, more than 650 clinical laboratories, and a catchment population of 41.9 million people (14.4% of the U.S. population) [15]. By 2004, the catchment population had expanded to 44.5 million (15.1% of the U.S. population) (Figure 10.12). The FoodNet population under surveillance is comparable to the U.S. population, with few limitations [80]. A demographic comparison of the FoodNet population under surveillance and the U.S. population, conducted in 2000, found little variation in age and gender distributions. However, among race/ ethnic groups, the Hispanic population in FoodNet was underrepresented at 6% compared to 12% of the U.S. population as indicated by census data. Figure 10.13, in which relative rates of listeriosis are shown in comparison with a 1996–1998 baseline estimate, summarizes trends in the incidence of listeriosis since the initiation of FoodNet surveillance. The use of an average annual incidence for the 3 years (1996 to 1998) as the baseline period provides the most stable, precise relative rate estimates [15]. To account for the increase in the number of FoodNet sites, the corresponding increase in the population under surveillance and variations in the incidence of listeriosis among sites, a main effects, log-linear Poisson regression (negative binomial) model was used to estimate statistically significant changes in incidence. As shown in

346

FIGURE 10.12 Map showing Emerging Infections Program sites (
) participating in the Foodborne Diseases Active Surveillance Network, United States, 2004. The population under surveillance is 44.5 million persons, representing 15.1% of the total population.

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Figure 10.13, the relative rate (RR) of listeriosis among the FoodNet population declined significantly by 45% from the 1996–1998 baseline period to 2002 (95% CI = 31%–57%, RR = 0.55). The relative rate increased in 2003 compared to 2002 (RR = 0.71, 29% decrease from 1996 to 1998, 95% CI = 12%–42%). These data may indeed reflect a true increase in the incidence of listeriosis in 2003. However, the widely publicized, multistate outbreak that occurred during late 2002 [70] may have resulted in an increased awareness of listeriosis among health providers, leading to enhanced diagnosis and reporting. This would, in turn, contribute to an apparent increase in incidence. Health officials hypothesized that a similar phenomenon may have contributed to statistically significant geographic differences in 1986 rates of perinatal listeriosis [68], estimated using active surveillance data collected following the large, highly publicized 1985 outbreak in Los Angeles County. The rate in Los Angeles County (24.3/100,000 live births) was over three times the combined rate in other surveillance areas. As shown in Figure 10.13, the relative rate of listeriosis declined again in 2004 to rates similar to 2002 (RR = 0.60, 40% decrease from 1996 to 1998, 95% CI = 25% to 52%) [15]. The incidence of 0.27 cases/100,000 persons approaches the 2005 National Health Objective of 0.25 cases/100,000 persons. Analyses of FoodNet data have revealed ethnic disparities in the incidence of listeriosis, which correlate well with nationwide trends discussed in Chapter 4. Among 523 listeriosis cases reported from 1996 to 2003 among individuals of known race/ethnicity, the incidence among Hispanics was significantly higher than in non-Hispanics (RR = 1.7, 95% CI = 1.2–2.4) (56). Among Hispanic casepatients, 58% were female and 63% were of childbearing age (15 to 39 years). These findings correspond well with the findings from outbreak investigations in which Hispanics were overrepresented among outbreak cases and infections were associated with the consumption of Hispanic-style cheeses. The substantial overall decline in the incidence of listeriosis can be attributed to several factors. The FDA and USDA have focused significant effort on regulatory measures, industry guidance, and initiatives on L. monocytogenes, including FDA and USDA–FSIS’s 1989 zero-tolerance rulings for ready-to-eat foods, the joint USDA–FSIS/FDA risk assessment focused on ready-to-eat foods, and the USDA’s more recent completion of a risk assessment for deli meats [67]. In October 2003, the USDA published an interim final rule (detailed in the discussion of meat and poultry productassociated outbreaks) requiring processors to enhance and verify measures to reduce the prevalence of L. monocytogenes in ready-to-eat products [8]. The food industry has responded with significant

Relative Rate

2

1 –29% (–42% –12%)

0.8 0.7 0.6

–40% (–52% –25%)

0.5 –45% (–57% –31%) 1996–1998

1999

2000

2001

2002

2003

2004

2005

Year

FIGURE 10.13 Relative rates of laboratory-diagnosed Listeria infections in humans compared with 1996–1998 baseline period, by year—Foodborne Diseases Active Surveillance Network, 1996–2004.

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effort to develop strategies to reduce the levels of L. monocytogenes in the food processing environment and prevent post-processing contamination of food items. For example, subsequent to the 2003 USDA interim rule, a survey of more than 2,900 establishments producing ready-toeat meat and poultry products found that more than 87% had made improvements to L. monocytogenes controls [6]. Improvements in the timeliness of listeriosis outbreak detection, greatly facilitated by the routine molecular characterization of L. monocytogenes human isolates, allows public health and regulatory officials to investigate and control outbreaks more efficiently. Enhanced consumer education efforts, such as the “Fight BAC” educational campaign (www.fightbac.org) developed by the Partnership for Food Safety Education in conjunction with the President’s National Food Safety Initiative and programs of the Food Safety Training and Education Alliance have likely contributed to a reduction in listeriosis incidence as well. FoodNet data have been used to provide the most accurate estimates of the incidence of listeriosis. However, these and estimates derived from passive surveillance data must be interpreted with limitations in mind. First, FoodNet captures exclusively laboratory-confirmed infections. A primary outcome of listeriosis during pregnancy is fetal loss; however, bacterial cultures are not routinely requested for spontaneously aborted fetuses or stillborn neonates. This results in reduced laboratory diagnosis [19]. Thus, whereas FoodNet surveillance provides the most robust estimates available, the true incidence of listeriosis in the United States is most certainly higher. Second, it is notable that the Hispanic population is underrepresented in the population under surveillance by FoodNet. Although it is clear that the incidence of listeriosis is disproportionately high among Hispanics compared to other racial-ethnic groups, it is likely that FoodNet data underestimate the true incidence for this population. This, in turn, would contribute to underestimation of listeriosis overall.

IMPROVED ESTIMATES

OF THE

BURDEN

OF

LISTERIOSIS

IN THE

UNITED STATES

In an ongoing effort to provide more accurate estimates of foodborne diseases including listeriosis, which are critical to guide prevention efforts and assess the effectiveness of food safety regulations, Mead et al. reported foodborne disease estimates in 1999 that were derived and validated using data from multiple sources [110]. Although L. monocytogenes causes only about 2,500 of the estimated 76,000,000 annual cases of food-related disease in the United States, it is responsible for an estimated 500 deaths annually, or one-third of the deaths caused by known foodborne pathogens. The hospitalization rate was estimated to be 92%, with a case fatality rate of 20%. Several data sources contributed to the listeriosis estimates. The annual number of food-related cases was calculated using an average of the 1996–1997 FoodNet incidence applied to the total U.S. population, and previous estimates resulting from a comparable sentinel surveillance system [143]. A multiplier of two was applied to account for underreporting based upon the assumption that the severity of listeriosis results in medical attention for most cases and, in turn, a higher rate of diagnosis and reporting as compared to a disease with less severity. Similarly, hospitalization rates were drawn from FoodNet data indicating that nearly 90% of listeriosis cases result in hospitalization. The case fatality rate was estimated using FoodNet data, previous estimates of the incidence of listeriosis in the United States [143], and outbreak data. Accounting for the demonstrated potential for nosocomial transmission [134], calculations were performed based upon the estimate that 99% of listeriosis cases are transmitted by food. Although some assumptions were required for generation of these estimates, such as those used to arrive at the multiplier accounting for underreporting, they are generally accepted as the most precise estimates of the burden of listeriosis currently available. L. monocytogenes is a foodborne pathogen of significant public health concern. Though relatively rare compared to other foodborne pathogens, it is responsible for almost one-third of foodborne disease-related deaths caused annually by known pathogens. Despite the challenges encountered during the investigation of listeriosis outbreaks, much has been learned from investigations

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resulting in the successful implication of food vehicles. These implicated foods tend to be those commercially prepared and consumed without cooking by the consumer. Environmental contamination of the processing facilities, which can be sustained for prolonged periods, often leads to contamination of the implicated processed foods. The incidence of listeriosis has been decreasing in the United States and other countries. Results of surveillance and successful outbreak investigations, as described in this chapter, have focused regulatory and food industry actions to control food contamination. However, despite documented progress, foodborne listeriosis outbreaks and sporadic cases leading to severe disease and death continue to occur. Further efforts are required to enhance surveillance, outbreak identification, investigation, regulatory interventions, industry controls, and consumer education to achieve food safety objectives nationally and internationally.

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and Behavior 11 Incidence of Listeria monocytogenes in Unfermented Dairy Products Elliot T. Ryser CONTENTS Introduction ....................................................................................................................................357 Incidence of Listeria spp. in Unfermented Dairy Products ..........................................................358 Raw Cow’s Milk...................................................................................................................358 Raw Ewe’s and Goat’s Milk ................................................................................................366 Pasteurized Milk and Other Unfermented Dairy Products..................................................367 Behavior of L. monocytogenes in Unfermented Dairy Products ..................................................376 Raw Milk ..............................................................................................................................377 Pasteurized and Intensively Pasteurized Milk .....................................................................379 Autoclaved Milk, Cream, and Chocolate Milk....................................................................380 Sweetened Condensed and Evaporated Milk.......................................................................387 Ultrafiltered Milk..................................................................................................................388 Growth of L. monocytogenes in Mixed Cultures ..........................................................................388 Nonfluid Dairy Products ................................................................................................................391 Ice Cream..............................................................................................................................391 Butter ....................................................................................................................................392 Nonfat Dry Milk...................................................................................................................393 References ......................................................................................................................................394

INTRODUCTION Recognition of raw milk as a potential source of Listeria monocytogenes led to speculation that consumption of such milk was at least partly responsible for the previously described listeriosis outbreak in post–World War II Germany. After this listeriosis epidemic, sporadic reports of individuals drinking raw milk, along with assurances that raw milk was being properly pasteurized, virtually eliminated the threat of any further outbreaks of milkborne listeriosis. Consequently, research in this area also decreased. However, in 1983, concerns about the possibility of milkborne listeriosis were rekindled when consumption of pasteurized milk was epidemiologically linked to an outbreak of listeriosis in Massachusetts. Two events, namely, publication of an article in the New England Journal of Medicine detailing this outbreak in Massachusetts and a report in June of 1985 that as many as 300 people in California had acquired listeriosis after eating Mexican-style cheese contaminated with L. monocytogenes, caused considerable concern in the United States about the presence of Listeria in dairy products. This problem subsequently took on international proportions with the 1987 report of another cheese-related outbreak in which consumption of tainted

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Vacherin Mont d’Or soft-ripened cheese was directly linked to numerous cases of listeriosis in Switzerland. Years later, one outbreak of Listeria gastroenteritis was traced to consumption of contaminated chocolate milk in the United States [201]. Despite considerable progress, L. monocytogenes outbreaks continue to plague the dairy industry, with soft cheeses, particularly the Mexican and surface-ripened varieties, being responsible for continued sporadic listeriosis outbreaks in North America and Europe. In response to questions raised by milk producers, dairy processors, health officials, and the general public, a plethora of work has been conducted worldwide since 1983 to determine the incidence and behavior of L. monocytogenes in unfermented (raw milk, pasteurized milk, chocolate milk, cream, butter, ice cream, other frozen dairy desserts), as well as fermented (cheese, yogurt, cultured milk) dairy products. The incidence and behavior of L. monocytogenes in unfermented dairy products will be dealt with in this chapter; similar information about fermented dairy products appears in Chapter 12.

INCIDENCE OF LISTERIA SPP. IN UNFERMENTED DAIRY PRODUCTS The dairy-related listeriosis outbreaks reported during the mid-1980s (see Chapter 10) prompted scientists worldwide to determine the extent of Listeria contamination in raw milk and in pasteurized dairy products including milk, ice cream, ice cream novelties, frozen desserts, nonfat dry milk, and casein. Listeria monocytogenes can readily enter dairy processing facilities in the raw milk supply, which can in turn lead to contamination of the factory environment. The occasional appearance of listeriae in pasteurized dairy products nearly always has been associated with contamination of the product after pasteurization. Thus, it is fitting to begin this discussion by examining the incidence of Listeria spp. in raw milk, which is a major source of this bacterium in dairy factory environments.

RAW COW’S MILK As you will recall from the discussion of animal listeriosis in Chapter 2, dairy cattle can intermittently shed L. monocytogenes in their milk as a consequence of listerial mastitis, encephalitis, or a Listeria-related abortion. Although milk from animals showing obvious signs of listeriosis is unlikely to reach consumers, the scientific literature contains numerous accounts in which mildly infected and apparently healthy dairy cattle, sheep, and goats have shed L. monocytogenes intermittently in their milk for many months. Thus, it appears that such asymptomatic carriers of listeriae pose the greatest threat to public health. The 1983 listeriosis outbreak in Massachusetts that was supposedly associated with drinking a particular brand of pasteurized milk raised numerous milk safety questions. The well-publicized outbreak of 1985 in which consumption of contaminated Mexican-style cheese was directly linked to at least 40 deaths in California prompted additional concerns about the safety of dairy products manufactured in the United States. Because raw milk is a potential source of L. monocytogenes, recalls of Listeria-contaminated pasteurized dairy products (i.e., milk, chocolate milk, ice cream) and imported soft-ripened cheeses prompted more than 80 surveys worldwide to determine the extent of Listeria contamination in raw milk. Results of these surveys, which will now be described in some detail, have been summarized in Table 11.1 and Table 11.2. The first large-scale survey of raw milk for Listeria spp. was prompted by the 1983 listeriosis outbreak in Massachusetts [127]. During the 3-week period immediately following the outbreak, Hayes et al. [151] examined 121 raw milk samples collected from milk trucks (40 samples), milk cooperatives (72 samples), and bulk tanks from four farms on which bovine listeriosis was diagnosed (9 samples), as well as 14 milk socks used to remove debris but not leukocytes from milk. All samples were analyzed for L. monocytogenes using a multiple two-stage enrichment procedure. Although investigators at the US Centers for Disease Control and Prevention (CDC) isolated the epidemic serotype along with other serotypes of L. monocytogenes from 15 of 121 (12.4%)

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TABLE 11.1 Incidence of Listeria spp. in Raw Milk Produced in the United States and Canada Location United States Northwest West Midwest Northeast Southeast California Massachusetts Massachusetts, Vermont Minnesota Nebraska South Dakota /Minnesota Ohio, Kentucky, and Indiana Pennsylvania Tennessee Wisconsin Total Canada Alberta Manitoba Ontario Ontario Total

Number of Samples

948 176 361 363 61 200 100 121 939 300 84 200 131 350 2511 292 50 55 7227 426 252 256 1720 445 315 3414

Number of Positive Samples (%) L. monocytogenes

56 7 18 32 6 14 0 15 15 9 0 8 12 13

(5.9) (4.0) (5.0) (8.8) (9.8) (7.0) (12.4) (1.6) (3.0) (4.0) (9.2) (3.7)

79 (3.1) 12 (4.1) 0 0 288 (4.0) 8 4 4 47 6 17 86

(1.9) (1.6) (1.6) (2.7) (1.3) (5.4) (2.5)

L. innocua

L. welshimeri

Others

Reference

ND ND ND ND ND 19 (9.5) 4 (4.0) ND ND 77 (25.7) 6 (7.1) 10 (5.0) ND 27 (7.7)

ND ND ND ND ND

ND ND ND ND ND

186 236 236 236 236 168 168 151 101 171 193 165 158 168

ND ND ND

0 0 ND ND 5 (1.7) 1 (1.2) 0 ND 6 (1.5) ND ND ND

0 1 (1.0)a ND ND 0 0 0 ND 3 (0.9)a ND ND ND

0 143 (11.1)

0 12 (0.9)

0 4 (0.3)

ND ND ND ND 43 (9.7) 26 (8.2) 69 (9.1)

ND ND ND ND 6 (1.3) 1 (0.3) 7 (0.9)

ND ND ND ND 0 0 0 (0)

102 210 103 237

123 235 93 227 112 223

Note: ND = Not determined (omitted from total). a

Two L. ivanovii and one L. seeligeri.

(including 1 of 9 bulk tank samples) [150] and 2 of 14 (14%) raw milk and milk sock samples, respectively, the epidemic phage type was never detected. Between October 1984 and August 1985, US Food and Drug Administration (FDA) officials surveyed 650 raw milk samples that were collected from bulk tanks in Massachusetts, Vermont, California, and the tristate area of Kentucky, Ohio, and Indiana. The samples were examined for Listeria spp. using the original FDA method [168]. Low levels of various Listeria spp., including L. monocytogenes, were detected in raw milk samples obtained from all states except California. Overall, 82 of 650 (12.6%) samples contained Listeria spp., with L. monocytogenes being found in 27 of 650 (4.2%) samples. Of the 27 L. monocytogenes strains isolated from raw milk, 16 were serotype 1, 10 were serotype 4, and 1 was nontypeable. In addition, only 2 of the 27 L. monocytogenes strains proved to be nonpathogenic to mice, and both were of serotype 4. In 1988, Donnelly et al. [101] used flow cytometry to analyze 939 raw milk samples obtained from 54 farms in California. Unlike the FDA study just described [168], string samples (milk pooled

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TABLE 11.2 Incidence of Listeria spp. in Raw Cow’s Milk Collected outside North America Number of Positive Samples (%) Location Europe Austria Belgium Czechoslovakia Denmark Finland France

Germany Hungary Ireland Italy

The Netherlands Poland Portugal Spain Sweden Switzerland Turkey United Kingdom Great Britain England/Wales Scotland

Northern Ireland Total

Number of Samples

L. monocytogenes

201 143 177 123 1,227,053 256 59 1787 1409 561 337 51 635 80 50 589 50 290 142 98 85 50 40 32 137 134 81 54 340 774 294 4046 340 77

0 9 6 4 278 13 1 60 85 21 14 10 2 3 2 29 4 8 0 2 0 0 0 0 6 2 6 3 23 28 3 14 2 14

350 2009 361 640 560 540 176 113 18,271

13 102 13 90 14 14 27 6 653

L. innocua

L. welshimeri

Reference

(4.4) (1.5) (7.4) (5.6) (6.8) (3.6) (1.0) (0.4) (0.6) (18.2)

2 (1.0) ND ND ND ND ND ND ND ND ND 5 (1.5) 10 (19.6) ND ND ND 20 (3.4) ND 16 (5.6) 1 (0.6) ND 0 1 (2.0) 0 2 (6.3) ND ND ND ND 24 (7.0) 21 (2.7) 7 (2.3) ND ND ND

ND ND ND ND ND ND ND ND ND 0 18 (35.3) ND ND ND ND ND 0 0 ND 0 0 0 ND ND ND ND ND 1 (0.3) 0 0 ND ND ND

2 (1.0) ND ND ND ND ND ND ND ND ND 0 9 (17.6) ND ND ND ND ND 2 (0.7) 0 ND 0 0 0 ND ND ND ND ND 6 (1.8) 0 0 ND ND ND

107 97 181 182 200 200 200 180 45 51 139, 140 139, 140 133, 232 207 207 206 157 96 226 164 136 234 175 130 78 161 211 146 240 135 241 73 73 220

(3.6) (5.1) (3.6) (14.1) (2.5) (2.6) (15.3) (5.3) (3.6)

ND ND ND ND 7 (1.3) ND 18 (10.2) ND 134 (3.4)

ND ND ND ND 0 ND 0 ND 19 (0.6)

ND ND ND ND 1 (0.2) ND 5 (2.8)a ND 35 (1.0)

137 188 145 125 124 124 149 138

(6.3) (3.3) (3.3) (0.02) (5.1) (1.7) (3.4) (6.0) (3.8) (4.2) (19.6) (0.3) (3.8) (4.0) (4.9) (8.0) (2.8) (2.0)

0

Others

(continued)

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TABLE 11.2 (CONTINUED) Incidence of Listeria spp. in Raw Cow’s Milk Collected outside North America Number of Positive Samples (%) Location Elsewhere Argentina Australia Brazil

Costa Rica Egypt Iran Japan

Jordan Korea Malaysia Mexico Morocco New Zealand South Africa Taiwan Total

Number of Samples

208 169 150 220 20 12 220 236 190 362 120 150 170 50 45 930 1300 100 30 71 982 80 5815

L. monocytogenes

1 1 0 11 0 1 0 7 4 3 0 6 2 1 2 18 162 0 3 0 67 5 304

(0.5) (0.6) (4.8) (8.3) (3.0) (2.1) (0.8) (0.4) (7.1) (2.0) (4.4) (1.9) (12.5) (10.0) (6.8) (6.3) (5.2)

L. innocua

L. welshimeri

Others

Reference

5 (2.4) ND 4 (2.7) 21 (9.5) ND 2 (16.7) 10 (4.5) 20 (8.5) ND ND ND 2 (1.3) ND ND ND 20 (2.1) 9 (0.7) 7 (7.0) ND 10 (14.1) ND ND 110 (3.1)

1 (0.5) ND 0 2 (0.9) ND 0 3 (1.4) 0 ND ND ND 0 ND ND ND 6 (0.6) 0 2 (2.0) ND 1 (1.4) ND ND 15 (0.4)

0 ND 0 1 (0.4) ND 0 1 (0.5)b 0 ND ND ND 0 ND ND ND 0 129 (9.9) 0 ND 7 (9.9) ND ND 138 (3.8)

162 110 154 185 83 129 70 108 205 244 231 219 121 147 74 85 238 169 109 229 243 84

Note: ND = Not determined (omitted from total). a

L. seeligeri. L. grayi.

b

from 25 to 40 cows), combination samples (milk pooled from 200 cows), and samples of raw milk from bulk tanks were tested for L. monocytogenes. Using this method, L. monocytogenes was detected in 15 of 939 (1.5%) samples. Researchers in Minnesota [193] and Wisconsin [103,237] failed to detect L. monocytogenes in raw milk during three small surveys. However, the pathogen was found in 4.0 and 2.8% of raw milk samples obtained from bulk storage tanks and tank trucks in Nebraska [166] and Pennsylvania [102], respectively, with approximately equal numbers of L. monocytogenes isolates being classified as serotype 1, 4, or nontypeable (non–serotype 1 or 4) in the latter study. More recent findings indicate a relatively stable incidence for L. monocytogenes in bulk tank raw milk, with 3.0 and 4.1% of such samples from Minnesota [171] and Tennessee [210], respectively, being positive. In the Minnesota survey, Listeria-positive samples also tended to have higher bacterial and somatic cell counts, which are indicative of less stringent sanitation and mastitis control practices. As part of the National Animal Health Monitoring System, a large-scale national survey was conducted in 2002 during which 861 raw milk bulk tank samples were analyzed for various microbial contaminants including L. monocytogenes [236]. The incidence of L. monocytogenes contamination was 4.0, 5.0, 8.8, and 9.8% for milk samples collected in the West, Midwest,

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Northeast, and Southeast (average of 6.5%), respectively, with 93% of the isolates belonging to serotypes 1/2a, 1/2b, and 4b. One year earlier, Muraoka et al. [186] reported an overall incidence rate of 5.9% for L. monocytogenes in bulk tank samples from the Pacific Northwest with strains of serotype 1/2a predominating. In one additional survey [149], milk filters rather than samples of bulk tank milk were collected from 404 farms throughout New York State from April 1998 to March 1999 and assessed for L. monocytogenes. Fifty-one (12.6%) of these milk filters through which an average of 1351 kg of milk had passed yielded L. monocytogenes with this substantially higher contamination rate compared to bulk tank milk related to presence of debris in the filters at the time of sampling. Additional observations from this study included regional differences in percentage of positive filters (6.5 to 19.0%), with the highest contamination rate found in central New York State and a higher isolation rate during spring (18%) compared to summer, fall, and winter (6–8%). The overall findings from Table 11.1 indicate that L. monocytogenes was present in 0–12.4% of the US raw milk samples examined. When the results are averaged, 4.0% of all raw milk processed in the United States can be expected to contain low levels (i.e., <10 CFU/mL) of L. monocytogenes at any given time, with the incidence of L. innocua being appreciably higher at 11.1%. Hence, it is imperative that all milk processors take special precautions to prevent the spread of L. monocytogenes from raw milk to production, packaging, and other sensitive areas within the factory. Turning to the Canadian raw milk supply (Table 11.1), Farber et al. [112] and Slade et al. [223], respectively, isolated L. monocytogenes from 6 of 445 (1.3%) and 17 of 315 (5.4%) raw milk samples obtained from bulk tanks located throughout the province of Ontario. Davidson et al. [93] also reported that a similar percentage of raw milk samples collected from four local dairies and 48 farms in Manitoba contained L. monocytogenes. In the study by Slade et al. [223], 14 of 17 (82%) L. monocytogenes isolates belonged to serotype 1, the remaining three strains being classified as serotype 4. Although subsequent work [126] demonstrated that none of these L. monocytogenes strains harbored plasmid DNA, 8 of 22 (36%) L. innocua strains did carry plasmids ranging in size from 10 to 44 MDa. Variable plasmid profiles among these isolates further suggest that contamination of raw milk on the farm is an ongoing process. Whereas these authors did not quantitate L. monocytogenes in any of the samples examined, Slade and Collins-Thompson [222] reported that positive raw milks from bulk tanks in southwestern Ontario always contained <5 L. monocytogenes CFU/mL when samples were analyzed by direct plating and a most probable number (MPN) enrichment procedure. Thus, the positive raw milk samples encountered in three Canadian studies likely contained only low levels of L. monocytogenes. In two subsequent surveys, L. monocytogenes was identified in 1.6 [235] and 1.9% [123] of all raw milk bulk tank samples examined in the province of Alberta. However, both surveys also indicated substantially higher contamination rates for commingled raw milk before processing with milk in 5 of 72 (6.9%) tank trucks [123] and in 4 of 15 (26.6%) milk silos [235] testing positive for L. monocytogenes. Finally, in the largest Canadian survey of raw milk for pathogens [227], L. monocytogenes was identified in 2.7% of 1720 bulk tank samples collected from dairy farms in Ontario. More importantly, these same authors estimated that the likelihood of L. monocytogenes contamination would increase to 8.0, 12.9, and 24.1% from the pooling of 3, 5, and 10 bulk tank samples, respectively, during multiple pickups. Consequently, all dairy facilities can expect to have low levels of L. monocytogenes present in the incoming raw milk. As was true for the three U.S. surveys [168] just discussed, L. innocua also was the most common Listeria sp. isolated from Canadian raw milk, with 8.2 and 9.7% of the samples being reported as positive. Overall, 2.5 and 9.1% of all Canadian raw milk samples contained L. monocytogenes and L. innocua, respectively, as compared to 4.0 and 11.1% of raw milk samples examined in the United States. Thus, the incidence of listeriae in raw milk from both countries appears to be similar. Public concern and economic hardships brought about by several recalls of French Brie cheese imported into the United States prompted French scientists to begin surveying raw milk for L. monocytogenes. Results from three such surveys [45,51,139] (see Table 11.2) conducted between

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1986 and 1988 indicated that 85 of 1409 (6.0%), 21 of 561 (3.8%), and 14 of 337 (4.2%) raw milk samples from French bulk tanks were positive for L. monocytogenes, with L. innocua being detected in about one-third as many samples in the latter study [139]. During January of 1986, 51 raw milk samples were submitted to the French Central Laboratory of Food Hygiene. Both L. monocytogenes and L. innocua were found in 19.6% of these samples. The unusually high incidence of listeriae observed in this survey, as compared to other studies described thus far, may be the result of nonrandom sampling or seasonal variations, the latter of which will be discussed shortly. Meyer-Broseta et al. [180] sampled 1787 raw milk tankers from 1997 to 2001 and reported an L. monocytogenes prevalence rate of 2.4 to 4.4% (average of 3.4%). This milk originated from 50 different dairy farms with up to six farms positive for L. monocytogenes during any given month. Levels of L. monocytogenes in positive bulk tank samples were low and never exceeded 210 CFU/mL. Frequent isolation of L. monocytogenes from dairy products prompted additional surveys of the raw milk supply throughout Europe and later elsewhere, the primary focus being given to countries with sizeable dairy industries (see Table 10.2). Results from two comprehensive year-long surveys in the United Kingdom [137] and Scotland [124] revealed an incidence of L. monocytogenes in raw milk similar to that observed in the United States and Canada, with actual Listeria populations in positive samples also estimated to be extremely low. As in North America, L. monocytogenes strains belonging to serotype 1/2 appear to predominate in European raw milk [124,125,145,212]. Although four subsequent surveys from the United Kingdom yielded similar findings [124,125,145,188], L. monocytogenes contamination rates of 14 to 15% have been reported from both Scotland [125] and Northern Ireland [148], suggesting considerable local variability. Harvey and Gilmour [148] also found that 33% of raw milk samples collected from processing centers harbored L. monocytogenes, which again emphasizes the impact of commingling milk. However, levels of listeriae in such milk again appear to be quite low, with actual numbers of L. monocytogenes typically being <10 CFU/mL in positive samples from Scotland [125]. In 1987, Beckers et al. [78] reported culturing L. monocytogenes from 6 of 137 (4.4%) raw milk samples obtained from farms in the Utrecht region of The Netherlands. As was true for raw milk tested in the United States and Canada, milk samples from The Netherlands again contained <100 L. monocytogenes CFU/mL. Similar findings have been reported from most other European surveys, with 3–5% of raw milk samples harboring low levels of L. monocytogenes. However, two nationwide surveys conducted in Switzerland during and after the outbreak involving Vacherin Mont d’Or cheese indicated a far lower incidence, with only 0.4 and 0.6% of the raw milk samples being positive for L. monocytogenes [73]. Several years earlier, Terplan [232] detected L. monocytogenes in only 2 of 635 (0.3%) raw milk samples obtained from farm bulk tanks in Würtemburg, Germany. Subsequent attempts to isolate this pathogen from 448 quarter-milk and 30 separator sludge samples failed, along with attempts to culture this organism from raw milk samples obtained from tank trucks and storage tanks. Findings from an exhaustive survey in Denmark of over 1 million raw milk samples demonstrated an L. monocytogenes contamination rate of only 0.2%, with several additional small-scale Italian surveys yielding negative results. Thus, when the Danish results are excluded, 3.6% of all European raw milk would be expected to contain low levels of L. monocytogenes as compared to 3.3% of the raw milk produced in North America with these contamination rates having remained relatively unchanged since the 1980s. In response to the aforementioned surveys and continued sporadic outbreaks of listeriosis traced to consumption of soft and surface-ripened cheeses, investigators in Australia, New Zealand, the Middle East, South America, South Africa, and the Orient also have begun assessing the incidence of L. monocytogenes contamination in their own raw milk supplies. In the first of these surveys, workers in New Zealand [229] collected and analyzed 71 raw milk bulk tank samples between August 1986 and March 1987 for listeriae using both warm and cold enrichment. Although L. monocytogenes was apparently absent from this milk as well as from milk examined in a later Australian survey, isolation of other Listeria spp. from these samples strongly suggests that such milk is unlikely to be completely free of L. monocytogenes. Similar negative findings

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have been obtained from surveys conducted in Brazil [83] and Costa Rica [70], with Arias et al. [70] also citing another survey in which 20% of hand-milked samples harbored L. monocytogenes. According to Vasquez-Salinas [238], 162 of 1300 raw milk samples obtained from four different dairy farms near Mexico City, Mexico, yielded L. monocytogenes. These findings support current safety concerns regarding the consumption of soft Hispanic-style cheeses prepared from raw milk. Working in Japan, Takai et al. [231] reported the absence of Listeria spp. from 120 raw milk samples, whereas L. monocytogenes was recovered from 6 of 150 (4.0%) and 3 of 362 samples in subsequent Japanese surveys [219,244]. Three more recent surveys have shown L. monocytogenes contamination rates of 2.0–4.4 [74,147] and 1.9% [85] for raw milk sampled in Korea and Malaysia, respectively. Given these findings, and additional reports of L. monocytogenes in raw milk from Egypt [1,108], Jordan [121], Iran [205], Turkey [143,220], Morocco [109], and South Africa [243], it is now clear that milkborne listeriosis constitutes a worldwide threat. However, confirmation of dairy-related listeriosis cases in such developing countries will likely remain very difficult given their lack of resources and understandable preoccupation with far more immediate health concerns. Examination of data summarized in Table 10.1 and Table 10.2 indicates that 4.0, 2.5, 3.6, and 5.2% of the raw milk produced in the United States, Canada, Europe, and elsewhere, respectively, can be expected to contain low levels of L. monocytogenes, with similar contamination rates also likely occurring in other parts of the world. Except for the European samples, L. innocua was isolated from raw milk more frequently than L. monocytogenes, with the former found in 11.1, 9.1, 3.4, and 3.1% of the samples analyzed for all Listeria spp. in the United States, Canada, Europe, and elsewhere, respectively. Although L. innocua is nonpathogenic, isolation of this organism and other listeriae from dairy products and dairy processing facilities is taken very seriously in the United States, because nonpathogenic listeriae and L. monocytogenes are assumed to occur in similar environmental niches. Use of L. innocua as a potential indicator of L. monocytogenes in the United States and elsewhere is supported by data from the raw milk surveys just described (see Table 10.1) in that isolation of listeriae other than L. monocytogenes from dairy products and processing facilities suggests that the factory environment may be contaminated with raw milk that can be expected to contain L. monocytogenes 3–4% of the time. Although long theorized, the spread of identical L. monocytogenes strains from the farm environment into the raw milk supply and ultimately to dairy processing facilities now has been confirmed using various strain-specific typing methods, including restriction fragment length polymorphism [148] and automated ribotyping [71]. In addition to assessing the general incidence of listeriae in raw milk, several of the surveys just described also dealt with seasonal variations in the incidence of L. monocytogenes and L. innocua in raw milk produced in the United States [102,165,168] and Canada [112]. However, because all the aforementioned studies differ in numbers and sizes of samples analyzed as well as the Listeria isolation procedures employed, it is difficult to make any definitive statement concerning the seasonal occurrence of Listeria in raw milk [124,223]. Nonetheless, several distinct trends can be observed from selected data in Figure 10.1. First, the overall incidence of L. monocytogenes in raw milk was highest in spring (5.8%), followed by winter (4.5%), fall (2.8%), and summer (2.5%). Second, a somewhat similar seasonal variation also can be observed for the incidence of L. innocua in raw milk, with the highest overall percentage of positive samples again occurring in winter (11.7%), followed by spring (10.2%), summer (6.0%), and fall (4.2%). These findings again suggest that L. innocua can be used as a potential indicator for the presence of L. monocytogenes. Although not fully understood, current herd management and feeding practices may be at least partly responsible for seasonal differences observed in isolation rates for L. monocytogenes and possibly L. innocua. During cold winter months, silage comprises a major component of the diet. While investigating one listeriosis outbreak, Donnelly [99] observed that 8 of 44 Holstein cows fed Listeria-contaminated silage shed the organism in their milk. Furthermore, milk from these animals was free of L. monocytogenes 1 month after feeding of contaminated silage ceased. Ruminants that ingest contaminated silage may either succumb to infection or carry L. monocytogenes

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Positive Samples (%)

20 16

A = Massachusetts, Vermont B = Ohio, Kentucky, Indiana C = Pennsylvania D = Nebraska E = Ontario, Canada F = England/Wales

365

NS= Not Sample LA= Listeria Absent = L. monocytogenes = L. innocua

12 8 4 NS A B C D E F Spring

A

BC D E F Summer

NS A B CD E F

NS LA A BC D E F

Fall

Winter

FIGURE 11.1 Seasonal variation in the incidence of L. monocytogenes and L. innocua in raw milk from the United States (from Doores, S. and J. Amelang. 1990. Personal communication; Hayes, P. S., J.C. Feeley, L.M. Graves, G.W. Ajello, and D.W. Fleming. 1986. Isolation of Listeria monocytogenes from raw milk. Appl. Environ. Microbiol. 51: 438–440; Liewen, M.B. and M.W. Plautz. 1988. Occurrence of Listeria monocytogenes in raw milk in Nebraska. J. Food Prot. 51: 840–841; Lovett, J., D.W. Francis, and J.M. Hunt. 1987. Listeria monocytogenes in raw milk: detection, incidence, and pathogenicity. J. Food Prot. 50: 188–192), Canada (from Farber, J.M., G.W. Sanders, and S.A. Malcom. 1988. The presence of Listeria spp. in raw milk in Ontario. Can. J. Microbiol. 34: 95–100), and England/Wales (from O’Donnell, E.T. 1995. The incidence of Salmonella and Listeria in raw milk from farm bulk tanks in England and Wales. J. Soc. Dairy Technol. 48: 25–29).

asymptomatically; however, if the animal lives, the organism can be shed for many months in feces and in milk from lactating animals. Extended survival of L. monocytogenes in fecal material, soil, and grass can perpetuate the infectious cycle shown in Figure 11.2, particularly when animals are wintered in cramped quarters. Once dairy cattle resume grazing on pastures during late spring, summer, and early fall, L. monocytogenes becomes dispersed over a wide area, which, in turn, weakens the infectious cycle by decreasing the likelihood that animals will come in contact with contaminated material.

Survival and/or growth of L. monocytogenes in silage

Ingestion by ruminants

Survival in soil and grass Winter confinement L. monocytogenes excreted in feces

L. monocytogenes secreted in milk

FIGURE 11.2 Infective cycle for maintaining L. monocytogenes in ruminants.

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Seasonal differences in the incidence of Listeria spp. in raw milk also may be related to breeding practices. Dairy cattle typically bear their young in late winter or early spring. During winter gestation, dairy cattle develop a weakened immune system as a direct result of pregnancy, which, in turn, makes these animals more susceptible to listerial infections and abortions. These events can then culminate in the shedding of L. monocytogenes in milk and fecal material. Increased environmental stress and changes in habitat that occur during winter, along with increased difficulties in providing proper herd hygiene, all can serve to decrease the natural defense system in dairy cattle, which again increases the likelihood of listerial infections. Once an asymptomatic animal begins shedding L. monocytogenes in feces, the organism is likely to spread quickly to other animals that are housed in close proximity to the shedder. In this way, confinement of dairy cattle may play an important role in increasing the number of animals that shed L. monocytogenes in their milk during late winter and early spring.

RAW EWE’S

AND

GOAT’S MILK

Surveys in Europe, Australia, and the United States also have demonstrated that raw milk from ewes and goats can occasionally contain L. monocytogenes (Table 11.3) with incidence rates ranging from 0 to 3.0% (average of 1.6%) and 0 to 4.0% (average 2.4%), respectively. These contamination levels, and that reported in India for buffalo milk, are similar to those seen for cow’s milk. Working in Vermont, Abou-Eleinin et al. (2) recovered Listeria spp. from 35 of 445 (7.9%) bulk tank samples of raw goat’s milk using three different enrichment methods. Seventeen samples contained L. monocytogenes and 8 contained both L. monocytogenes and L. innocua. Listeria contamination

TABLE 11.3 Incidence of L. monocytogenes in Raw Ewe’s and Goat’s Milk Type of Milk Ewe

Total Goat

Total Buffalo a

Location

Number of Samples

England/Wales England/Wales India Italy Italy Portugal Spain Spain Turkey

56 26 23 40 34 119 1052 202 302 1854 450 480 100 60 64 24 39 25 1445 2756 64

United States England/Wales England/Wales Italy India Italy Portugal Portugal Spain India

Number of Samples Positive (%) (1.8) 0 0 0 0 2 (1.7) 23 (2.2)a 6 (3.0) 0 32 (1.7) 17 (3.8)b 4 (0.8) 0 0 1 (1.6) 0 0 1 (4.0) 37 (2.6) 61 (2.2) 4 (6.3)

Also 21 L. innocua, 4 L. welshimeri, 3 L. seeligeri, 3 L. grayi, and 2 L. ivanovii. Eight L. monocytogenes and L. innocua, and twenty-six L. innocua.

b

Reference 145 167 76 81 88 146 208 240 91 2 145 167 129 76 81 146 92 134 75

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rates were higher in winter (14.3%) and spring (10.4%) than in fall (5.3%) and summer (0.9%), with similar observations reported for cow’s milk. Overall, 62.6 and 37.4% of those L. monocytogenes that were further characterized belonged to serovars 1 and 4, respectively. Automated ribotyping of selected isolates also indicated five distinct ribotypes, two clinically important ribotypes of which were eventually traced back to the farm environment.

PASTEURIZED MILK

AND

OTHER UNFERMENTED DAIRY PRODUCTS

The dairy industry has long been considered as the most regulated food industry in the United States. The FDA was given responsibility under the Food, Drug and Cosmetic Act and the Public Health Service Act to assure the public that this country’s milk supply is both uniformly safe and wholesome. Dairy sanitation laws and regulations, including microbiological criteria for some dairy products, enforced by the FDA and state agencies are based almost exclusively on the Public Health Service/FDA Grade A Pasteurized Milk Ordinance. In the United States, pasteurized milk and other unfermented dairy products prepared from pasteurized milk, including ice cream, butter, and nonfat dry milk, generally have earned a reputation for being both safe and nutritious, with dairy products accounting for <1.5% of all foodborne illnesses [77]. Pasteurized milk is responsible for about 5% of all reported dairy-related illnesses [178]. Despite these impressive findings, public confidence in the safety of pasteurized milk began to erode in July 1982 following an outbreak of yersiniosis in Tennessee, Arkansas, and Mississippi [230] and again in 1983 when the CDC claimed that consumption of pasteurized milk was responsible for 49 cases of listeriosis in Massachusetts. In 1985, the dairy industry was dealt another blow when at least 16,000 culture-confirmed cases of salmonellosis were associated with drinking a particular brand of pasteurized milk produced in the Chicago area [216]. These three outbreaks, along with the previously discussed listeriosis outbreak in California linked to consumption of contaminated Mexican-style cheese, prompted the FDA to take corrective action in the form of a large-scale testing program commonly referred to as the FDA Dairy Initiative Program [159] (Figure 11.3). This program, begun in April of 1986 in cooperation with individual

June to August, 1983: Listeriosis outbreak in Massachusetts associated with pasteurized milk

April 1986:

October 1986:

October 1987:

March to April, 1985: Salmonellosis outbreak in Chicago linked to pasteurized milk

January to June, 1985: Listeriosis outbreak in California linked to Mexican-style cheese

Begin dairy iniciatives. Portion of IMS and non-IMS inventory sampled for Listeria, Salmonella, Yersinia and Campylobacter. Plant inspections in cooperation with state agencies. 1174 samples analyzed during fiscal 1986.

Continue sampling except omit Campylobacter. 1444 samples analyzed during fiscal 1987.

Continue sampling remainder of inventory and the following composite samples – Fluid milk: Listeria, Salmonella, and Staphylococcus aureus.

FIGURE 11.3 FDA Dairy Initiatives Program. IMS, interstate milk shipment. (Adapted from Kozak, J.J. 1986. FDA’s dairy program initiatives. Dairy Food Sanit. 6: 184–185.)

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state agencies and members of the National Conference on Interstate Milk Shipments, was designed to examine every interstate milk shipment (IMS) pasteurization facility in the United States for potential safety problems related to pasteurization, postpasteurization contamination, cleaning and sanitizing regimens, equipment maintenance, and educational/training programs for dairy factory personnel. As part of the FDA Dairy Initiative Program, the agency also established the Microbiological Surveillance Program, which was designed to detect L. monocytogenes, Salmonella spp., Yersinia enterocolitica, Campylobacter jejuni, and C. coli (Campylobacter omitted in 1987) as well as Staphylococcus aureus, which was added in 1987 for dry and fluid milk [18]. Dairy products tested under this program included fluid milk, nonfat dry milk, cream, butter, ice cream, ice milk, and other dairy commodities over which the FDA has jurisdiction. Analysis of cheeses (except cottage cheese) was covered under a series of separate programs, which will be discussed in Chapter 12 concerning fermented dairy products. Under the provisions of this program, FDA inspectors collected 30 retail-sized containers of as many as five different products available from dairy factories at the time of inspection. Duplicate 25-g or 25-mL samples obtained after combining 30 retailsized samples per product were then analyzed for L. monocytogenes and other listeriae using the original and later versions of the FDA procedure. Because raw milk containing L. monocytogenes will periodically enter virtually every dairy factory in the United States, it is logical to assume that finished products also may become contaminated with this pathogen. During the first 2 years of the FDA Dairy Initiative Microbiological Surveillance Program (Table 11.4), L. monocytogenes was isolated from 2 of 350 samples of pasteurized whole milk and 5 of 415 samples of chocolate milk, which suggests that

TABLE 11.4 Incidence of Listeria spp. in Unfermented Dairy Products Manufactured in the United States during 1986 and 1987 Number of Positive Samples (%) Product Whole milk Low-fat milk Skim milk Chocolate milk Cream Half-and-half Ice milk Ice cream Novelty ice cream Butter Nonfat dry milk Casein/Milk protein hydrolysate Other productsc Total a b c

Number of Samples Tested

L. monocytogenes

350 182 98 415 52 42 99 659 351 30 44 15 171 2518

2 (0.57) 0 0 5 (1.20) 0 0 3 (3.03) 23 (3.03) 30 (8.55) 0 0 0 0 63 (2.50)

L. innocua 0 2 (1.10) 0 1 (0.24) 0 0 0 12a (1.82) 10b (2.85) 0 0 0 0 25 (0.99)

L. seeligeri also detected in 7 of 659 (1.06%) samples. L. grayi also detected in 1 of 351 (0.28%) samples. Dairy blend whey, eggnog.

Source: Archer, D.L. 1988. Review of the latest FDA information on the presence of Listeria in foods. WHO Working Group on Foodborne Listeriosis, Geneva, February 15–19; Kozak, J., T. Balmer, R. Byrne, and K. Fisher. 1996. Prevalence of Listeria monocytogenes in foods: incidence in dairy products. Food Control 7: 215–221.

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approximately 0.67% of the pasteurized milk available in the United States could contain this pathogen unless factories take corrective action to reduce this value. In contrast, L. monocytogenes was isolated from 3 of 99, 23 of 659, and 30 of 351 samples of ice milk, ice cream, and novelty ice cream, respectively. Only two of the positive ice cream samples were analyzed quantitatively for L. monocytogenes. One contained an average of 15 L. monocytogenes CFU/g, whereas the other sample contained between 1 and 5 CFU/g. Thus, during the time of this survey, approximately 5% of frozen dairy products manufactured in the United States presumably contained low levels of L. monocytogenes. Furthermore, L. innocua was isolated from three of four product categories (see Table 11.4) that also contained samples positive for L. monocytogenes. Because both organisms likely occupy similar niches in the natural environment and dairy processing facilities, isolation of L. innocua from a dairy product should raise immediate concerns about the possible presence of L. monocytogenes. Greater success in isolating L. monocytogenes from chocolate rather than whole milk is likely related to the organism’s ability for enhanced growth in this product as compared to other fluid dairy products. Reasons for increased growth of Listeria in chocolate milk will be discussed shortly in conjunction with the behavior of listeriae in autoclaved fluid dairy products. The higher incidence of L. monocytogenes in frozen rather than fluid dairy products coincides with the relatively complex handling of ice milk and ice cream, particularly ice cream novelties, during manufacture and packaging. This, in turn, suggests that these products are most likely contaminated after pasteurization through either direct or indirect contact with listeriae within the dairy factory environment. This hypothesis is supported by frequent isolations of L. monocytogenes from many areas within dairy factories, including floors, ceilings, drains, and coolers. In addition, this organism also has been found in air and condensate and on various pieces of equipment, including conveyor belts. A detailed discussion of the incidence of L. monocytogenes in foodprocessing facilities, including dairy factories, can be found in Chapter 17. Inability of the FDA to detect L. monocytogenes in skim and low-fat milk as well as half-andhalf, cream, and butter may have resulted from the separation processes used for adjusting milkfat content that these products undergo. Such centrifugal separation processes tend to decrease levels of listeriae, particularly if leukocytes containing the organism are still present after initial clarification of the milk. Failure to isolate L. monocytogenes from nonfat dry milk and casein/protein hydrolysates may be partly related to the heat treatments necessary to manufacture these products. This theory is supported by the work of Doyle et al. [104], who demonstrated that populations of L. monocytogenes decreased by greater than 90% during conversion of skim milk into nonfat dry milk via spray drying. However, failure to isolate L. monocytogenes from dried dairy products also may result from the generally recognized inability of the FDA method to detect cells of L. monocytogenes that have been sublethally injured during thermal processing. Thus, the methodology employed to detect L. monocytogenes will predetermine whether or not the organism can be isolated from a particular food. According to the Food, Drug and Cosmetic Act of 1938, a food may be considered adulterated and therefore unfit for human consumption if the product contains poisons or other harmful substances (e.g., pathogenic microorganisms) at detrimental concentrations. Although the oral infective dose for L. monocytogenes is known to vary widely based on individual susceptibility, evidence from the California listeriosis outbreak involving Mexican-style cheese demonstrated that the number of L. monocytogenes cells needed to induce this life-threatening illness can in some instances be quite low—perhaps as few as several hundred to a few thousand total cells for certain segments of the population. Several independent studies involving immunocompromised mice demonstrated LD50 values (the dose of cells that is lethal to 50% of a given population) in the range of approximately 10 [142] and 4 to 480 [89,228] L. monocytogenes cells when the pathogen was administered orally and intraperitoneally, respectively. However, more recent work with rhesus monkeys suggests an oral infectious dose generally >106 L. monocytogenes

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cells (225). Although the FDA is morally obligated to uphold its policy of “zero tolerance” for L. monocytogenes in ready-to-eat foods, results from several risk assessments to be discussed in Chapter 18 indicate that the current zero tolerance policy will likely be modified to allow L. monocytogenes at levels up to 100 or 1000 CFU/g in certain products such as ice cream in which the organism is unable to grow. In accordance with Title 21 of the United States Code of Federal Regulations, Section 7.40 [128], the FDA can request that firms voluntarily recall any product that contains, or is suspected of containing, L. monocytogenes. These recalls can be classified into one of three categories: Class I, Class II, or Class III. A Class I recall is the most serious and is defined by the FDA as “a situation in which there is a reasonable probability that the use of or exposure to a violative product will cause serious adverse health consequences or death.” Thus far, all recalls issued for products contaminated with L. monocytogenes have been categorized as Class I. In the unlikely event that a firm fails to comply with the FDA’s request to recall a product containing L. monocytogenes, FDA officials can (1) initiate a seizure request in the US District Court to have the product removed from commerce (Title 21 Code of Federal Regulations, Section 334) or (2) obtain a legal injunction to halt production and distribution of the contaminated product (Title 21 Code of Federal Regulations, Section 332). In addition, the FDA also can take criminal action against individuals of a company who are responsible for commercial distribution of a contaminated product. Adoption of the FDA Dairy Initiative Microbiological Surveillance Program in April 1986 prompted the first of at least 58 recalls for milk and unfermented dairy products contaminated with L. monocytogenes (Table 11.5). Just 1 month after the surveillance program began, a California firm voluntarily recalled an unknown quantity of ice milk mix contaminated with L. monocytogenes. During the same month, approximately 1 million gal of fluid dairy products that comprised milk, chocolate milk, half-and-half, whipping cream, ice milk mix, ice milk shake mix, and ice cream mix also were recalled in Texas [13,14]. However, other than this single incident, the remaining recalls have been primarily confined to frozen dairy products such as ice cream, ice cream novelties, ice milk, and sherbet, with only six additional recalls thus far being traced to nonfrozen dairy products (i.e., butter). In July 1986, a large recall of Listeria-contaminated ice cream bars received considerable media attention, which in turn did much to enhance the state of hysteria concerning the presence of L. monocytogenes in dairy products [7]. The following month, another nationwide recall was issued for frozen dairy products. As a result of this recall, approximately 1 million gal of products possibly contaminated with L. monocytogenes and including ice cream (132 flavors), ice milk (16 flavors), sherbet (9 flavors), and gelati-da products (6 flavors) were reportedly buried at a Minnesota landfill site. One year later, a similar recall was issued for contaminated ice cream, ice milk, and sherbet manufactured in Iowa [28,32]. By the end of 1987, well over 500 Listeriacontaminated dairy products were voluntarily recalled in the United States at a total cost to the dairy industry of well over $70 million [31,177]. Although corrective measures instituted by the dairy industry in response to governmental pressure sharply reduced both the incidence of Listeria contamination in processing facilities and the number of Class I recalls issued for L. monocytogenescontaminated dairy products since 1988 [51], recalls involving frozen desserts—and most recently butter—have continued to negatively impact the industry. During the 6-year period from 1990 to 1995, 17 of 39 (44%) dairy-related L. monocytogenes recalls involved unfermented dairy products, with Listeria being responsible for 31% of all dairy-related recalls issued for any reason [217]. Unfortunately, product losses are also being substantially underreported, because, under certain circumstances manufacturers can retrieve their own product without issuing a formal Class I recall. In one such instance, a manufacturer of Listeria-contaminated ice cream was not required to issue a formal recall, because the product had not yet reached the consumer [36]. Additional cases also likely occurred in which contaminated products moved only as far as the company’s warehouse and were recalled internally by the manufacturer.

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TABLE 11.5 Chronological List of Voluntary Class I Recalls Issued in the United States from 1986 to 2005 for Milk and Unfermented Dairy Products Contaminated with L. monocytogenes Type of Dairy Product Ice milk mix Milk (2 and 1/2% fat), chocolate milk, chocolate milk (2% fat), half-and-half, whipping cream, vanilla and chocolate ice cream mix, ice milk shake mix, and ice cream mix Ice cream bars Ice cream, sherbet, glacee Ice cream (132 favors), ice milk (16 flavors), sherbet (9 flavors), gelati-da products (6 flavors) Ice cream and ice cream novelties—bars, drumsticks, slices, sundaes Ice cream Ice cream Ice cream Ice cream, ice milk, sherbet

Month/Year State of of Recall Manufacture

Distribution

Quantity

Reference

5/86 5/86

California Texas

Arizona, Nevada, California Texas

Unknown ∼1 million gal

7/86 7/86

Virginia Wisconsin

Eastern United States Minnesota, Wisconsin

Large but unknown 8,000 gal

8/86

Minnesota

Illinois, Indiana, Iowa, Kentucky, Michigan, Minnesota, Missouri, North Dakota, Ohio, South Dakota, Wisconsin

∼1 million gal

8/86

Iowa

Illinois, Iowa, Minnesota, Missouri, North Dakota, Wisconsin

Unknown

9/86 12/86 12/86 1/87

Wisconsin New York West Virginia Iowa

Wisconsin New York West Virginia, Ohio Arkansas, Delaware, Illinois, Iowa, Kansas, Maryland, Minnesota, Missouri, Nebraska, Oklahoma, Pennsylvania, South Dakota, Wisconsin New York, Pennsylvania California, Oregon

Unknown ∼835 gal 450 gal ∼1 million gal

∼316 gal ∼60,000 gal

29,32 22,42

Connecticut, Florida, Maryland, New Jersey, New York, North Carolina, Ohio, Pennsylvania, Virginia Colorado, Iowa, Kansas, Nebraska, North Dakota, South Dakota, Wyoming

20,400 boxes

23,30

Ice cream Ice cream (48 flavors), ice milk (6 flavors), sherbet (5 flavors) Ice cream nuggets

4/87 7/87

New York California

7/87

Maryland

Ice cream, ice milk

7/87

Nebraska

∼30,000 gal

11,13 13,14

7 10 12,15

8

9 27,32 17,28 17,21,30, 32

19

(continued)

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TABLE 11.5 (CONTINUED) Chronological List of Voluntary Class I Recalls Issued in the United States from 1986 to 2005 for Milk and Unfermented Dairy Products Contaminated with L. monocytogenes Type of Dairy Product

Month/Year State of of Recall Manufacture

Ice cream sundae cones

8/87

Florida

Chocolate ice cream Ice cream nuggets Ice cream novelties—bars, sandwiches, pieces, slices, sundae cones Ice cream bars

8/87 9/87 9/87

Kentucky Maryland Ohio

10/87

Ohio

Chocolate ice cream Ice cream, sherbet, ice Ice cream

2/88 7/88

Georgia Ohio

8/88

Connecticut

Ice cream Ice cream pies

8/88 9/88

Connecticut Connecticut

Ice Ice Ice Ice Ice

cream cream bars cream cream bars cream bars

9/88 12/88 2/89 11/89 2/90

Pennsylvania New York Connecticut Wisconsin New Mexico

Sherbet, ice milk, ice cream Ice cream Ice cream novelties, frozen novelties Ice cream, ice milk Butter

6/90

Ohio

8/90 5/91

Ohio Tennessee

6/91 6/91

Butter

6/91

Butter, butterine

3/92

Illinois North Carolina, Tennessee North Carolina, Tennessee Wisconsin

Ice milk, ice cream

10/92

Wisconsin

Distribution Alabama, Arizona, British West Indies, Florida, Louisiana, Mississippi, North Carolina, Ohio, Puerto Rico, South Carolina, Tennessee, Virginia, West Virginia Florida, Puerto Rico Nationwide Nationwide

Michigan, Ohio, Pennsylvania, West Virginia Georgia, North Carolina Ohio Connecticut, New York, Massachusetts Connecticut Connecticut, New York, Massachusetts Pennsylvania New York Connecticut Wisconsin Alabama, Illinois, Pennsylvania, Texas Indiana, Kentucky, Michigan, Ohio Indiana, Kentucky, Ohio Alabama, Georgia, Kentucky, Tennessee, Virginia, West Virginia Illinois, Indiana, Wisconsin Pennsylvania

Pennsylvania

Quantity

Reference

Unknown

24

∼956 gal Unknown Unknown

16 25 26

51,780 bars

20

Unknown >1083 gal

34,37 33

5.6–8.4 gal

39

30 gal 1700 pies

38 35

215 gal ∼128 gal Unknown ∼365 gal ∼1000 gal

40 43 44 46 48

Unknown

50

∼778 gal Unknown

49 47

>2275 gal ∼18,165 lb

53 52

∼18,428 lb

52

Arizona, California, Florida, Unknown Illinois, Maryland, Massachusetts, Michigan, Minnesota, Mississippi, Missouri, New Jersey, Ohio, Wisconsin Wisconsin ∼1125 gal

54

55 (continued)

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TABLE 11.5 (CONTINUED) Chronological List of Voluntary Class I Recalls Issued in the United States from 1986 to 2005 for Milk and Unfermented Dairy Products Contaminated with L. monocytogenes Type of Dairy Product

Month/Year State of of Recall Manufacture

Ice cream bars Chocolate milk Whipping cream Butter

6/93 7/94 8/94 8/94

Ohio Wisconsin Wisconsin California

Butter Ice cream bars Ice cream novelties

9/94 9/94 10/95

Wisconsin Wisconsin Ohio

Ice cream, frozen yogurt, sherbet, sorbet, ice cream mix Ice cream

10/95

Ohio

12/95

California

Ice cream Frozen yogurt

12/95 12/95

Michigan Ohio

Ice cream, sherbet Chocolate ice cream Frozen strawberry yogurt Ice cream bars

1/96 7/97 7/97

Ohio Michigan Pennsylvania

10/97

Texas

Ice cream sandwiches Ice cream

1/98

California

3/98

North Carolina

Heavy whipping cream, half-andhalf, milk, chocolate milk Chocolate ice cream Cream flan dessert

1/99

Minnesota

2/99 4/01

Ohio Minnesota

Butter

2/04

Wisconsin

Distribution Ohio, Kentucky, New England Michigan, Wisconsin Michigan, Wisconsin Maine, Michigan, New Hampshire, Wisconsin Pennsylvania, Wisconsin Michigan, Wisconsin Illinois, Indiana, Iowa, Kentucky, Maryland, Michigan, Ohio, North Carolina, Pennsylvania Georgia, Indiana, Kentucky, Michigan, North Carolina, Ohio, Pennsylvania, South Carolina, Tennessee, Virginia Arizona, Hawaii, California, New Mexico, Nevada Michigan Connecticut, Delaware, Florida, Maine, Maryland, Massachusetts, Michigan, New Hampshire, New Jersey, New York, Ohio, Pennsylvania, Rhode Island, Vermont, Virginia Ohio Michigan Pennsylvania

Quantity 34,752 bars Unknown Unknown 36 lb

56 168 168 57

Unknown 167 gal Large but unknown

168 58 59

∼420,000 gal

62

>500 gal

63

Unknown ∼12,381 gal

63 60

2,864 gal Unknown ∼42 gal

61 64 65

Arkansas, California, Florida, 29,814 cases Georgia, Illinois, Kansas, Maryland, New York, Ohio, Oregon, Texas, Washington State California Unknown Delaware, Florida, Georgia, Kentucky, Maryland, North Carolina, Pennsylvania, South Carolina, Tennessee, Virginia, West Virginia Nationwide

Ohio Florida, Illinois, Kentucky, Michigan, Minnesota, New York, Oklahoma, Pennsylvania, Washington State Nationwide

Reference

66

67

130,000 gal

68

500,000 gal

122

244 gal 375 lb

122 122

4,000,000 lb

122

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Despite millions of gallons of frozen dairy products that have been recalled both formally and internally, it must be stressed that only one case of listeriosis has been positively linked to consumption of a contaminated frozen dairy product—in Belgium [6] (see Chapter 10). On this basis, the International Ice Cream Association and the Milk Industry Foundation contended that a Class I recall is too harsh a response for a frozen dairy product containing presumably very low levels of L. monocytogenes. The US government has developed one of the most stringent policies regarding presence of L. monocytogenes in ready-to-eat foods, whereas most other countries have adopted more relaxed policies (i.e., not >100 or 1000 CFU/g or mL), particularly for products in which Listeria is unable to grow. Until recently, the Canadian government confined its recalls to only those foods that have been linked to major outbreaks of listeriosis, with the role of pasteurized milk in foodborne listeriosis still being highly debated [157]. Hence, no recalls were issued when investigators at the Health Protection Branch of Health and Welfare Canada (analogous to the U.S. FDA) identified L. monocytogenes in 1 of 394 (0.25%) and 1 of 51 (2.0%) samples of ice cream and ice cream novelties, respectively [111], during their own federal inspection program. Although subsequent investigations were presumably conducted to identify (1) the source of contamination, (2) proper corrective measures, and (3) possible links to human illness, Canadian officials maintained that recalling the two contaminated lots would be inappropriate without proof that consumption of Listeria-contaminated ice cream could lead to listeriosis. Many individuals and most manufacturers have argued in favor of the more relaxed Canadian position. When one considers the numerous recalls of Listeria-contaminated ice cream in the United States, the fact that worldwide only one case of listeriosis has been positively linked to ice cream containing unusually high numbers of listeriae, the inability of L. monocytogenes to grow in this product during frozen storage, and the normal exposure rate of the human population to listeriae, it is clear that the risk of contracting listeriosis from contaminated ice cream is extremely low, as will be discussed later in regard to several risk assessments. Although current regulations mandate immediate removal of fluid dairy products and cheeses that support growth of L. monocytogenes, a scientifically valid argument can now be made against recalling certain dairy products in which listeriae will not proliferate, such as ice cream and dried goods which, if contaminated, typically contain very low numbers of listeriae as postpasteurization contaminants. As a result of several large recalls of French Brie cheese and a listeriosis outbreak in Switzerland that was traced to consumption of Vacherin Mont d’Or soft-ripened cheese, European scientists have logically focused their attention on the incidence of listeriae in soft cheese. However, numerous recalls of unfermented dairy products in the United States also have heightened public health concerns about the presence of listeriae in pasteurized dairy products manufactured outside North America [41]. In one of the first European surveys of finished products reported in 1988, researchers in Germany [232] failed to isolate Listeria spp. from pasteurized milk (39 samples), nonfat dry milk (11 samples), casein/caseinate (30 samples), and various dried products, including baby food (Table 10.6). During the same year, investigators in Hungary [115] and The Netherlands [79] also failed to recover L. monocytogenes from samples of pasteurized milk, with similar negative findings being obtained from most other subsequent surveys of pasteurized milk and cream produced elsewhere (Table 11.6). However, L. monocytogenes was eventually demonstrated in 11 of 1039 (1.1%), 4 of 115 (3.5%), and 1 of 95 (1.1%) pasteurized milk samples examined in the United Kingdom [138,145,215] for a combined contamination rate of 1.3%, these findings generally being similar to those observed in the United States. According to Garayzabal et al. [131], 21.4, 89.2, 10.7, and 3.6% of pasteurized milk samples from one particular milk processing facility in Madrid contained L. monocytogenes, L. grayi, L. innocua, and L. welshimeri, respectively. These authors [132,209] previously reported similar Listeria contamination rates for raw milk entering the same processing facility. Further, after pasteurization these same samples had a total mesophilic aerobic plate count of 2.5 × 107 CFU/mL, which is well above the maximum allowable limit of 1 × 104 CFU/mL for properly pasteurized milk in the United States. Hence, improper pasteurization caused

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TABLE 11.6 Incidence of Listeria spp. in Pasteurized Dairy Products Produced outside the United States and Canada Number of Positive Samples (%) Product

Country of Origin

Milk

Australia Brazil Czechoslovakia Germany Hungary Italy Korea

Chocolate milk Flavored milk Ice cream

Cream

Butter Nonfat dry milk Casein/caseinate Dry infant formula

Morocco The Netherlands Poland Portugal Turkey United Arab Emirates United Kingdom England/Wales Scotland Northern Ireland Hungary Australia Australia Chile Costa Rica England/Wales Korea Turkey Australia England/Wales Hungary Morocco Hungary Italy Germany Germany Germany

Note: ND = Not determined. a

Seven non-L. monocytogenes isolates. One non-L. monocytogenes.

b

Number of Samples

L. monocytogenes

L. innocua

L. welshimeri

Other

Reference

77 33 220 20 30 15 39 100 50 348 26 50 20 41 73 28 22 182

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 ND 2(0.9) 0 ND ND 0 ND 0 0 ND 0 ND 0 0 ND ND 0

0 ND 0 0 ND ND 0 ND 0 0 ND 0 ND 0 0 ND ND 0

0 ND 0 0 ND ND 0 ND 0 0 ND 0 ND 0 7a ND ND 0

144 72 185 83 181 182 232 155 115 136 74 234 109 79 211 179 220 141

ND ND ND 0 ND ND ND ND ND ND 6 (12.0) 0 ND ND ND 0 ND 0 0 0

ND ND ND 0 ND ND ND ND ND ND 0 0 ND ND ND 0 ND 0 0 0

ND ND ND 0 ND ND ND ND ND ND 0 0 ND ND ND 1b ND 0 0 0

145 215 138 155 239 72 90 184 137 74 86 144 145 155 109 155 202 232 232 232

1039 115 95 60 206 166 603 50 40 132 50 12 40 15 20 15 130 11 30 120

11 4 1 7 1 23 21 1 8 5

1 5

(1.1) (3.5) (1.1) (11.6) (0.5) (13.9) (3.5) (2.0) 0 (6.1) (10.0) 0 0 0 0 (6.7) (3.8) 0 0 0

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by leaking pasteurizer plates, as suggested by Northolt et al. [187], or postpasteurization contamination from the factory environment appear to be most likely responsible for the unusually high incidence of listeriae in “pasteurized” milk samples from this particular dairy factory. Although results from these aforementioned surveys of pasteurized milk, cream, and dried products are very encouraging, the isolation methods used in these studies were generally unable to detect sublethally injured listeriae. Hence, the true incidence of listeriae in pasteurized milk, cream, and dried products may well be somewhat higher. To enhance recovery of injured cells, the International Dairy Federation has recommended that such dairy products undergo preenrichment in a nonselective medium (i.e., buffered peptone water) before primary enrichment in various selective broths and plating on Listeria-selective media [39,233]. Further details concerning recovery of sublethally injured listeriae can be found in Chapter 7. Results from a 1989 International Dairy Federation survey [157] indicated that public health issues regarding the presence of listeriae in pasteurized milk were clearly spreading beyond the continental boundaries of Europe and North America, with the many aforementioned surveys from Table 10.6 attesting to these concerns. More recently, the safety of several additional dairy products, including flavored milks, chocolate milk, ice cream, and particularly butter, has attracted international attention with the FDA Initiatives Program, the many Class I recalls of Listeria-contaminated dairy products, and fears of international trade embargoes fueling these concerns. Following the 1987 discovery of L. monocytogenes in Australian ricotta cheese, New Zealand and Australian officials instituted Listeria-monitoring programs for casein/caseinate products as well as high-moisture cheese, pasteurized milk, ice cream, and milk powders. Results from one 10-month survey begun in April 1988 [239] revealed the presence of L. monocytogenes in 1 of 206 (0.48%) samples of pasteurized flavored/unflavored milk processed in and around Melbourne. Subsequent identification of heat-labile alkaline phosphatase in the contaminated product (pasteurized milk to which a pasteurized flavored syrup was added) suggested that improper pasteurization was most likely responsible for the presence of L. monocytogenes in the final product. However, unsatisfactory storage of the flavored syrup also may have contributed to contamination. In keeping with Listeria policies developed in the United States and Canada, Australian officials withdrew the affected product from the marketplace and prohibited the sale of all subsequently produced product until 12 consecutive lots of Listeria-free pasteurized flavored milk could be produced from the same product line. As in the United States, recent foreign surveys also have shown a higher incidence of L. monocytogenes in chocolate milk (11.6%), ice cream (2.0–13.9%), and butter (3.8–6.7%) as compared to pasteurized milk and dried products that are seldom contaminated (Table 11.6). The increased incidence of listeriae in ice cream and butter is clearly the result of postpasteurization contamination during handling and packaging, as evidenced by the highest contamination rates in ice cream bars and novelties. The fact that Listeria spp. are more commonly found in chocolate milk, as opposed to unflavored milk, is also not surprising given that the added ingredients can serve as another source of listeriae.

BEHAVIOR OF L. MONOCYTOGENES IN UNFERMENTED DAIRY PRODUCTS Although the psychrotrophic nature of L. monocytogenes and the ability of both normal and diseased animals to shed this pathogen in their milk have been recognized for many years, behavior of L. monocytogenes in raw milk and unfermented dairy products did not receive serious attention until 1983 when an outbreak of “milkborne” listeriosis was reported in Massachusetts. Research efforts prompted by this and two other dairy-related outbreaks in the United States and Switzerland have given us an understanding of the behavior of L. monocytogenes in raw and pasteurized milk as well as in chocolate milk, cream, nonfat dry milk, and butter. The remainder of this chapter will describe results from these studies along with information concerning behavior of this organism in ultrafiltered milk and ice cream mix.

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RAW MILK Despite longstanding recognition of L. monocytogenes as a raw milk contaminant, relatively few studies assessing the behavior of this organism in raw milk can be found in the literature. In 1958, Dedie [95] found that L. monocytogenes survived 210 days in naturally contaminated raw milk stored in an ice chest. Thirteen years later, Dijkstra [98] reported results from a much longer storage study in which 36 samples of naturally contaminated raw milk (obtained from cows that experienced Listeria-related abortions) were held at 5°C and examined for viable L. monocytogenes over a period of 9 years. Although 4 of 36 (11%) samples were free of L. monocytogenes within 6 months, the pathogen was still detected in 16 of 36 (44%) samples following 2 years of refrigerated storage. The number of samples from which listeriae could be isolated continued to decrease, with 9 of 36 (25%) samples being positive after 4 years of storage. However, the pathogen was still present in 4 of 36 (11%) raw milk samples after 8–9 years of storage. These early findings emphasize the importance of establishing proper cleaning and sanitizing programs for all phases of milk production. If routinely used, such programs will likely prevent this organism from finding an appropriate niche within the farm or dairy factory environment and greatly reduce the threat of this pathogen surviving long term. The studies just described adequately demonstrate that L. monocytogenes can persist in raw milk for long periods; however, until several outbreaks of milkborne and cheeseborne listeriosis were reported in the 1980s, little attention had been given to the potential for growth of L. monocytogenes in raw milk. In 1988, Northolt et al. [187] examined the behavior of listeriae in samples of freshly drawn raw milk that were inoculated to contain approximately 500 L. monocytogenes CFU/mL and incubated at 4 and 7°C. As shown in Figure 11.4, Listeria populations decreased approximately 4- and 8.5-fold in raw milk during the first 2 days of incubation at 4 and 7°C, respectively. These authors suggested that naturally occurring antibacterial substances in raw milk (i.e., lactoperoxidase and lysozyme) may have partially inhibited growth of listeriae during the first 2 days of incubation as has been more recently confirmed by two other investigators [199,245]. However, in a Canadian study that will be discussed shortly [113], no such decrease was observed when incubated samples of naturally contaminated raw milk were surface-plated on FDA Modified McBride Listeria Agar. Hence, a more likely explanation is that the plating medium—Trypaflavine Nalidixic Acid Serum Agar—used by Northolt et al. [187] was less than ideal for recovering listeriae, as also was observed during concurrent work with pasteurized milk. Although L. monocytogenes failed to grow in raw milk samples incubated at 4°C for up to 7 days, Listeria populations increased approximately 10-fold during this period when the incubation temperature was raised to 7°C. Following 3 days of incubation at 4 and 7°C, Listeria populations began doubling every 3.5 and 1.0 days, respectively. Two years later, Wenzel and Marth [242] reported that populations of L. monocytogenes strain V7 remained constant in inoculated raw milk during 5 days of storage at 4 and 7°C, with numbers of listeriae also being unaffected by the presence of a commercial raw milk lactic acid bacteria inoculant designed to suppress growth of primarily Gram-negative psychrotrophic bacteria. L. monocytogenes failed to grow during 3–5 days of incubation at 7°C. It appears that the 3-day period during which raw milk is sometimes held in farm bulk tanks is insufficient to allow growth of the organism. However, the temperature of raw milk in farm bulk tanks fluctuates every time freshly drawn raw milk at 37°C is commingled with bulk tank milk at 4°C from previous milkings. In 1985, Oz and Farnsworth [190] found that raw milk in farm bulk tanks attained temperatures of 30–31°C, 10–14°C, 12°C, and 9°C when freshly drawn raw milk was added after the first, second, third, and fourth milking periods, respectively. Moreover, 6 h were generally needed for the milk to cool to 4°C after each milking period. In view of these findings, it appears that temperatures obtained after adding warm milk to farm bulk tanks may be sufficient to allow at least limited growth of L. monocytogenes, particularly when raw milk from early milkings enters the bulk tank. Although the temperature of bulk tank milk will eventually decrease to 4°C, exposure

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104

L. monocytogenes CFU/mL

7°C

103

4°C

102

Raw Milk 0

0

2

4

6

Days

FIGURE 11.4 Growth of L. monocytogenes strains in raw milk incubated at 4 and 7°C (enumerated on Trypaflavine Nalidixic Acid Serum Agar). (Adapted from Northolt, M.D., H.J. Beckers, U. Vecht, L. Toepoel, P.S.S. Soentoro, and H.J. Wisselink. 1988. Listeria monocytogenes: heat resistance and behavior during storage of milk and whey and making of Dutch types of cheese. Neth. Milk Dairy J. 42: 207–219.)

to temperatures as high as 9°C when raw milk is trucked to processing facilities during summer [114] also may lead to some multiplication of the pathogen. Discovery of a naturally infected cow in Canada that shed freely suspended and phagocytized cells of L. monocytogenes in milk (maximum of 104 CFU/mL in milk from one of four quarters of the mammary gland) continuously for nearly 3 years provided Farber et al. [113] with a unique opportunity to study growth of L. monocytogenes in naturally rather than artificially contaminated raw milk during extended storage. When raw milk from this cow was analyzed for numbers of L. monocytogenes, no appreciable growth of the pathogen was observed during the first 3 days and 1 day of incubation at 4 and 10°C, respectively (Figure 11.5). The delay in onset of growth was less than 1 day at 15°C. Immunological staining of milk smears indicated that some multiplication of L. monocytogenes had occurred within macrophages after 1 and 2 days of incubation at 15 and 10°C, respectively, with 10–50% of the macrophages containing 1–20 intracellular listeriae. Nonetheless, as previously noted by Doyle et al. [105], rapid deterioration of macrophages shortly thereafter was followed by appearance of freely suspended listeriae in milk with few intact macrophages remaining after 5 days, regardless of incubation temperature. Following the lag phase, L. monocytogenes entered a period of logarithmic growth, with generation or doubling times of 25.3, 10.8, and 7.4 h being calculated for raw milk samples held at 4, 10, and 15°C, respectively.

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7.0

10

L. monocytogenes log CFU/mL

Incidence and Behavior of Listeria monocytogenes in Unfermented Dairy Products

6.0

4°C 10°C 15°C

5.0

4.0 0

2

4

6

8

10

12

14

FIGURE 11.5 Growth of L. monocytogenes in naturally contaminated raw milk during incubation at 4, 10, and 15°C. (Adapted from Farber, J.M., G.W. Sanders, and J.I. Speirs. 1990. Growth of Listeria monocytogenes in naturally-contaminated raw milk. Lebensm. Wiss. Technol. 23: 252–254.)

Although maximum L. monocytogenes populations were approximately 2 × 107 CFU/mL after 10, 7, and 3 days of incubation at 4, 10, and 15°C, respectively, the highest achievable population in raw milk was independent of incubation temperature (Figure 11.6). As in the previous study by Northolt et al. [187], these findings again stress the importance of maintaining raw milk at 4°C during storage and transport to milk processing facilities. Despite an increasing market for both ewe’s and goat’s milk and the use of these milk types in a wide range of ethnic and specialty cheeses and, to a lesser extent, yogurt, most work on growth and survival of Listeria in raw milk has been conducted using cow’s milk. Reports of ovine listeriosis in Europe dating back to the 1940s prompted an early study by Ikonomov and Todorov [156] to examine the behavior of L. monocytogenes in raw ewe’s milk inoculated with the pathogen. In their work, L. monocytogenes remained viable for long periods and persisted in the milk even after coagulation at 10 and 20°C. Information on the behavior of L. monocytogenes in goat’s milk is limited to one 2002 report by Leuchner et al. [164a] in which samples of raw and pasteurized goat’s milk were inoculated with a 3-strain “cocktail” of L. monocytogenes at 103 CFU/mL and then stored at 4, 10, and 15°C for 58 days. The pathogen survived at least 58 days when raw goat’s milk was held at 4 and 10°C and up to 44 days when the same milk was stored at 15°C. Populations of Listeria in pasteurized milk increased about 3 logs after 58 days of storage at 4°C and about 4 logs after 58 days at 10 and 15°C. Given these findings and the fact that many varieties of ethnic- and specialty-type cheeses are now being manufactured by many small cheese makers from ewe’s or goat’s milk, the safety of these cheeses is likely to receive increased attention in the future.

PASTEURIZED

AND INTENSIVELY

PASTEURIZED MILK

In addition to defining the growth pattern of L. monocytogenes in artificially contaminated raw milk (Figure 10.4), Northolt et al. [187] also examined behavior of this organism in pasteurized (72°C/15 sec) and intensively pasteurized whole milk (Figure 11.6). Although L. monocytogenes failed to grow in raw milk incubated at 4°C (Figure 11.4), Listeria populations in pasteurized milk increased nearly 10-fold during 7 days of incubation at the same temperature. The organism also grew markedly faster in pasteurized than in raw milk when both products were incubated

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3

7°C

L. monocytogenes CFU/mL

7°C 2

4°C

4°C

1

Intensively Pasteurized Milk

HTST-Pasteurized Milk

0

2

4

6

0

2

4

6

FIGURE 11.6 Growth of L. monocytogenes in high-temperature, short-time (HTST)-pasteurized and intensively pasteurized milk incubated at 4 and 7°C. —: Enumerated from samples at 4°C on Trypaflavine Nalidixic Acid Serum Agar; ---: enumerated on Nutrient Agar. (Adapted from Northolt, M.D., H.J. Beckers, U. Vecht, L. Toepoel, P.S.S. Soentoro, and H.J. Wisselink. 1988. Listeria monocytogenes: heat resistance and behavior during storage of milk and whey and making of Dutch types of cheese. Neth. Milk Dairy J. 42: 207–219.)

at 7°C. In contrast to their data for raw and pasteurized milk, lag times for L. monocytogenes were reduced considerably when the organism was grown in intensively pasteurized milk incubated at 4 and 7°C. Further, numbers of listeriae in intensively pasteurized milk increased approximately 100-fold following 3 and 6 days of incubation at 7 and 4°C, respectively. When L. monocytogenes was later grown in ultrahigh temperature (UHT) sterilized milk, Rajikowski et al. [204] reported generation times of 4.7, 1.7, 1.0, and 0.9 h for samples incubated at 12, 19, 28, and 37°C, respectively. Hence, these findings suggest that the growth rate for L. monocytogenes in milk is directly related to the degree of heat applied to milk, as was also reported by Mathew et al. [176]. Further work is needed to define more clearly the effect of competing microorganisms on growth of listeriae in raw and pasteurized milk as compared to intensively pasteurized and UHT-sterilized milk, with biochemical changes that occur in milk during thermal processing (i.e., protein denaturation, enzyme inactivation, carmelization) also likely influencing listeriae growth in these products. However, the aforementioned studies all indicate the potential for L. monocytogenes to reach potentially hazardous levels in pasteurized milk during the now normal 2-week refrigerated shelf life, with at least one risk assessment to be discussed in Chapter 18 also suggesting that consumption of pasteurized milk is likely responsible for the largest percentage of listeriosis cases.

AUTOCLAVED MILK, CREAM,

AND

CHOCOLATE MILK

Except for the three studies just mentioned [113,187,176] and an initial attempt by Pine et al. [198] to quantify growth of L. monocytogenes in inoculated samples of pasteurized milk, all remaining

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9

Strain CA log10 CFU/mL

8

7

6 Skim Milk 5

Whole Milk Chocolate Milk

4

Cream 3

2 0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

Days

FIGURE 11.7 Growth of L. monocytogenes strain California in fluid dairy products at 4°C. (Adapted from Rosenow, E.M. and E.H. Marth. 1987. Growth of Listeria monocytogenes in skim, whole and chocolate milk, and in whipping cream during incubation at 4, 8, 13, 21, and 35°C. J. Food Prot. 50: 452–459.)

work dealing with behavior of Listeria in fluid dairy products has been done using autoclaved samples. Although using such sterile products as growth media for listeriae offers several major advantages, including the ability to accurately quantify both stressed and unstressed listeriae on nonselective plating media in the absence of other microbial competitors, readers should keep in mind that growth rates of L. monocytogenes are somewhat faster in autoclaved than in pasteurized, and especially in raw milk, products. Nevertheless, L. monocytogenes clearly can grow to dangerously high levels in all three types of milk during extended refrigeration. In 1987, Rosenow and Marth [213] published results from a definitive study in which autoclaved (121°C/15 min) samples of whole, skim, and chocolate milk, as well as whipping cream, were each inoculated separately with four strains of L. monocytogenes (Scott A, V7, V37CE, or California), incubated at 4, 8, 13, 21, or 35°C, and examined for numbers of listeriae at suitable intervals by surface-plating appropriate dilutions on Tryptose Agar. Growth rates for L. monocytogenes were generally similar in all four products at a given temperature and increased with an increase in incubation temperature. At 4°C, listeriae grew after an initial delay of approximately 5–10 days depending on the bacterial strain and type of product (see Figure 11.7). All four strains generally attained maximum populations of 107 CFU/mL after 30–40 days of incubation, with little change in numbers occurring after 30–40 days of additional storage. Overall, chocolate milk supported development of the highest Listeria populations followed by skim milk, whole milk, and whipping cream. Generation times at 4°C ranged between 28.16 and 45.55 h with average generation times for L. monocytogenes in all four products shown in Table 11.7. Although these results clearly demonstrate the ability of L. monocytogenes to reach potentially hazardous levels in fluid dairy products held at 4°C, more recent data suggest that slow growth of this organism can even occur in milk held at 0°C. Thus, the only way to avoid a public health problem with fluid dairy products is to prevent L. monocytogenes from entering such products before, during, and after manufacture. Increasing the incubation temperature from 4 to 8°C decreased the lag period to 1.5–2 days (Figure 11.8) and nearly tripled the growth rate for L. monocytogenes in all four products (Table 11.7) [213,214]. After 10–14 days of incubation, the growth curves at 4 and 8°C were similar,

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TABLE 11.7 Generation Times for L. monocytogenes in Autoclaved Samples of Various Dairy Products Generation Time (h) at 4°Ca 33.27 34.52 33.46 36.30

Product Whole milk Skim milk Chocolate milk Whipping cream

8°Ca 13.06 12.49 10.56 11.93

13°Ca 5.82 6.03 5.16 5.56

21°Cb 1.86 1.92 1.72 1.80

35°Cb 0.692 0.693 0.678 0.683

a

Average generation times for four strains of L. monocytogenes. Strain V7 only.

b

Source: Adapted from Meyer-Broseta, S., A. Diot, S. Bastian, J. Riviere, and O. Cerf. 2003. Estimation of low bacterial concentration: Listeria monocytogenes in raw milk. Int. J. Food Microbiol. 80: 1–15.

with highest Listeria populations again being found in chocolate milk. Theoretical calculations based on these data indicate that Listeria populations could increase from 10 to 4.2 × 106 organisms/qt (947 mL) of milk during 10 days of storage at 8°C (46°F), a temperature that commonly occurs in some home and commercial refrigerators. These findings, which have since been confirmed by Siswanto and Richard [221] using skim milk, raise additional safety concerns about reclaiming and reprocessing returned products that have likely undergone some degree of temperature abuse. As is true for 8°C, 13°C (55°F) also represents a temperature that dairy products occasionally encounter during transportation and storage. Following a 12-h lag period, all four Listeria strains grew nearly twice as fast at 13°C as at 8°C (see Table 11.7) and generally attained levels of

9

.

Strain CA log10 CFU/mL

8.

7.

6. Skim Milk 5.

Whole Milk Chocolate Milk

4.

Cream 3.

2. 0

2

4

6

8

10

12

14

16

18

20

22

Days

FIGURE 11.8 Growth of L. monocytogenes strain California in fluid dairy products at 8°C. (Adapted from Rosso, L., S. Bajard, J.P. Flandrois, C. Lahellec, J. Fournaud, and P. Veit. 1996. Differential growth of Listeria monocytogenes at 4 and 8°C: Consequences for the shelf life of chilled products. J. Food Prot. 59: 944–949.)

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106 CFU/mL in all four products by the third day [213]. These generation times are somewhat longer than those observed by Farber et al. [113] when naturally contaminated raw milk was incubated at 4 (25.3 h), 10 (10.8 h), and 15°C (7.4 h). L. monocytogenes also attained maximum populations that were approximately 10-fold lower in raw than in sterile milk, which in turn suggests possible depletion of essential nutrients by raw milk contaminants or production of substances inhibitory to growth of the pathogen. Maximum Listeria populations of 109 CFU/mL were again observed in chocolate milk, with numbers generally being 10-fold lower in skim milk, whole milk, and whipping cream [213]. Increasing the incubation temperature to 21°C doubled the growth rate (see Table 10.7) and led to maximum Listeria populations of 108–109 CFU/mL within 48 h. As expected, L. monocytogenes grew most rapidly at 35°C, with populations of 108–109 CFU/mL being observed after only 24 h of incubation. In another study examining the influence of temperature and milk composition on growth of listeriae, Donnelly and Briggs [100] found that five L. monocytogenes strains began growing in inoculated samples of autoclaved (121°C/10 min) whole, skim, and reconstituted nonfat dry milk (11% total solids) after approximately 24–48, 2–24, 4–12, and 0.5–4.0 h of incubation at 4, 10, 22, and 37°C, respectively. Although growth rates for all Listeria strains were primarily determined by the incubation temperature, two strains of L. monocytogenes serotype 4b grew considerably faster in whole rather than skim or reconstituted nonfat dry milk during incubation at 4 and 10°C. These observations led Donnelly and Briggs [100] to suggest a possible relationship between levels of milk fat and growth rate of L. monocytogenes in milk during refrigerated storage. Furthermore, these authors suggested that enhanced psychrotrophic growth in whole milk may be related to a listerial lipase produced by both hemolytic strains of L. monocytogenes serotype 4b. Unlike both of these strains, the three remaining L. monocytogenes strains of serotypes 1 and 3 failed to exhibit enhanced growth in whole milk at 10°C and had little if any hemolytic activity on McBride Listeria Agar containing sheep blood. In contrast to what might be expected from the study just described, Rosenow and Marth [213] failed to observe any significant difference in growth rates among four strains of L. monocytogenes (two serotype 4b, two serotype 1) when they were incubated in autoclaved samples of whole and skim milk at 4, 8, 13, 21, and 35°C. The pathogen also attained lower maximum populations in whipping cream than in whole, skim, or chocolate milk at all incubation temperatures. In support of these findings, Marshall and Schmidt [174] failed to observe enhanced growth of L. monocytogenes strain Scott A (serotype 4b) in whole rather than skim milk during 8 days of incubation at 10°C. Finally, in a study to be discussed in greater detail in Chapter 12 [218], four strains of L. monocytogenes (three serotype 4b and one serotype 1) frequently attained higher maximum populations in whey samples that were defatted by centrifugation, filter sterilized, and incubated at 6°C than would be expected to occur in autoclaved skim milk, whole milk, or whipping cream after prolonged incubation at 8°C. Thus, although some L. monocytogenes strains are lipolytic as reported by Marshall and Schmidt [174], one must presently conclude that psychrotrophic growth of L. monocytogenes is not generally enhanced by the normal level of milk fat found in fluid milk. Recognizing the vital importance of carbohydrates in microbial metabolism, researchers at the CDC [198] attempted to define growth of Listeria spp. in terms of sugar utilization. An initial experiment using aerobically incubated broth media indicated that five strains of L. monocytogenes and one strain each of L. innocua, L. seeligeri, and L. ivanovii utilized only the glucose moiety of lactose, whereas single strains of L. grayi and L. murrayi utilized both the glucose and galactose of lactose. Overall, maximum cell populations, as determined by optical density, were directly proportional to the concentration of glucose (0.125%) in the growth medium. However, marked differences were observed in the ability of L. monocytogenes and L. innocua to utilize lactose, with three strains of L. monocytogenes (isolated from Mexican-style cheese in connection with the 1985 listeriosis outbreak in California) unable to grow in a medium containing lactose as the only carbohydrate. Although these observations agree with several reports [117,173,174] indicating that the pH of fluid milk is unaffected by L. monocytogenes growth, Quinto et al. [203] did report a

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sharp pH decrease in such milk after 16 and 24 days of incubation at 14 and 7°C, respectively, these differences being most likely related to strain variation. Growth of L. monocytogenes in autoclaved samples of whole and skim milk was generally similar to that previously observed by Rosenow and Marth [213], with maximum populations of 5 × 108 CFU/mL developing after extended incubation at 5 and 25°C. Except for L. seeligeri, the behavior of L. innocua and L. ivanovii did not differ markedly from that of L. monocytogenes in these samples (Figure 11.9). However, as noted by Northolt et al. [187], higher maximum populations and increased survival rates were again observed when these organisms were grown in autoclaved rather than pasteurized whole milk. Examination of milk by gas-liquid chromatography indicated that lactic, acetic, isobutyric, isovaleric, and 2-hydroxy isocaproic acids were formed during incubation. Because this milk initially contained 81–85 mg of glucose/L, the aforementioned acids likely resulted, at least in part, from fermentation of glucose. Considerably lower populations of L. monocytogenes as well as L. innocua, L. grayi, and L. murrayi also developed in glucoseoxidase-treated (an enzyme that degrades glucose) rather than untreated milk during both aerobic and anaerobic incubation, and so it is evident that glucose is one of the major substrates for growth of listeriae in milk. However, when incubated anaerobically in glucose-oxidase-treated milk, two lactose-negative L. monocytogenes isolates from Mexican-style cheese still attained final populations of 108 CFU/mL, thus suggesting the involvement of other as yet unidentified growth factors.

L. monocytogenes L. seeligeri L. ivanovil L. innocua

Listeria log10 CFU/mL

9.0

8.0

7.0

6.0 0

4

8 12 16 20 24 Days

FIGURE 11.9 Growth of Listeria spp. in pasteurized (open symbols) and autoclaved whole milk (solid symbols) incubated at 5°C. (Adapted from Pine, L., G. B. Malcolm, J.B. Brooks, and M.I. Daneshvar. 1989. Physiological studies on the growth and utilization of sugars by Listeria species. Can. J. Microbiol. 35: 245–254.)

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10 9

Strain V7 log10 CFU/mL

8 7 6 2% milk (m) 5

2%m+sugar (s)

4

2%m+cocoa(c)+carr. 2%m+c+s+carr.

3 2 0

20

40

60

80

100

120

140

160

180

200

Hours

FIGURE 11.10 Growth of L. monocytogenes strain V7 in 2% fat milk with added sugar, cocoa, and carrageenan (carr.) at 13°C. (Adapted from Rosenow, E.M. and E.H. Marth. 1987. Addition of cocoa powder, cane sugar, and carrageenan to milk enhances growth of Listeria monocytogenes. J. Food Prot. 50: 726–729, 732.)

In the aforementioned study by Rosenow and Marth [213], maximum populations of L. monocytogenes were typically about 10-fold higher in chocolate milk than in other fluid dairy products. To explain the enhanced growth of L. monocytogenes in chocolate milk, several investigators at the University of Wisconsin examined the effect of major chocolate milk constituents (i.e., cocoa, sugar, and carrageenan) on growth of this organism in autoclaved skim milk and laboratory media. Rosenow and Marth [212] found that growth of L. monocytogenes at 13°C was only slightly enhanced in skim milk containing 5% cane sugar, and that the organism attained higher final populations when commercial cocoa power (1.3%) and carrageenan stabilizer (0.5%) were used in place of cane sugar (Figure 11.10). Carrageenan also enhanced the growth rate of L. monocytogenes in the presence of cocoa; however, the organism attained similar maximum populations regardless of the presence or absence of carrageenan. These findings suggest that carrageenan may be more important in increasing contact between cocoa particles and Listeria than as a source of nutrients. Highest final populations and shortest generation times were observed when L. monocytogenes was grown in skim milk containing cocoa, sugar, and carrageenan. In addition, maximum Listeria populations obtained in skim milk containing all three ingredients (see Figure 11.10) were similar to populations observed in initial work with commercially produced chocolate milk (see Figure 11.7 and Figure 11.8). Subsequently, Pearson and Marth [194] examined growth of L. monocytogenes strain V7 at 13°C in skim milk containing various concentrations of cocoa, sugar, and carrageenan. Because some Listeria strains can utilize sucrose, it is not surprising that L. monocytogenes developed significantly higher final populations (see Figure 11.11) and had shorter generation times (5.05 vs. 5.17 h) as the concentration of cane sugar (sucrose) in skim milk was increased from 0 to 12%. (Peters and Liewen [197] also reported that addition of 7% sucrose to ultrafiltered (concentrated) skim milk caused maximum L. monocytogenes populations to increase rather than decrease.) A near-linear relationship between increasing sugar concentration and maximum attainable populations of L. monocytogenes also was observed for all but one combination of sugar, cocoa, and

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8.90

Maximum population (log10 CFU/mL)

8.80 8.70 8.60

8.50 8.40

8.30 0

3

9 6 Cane Sugar (%, W/V)

12

FIGURE 11.11 Maximum L. monocytogenes populations in skim milk alone (
), skim milk + carrageenan (), skim milk + cocoa (), and skim milk + cocoa + carrageenan () with 0, 6.5, and 12.0% cane sugar after 36 h of incubation at 13°C. Any two points differing by 0.07 log10 CFU/mL are significantly different (P < .05). (Adapted from Pearson, L.J. and E.H. Marth. 1990. Behavior of Listeria monocytogenes in the presence of cocoa, carrageenan, and sugar in a milk medium incubated with and without agitation. J. Food Prot. 53: 30–37.)

carrageenan tested; that was 12% sugar and 0.03% carrageenan (see Figure 11.11). Although addition of 0.03% carrageenan significantly lengthened generation times and decreased maximum populations compared to those observed in skim milk without carrageenan, L. monocytogenes achieved highest populations in skim milk containing 0.75% cocoa with or without carrageenan, which in turn indicates that the apparent ability of cocoa to stimulate growth of this organism in skim milk containing 0–12% sugar is independent of carrageenan. Because cocoa contains only trace amounts of fermentable carbohydrates, these authors theorized that cocoa enhanced growth of L. monocytogenes in skim milk by providing increased levels of peptides and amino acids, particularly valine, leucine, and cysteine, which are reportedly essential for growth. Additional work showed that agitation, combined with the presence of cocoa, sugar, and/or carrageenan in skim milk, enhanced growth of the pathogen at 30°C when compared to growth in the same medium that was incubated quiescently. However, growth of Listeria in skim milk alone was better without rather than with agitation. Thus, agitation most likely increased the availability of extractable nutrients from cocoa, which in turn led to enhanced growth of the pathogen. In 1968, anthocyanins in cocoa were reported to inhibit growth of salmonellae in laboratory media; however, the inhibitory effect of cocoa could be neutralized with casein [82]. These early findings prompted Pearson and Marth [196] to investigate the effect of cocoa with and without casein on growth of L. monocytogenes strain V7. Using Modified Tryptose Phosphate Broth containing 0.2% tryptose, addition of 0.75–10% cocoa increased the generation time for L. monocytogenes at 30°C (1.02–1.12 h) as compared to samples without cocoa (0.94 h). However, the pathogen generally attained higher populations when grown in media with (1.1–1.5 × 109 CFU/mL) rather than without (6.4 × 108 CFU/mL) cocoa. Interestingly, when the same medium was inoculated to contain 105 L. monocytogenes CFU/mL and agitated, the pathogen decreased to nondetectable levels in samples containing 5–10% cocoa after 15–24 h of incubation at 30°C. Nevertheless, the

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organism readily grew in the presence of 0.75% cocoa and attained higher maximum populations in media with (1.9 × 109 CFU/mL) rather than without (7.6 × 108 CFU/mL) cocoa during agitated incubation at 30°C. As previously reported for salmonellae, the presence of 1.5 or 3.0% casein neutralized the inhibitory effect of cocoa toward L. monocytogenes, the pathogen exhibiting shorter lag phases and higher maximum populations in media containing both casein and 5.0% cocoa rather than cocoa alone, and incubated quiescently at 30°C. However, results obtained during agitated incubation of cultures containing 5% cocoa were far more dramatic, with L. monocytogenes populations of 2.9 × 109 rather than <10 CFU/mL developing in samples with rather than without 2.5% casein. Hence, these findings suggest that the behavior of L. monocytogenes in laboratory media containing cocoa partially depends on the concentration of one or more inhibitory substances that can be neutralized by casein and that are more readily extracted during agitated rather than quiescent incubation. Next, Pearson and Marth [195] determined if theobromine and caffeine (i.e., two methylxanthine compounds in cocoa that reportedly possess different degrees of antimicrobial activity) were responsible for the previously observed antilisterial activity of cocoa. Overall, addition of 2.5% theobromine to both Modified Tryptose Phosphate Broth and autoclaved skim milk with and without 0.5% caffeine did not markedly influence the behavior of L. monocytogenes during incubation at 30°C. This suggests that theobromine is not responsible for suppressing or enhancing growth of listeriae in chocolate milk. Unlike theobromine, addition of 0.5% caffeine to Modified Tryptose Phosphate Broth doubled or tripled the length of the organism’s lag phase, nearly doubled the organism’s generation time, and led to maximum Listeria populations approximately 10-fold lower than those obtained in caffeine-free media. Similar trends also were observed when autoclaved skim milk instead of Modified Tryptose Phosphate Broth served as the growth medium. Thus, although caffeine in cocoa may contribute to inhibition of L. monocytogenes in a broth medium, failure of casein in skim milk to neutralize the inhibitory effect of cocoa indicates that caffeine also is not responsible for inhibition of listeriae as observed in the previous study. Such efforts to identify Listeria-active components within cocoa should be continued to better understand the behavior of this pathogen in chocolate milk.

SWEETENED CONDENSED

AND

EVAPORATED MILK

Thus far Listeria spp. have not yet been isolated from commercially produced sweetened condensed milk (i.e., a nonsterile concentrated fluid milk product containing approximately 64% sucrose or glucose in the water phase, 8.5% milk fat, and 28% total milk solids) or evaporated milk (an unsweetened commercially sterile concentrated fluid milk product containing approximately 7.9% milk fat and 25.9% total milk solids). However, given the widespread incidence of Listeria in foodprocessing facilities, it is conceivable that listeriae could enter both of these products as postprocessing contaminants. Such concerns prompted Farrag et al. [118] to examine the fate of three L. monocytogenes strains in samples of commercially produced sweetened condensed and evaporated milk that were inoculated to contain three different levels (103–107 CFU/mL) of the pathogen. Regardless of initial inoculum, Listeria populations in sweetened condensed milk decreased 1.2 and 1.6–3.4 orders of magnitude following 42 days of storage at 7 and 21°C, respectively. This behavior was not surprising because addition of sugar to this product during manufacture reduces its water activity (aw) to 0.83, which is well below the minimum aw value of 0.90 reported for growth of L. monocytogenes. Unlike sweetened condensed milk, the relatively high aw value for evaporated milk (0.986) allowed profuse growth of listeriae, with lowest inoculum levels increasing approximately 4 orders of magnitude after 7 and 14 days of incubation at 21 and 7°C, respectively. In addition, no decrease in numbers of listeriae was noted during continued incubation at either temperature. Thus, because L. monocytogenes can survive >42 days in sweetened condensed milk and grow rapidly in evaporated milk, special precautions should be taken to prevent listeriae from entering these products during packaging, storage, and subsequent use.

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ULTRAFILTERED MILK Ultrafiltration, a mechanical process by which milk is filtered under pressure and concentrated, results in major compositional changes in the finished product when compared to the starting material. During ultrafiltration, 94–100% of the milk proteins and protein-bound vitamins (i.e., vitamin B12 and folic acid) remain in the retentate along with milk fat, whereas lactose is equally divided between the retentate and permeate. Increased use of ultrafiltered milk in cheesemaking prompted El-Gazzar et al. [106] to investigate the growth characteristics of L. monocytogenes in 2X and 4X retentate as well as the corresponding permeate obtained from ultrafiltered pasteurized milk. When samples were inoculated to contain about 104 CFU/mL of L. monocytogenes strain V7 or CA and incubated at 4°C, growth of both organisms was enhanced 10- to 100-fold in retentate as compared to unfiltered skim milk. Increasing the concentration of ultrafiltered skim milk retentate from 2X to 5X also resulted in faster growth, the pathogen attaining a population of 106 CFU/mL in 5X and 2X retentate after approximately 7 and 10–12 days of refrigerated storage, respectively. L. monocytogenes also grew to dangerous levels in permeate with maximum levels of 106 CFU/mL as compared to 108 CFU/mL in retentate following 30 days of incubation. When identical tyndalized samples were incubated at 32 and 40°C, both Listeria strains grew similarly in skim milk and retentate, with populations of 108 CFU/mL generally being reported after 24 h of incubation. However, as was true for samples incubated at 4°C, maximum numbers of listeriae were again 10to 100-fold lower in permeate than in retentate and unfiltered skim milk. Hence, the same care should be given to production of unfiltered milk to prevent contamination and subsequent growth of Listeria in the product during cold storage.

GROWTH OF L. MONOCYTOGENES IN MIXED CULTURES Except for several early works assessing the behavior of L. monocytogenes in raw and pasteurized milk, all studies described thus far have dealt with Listeria growth in the absence of competitive microorganisms. Although results from these studies have been of great value to the dairy industry, one should remember that pasteurized dairy products are not sterile. Psychrotrophic bacteria belonging to the genera Pseudomonas and Flavobacterium are typically present in raw milk and, similar to L. monocytogenes, can grow in milk at refrigeration temperatures both before and after milk is pasteurized. Although readily destroyed during pasteurization, these organisms universally appear in pasteurized dairy products as postpasteurization contaminants, often at levels >100 CFU/mL. Because L. monocytogenes is thought to enter dairy products primarily after pasteurization, products that contain low levels of listeriae (probably <10 CFU/mL) will likely contain higher populations of other psychrotrophs. Although the ability of psychrotrophic pseudomonads to stimulate growth of nonpathogenic as well as pathogenic bacteria in dairy products has been recognized for more than 25 years, data concerning the behavior of L. monocytogenes in mixed cultures are of more recent origin. After initial work [116] with Tryptose Broth demonstrated that growth of L. monocytogenes was slightly inhibited by the presence of Pseudomonas fluorescens, Farrag and Marth [117] examined associative growth of L. monocytogenes (strains Scott A, CA, and V7) with P. fluorescens (strains P26 and B52) in autoclaved (121°C/15 min) skim milk that was inoculated to contain equal populations (105 CFU/mL) of both organisms and incubated at 7 or 13°C for 56 days. Growth of L. monocytogenes was generally enhanced by the presence of P. fluorescens after 7 days of incubation, the pathogen attaining populations of 107 CFU/mL in mixed cultures. However, continued incubation at 7°C led to lower numbers of listeriae in mixed rather than pure cultures, populations of strain V7 being inhibited approximately 8-fold by P. fluorescens B52 following 56 days of storage. Farrag and Marth [117] later showed that inactivation of strain CA was affected by initial levels of P. fluorescens P26, with highest populations being most detrimental to Listeria

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survival. However, populations of L. monocytogenes strains Scott A and V7 remained unaltered in samples that were initially inoculated to contain P. fluorescens P26 at levels of 103–106 CFU/mL. When Farrag and Marth [117] increased the incubation temperature to 13°C, growth of L. monocytogenes was neither enhanced nor inhibited by either Pseudomonas strain during the first 7 days of incubation. However, after 56 days of incubation, final populations of Listeria were as much as 20-fold lower in mixed rather than pure culture. Although these findings and those of Quinto et al. [203] indicate that P. fluorescens was more detrimental to survival of listeriae in skim milk stored at 13 than 7°C, growth and survival of P. fluorescens was not appreciably affected by the presence of L. monocytogenes, with pseudomonads consistently reaching populations of 108–109 CFU/mL. In a similar study, Marshall and Schmidt [173] found that growth of L. monocytogenes strain Scott A (used in the previous study) in autoclaved skim and whole milk was not enhanced by the presence of Pseudomonas fragi during 8 days of incubation at 10°C. Similarly, growth of P. fragi also was unaffected by L. monocytogenes. Some researchers have speculated that psychrotrophic pseudomonads may be able to utilize some of the nutrients in milk faster than L. monocytogenes, thus suppressing growth of listeriae in milk during refrigerated storage. Marshall and Schmidt [173] investigated this theory by inoculating samples of autoclaved whole milk, skim milk, and reconstituted nonfat dry milk (10% solids) with P. fragi or P. fluorescens (strain T25, P26, or B52); incubating the samples for 3 days at 10°C to obtain 106–107 P. fragi CFU/mL or 104–106 P. fluorescens CFU/mL; inoculating these Pseudomonas cultures with L. monocytogenes; and then incubating the samples for an additional 8 days at 10°C. Throughout this study, addition of listeriae to all milks preincubated with P. fluorescens or P. fragi did not significantly affect growth or survival of either pseudomonad. However, as shown in Figure 11.12, L. monocytogenes grew faster and attained higher final populations in samples of whole milk that were preincubated with either of the two pseudomonads than in whole milk that was not treated with pseudomonads. L. monocytogenes behaved similarly in both whole and skim milk, with average generation times of approximately 7 and 8 h in milks preincubated with P. fluorescens and P. fragi, respectively (Figure 11.13). Although accelerated growth of Listeria was observed in reconstituted nonfat dry milk preincubated with either pseudomonad, generation times for listeriae in either of the two mixed cultures generally did not differ significantly. As was true for whole and skim milk, L. monocytogenes attained populations of 1 × 107 to 5 × 107 CFU/mL in reconstituted nonfat dry milk, with highest numbers occurring in milk preincubated with P. fluorescens rather than P. fragi. Flavobacterium is another genus of Gram-negative psychrotrophic bacteria that is frequently recovered from raw milk, pasteurized milk, and butter. Hence, Farrag and Marth [119] also examined behavior of L. monocytogenes in the presence of flavobacteria in skim milk at 7 and 13°C. Growth of L. monocytogenes strains Scott A, CA, and V7 in autoclaved skim milk was enhanced by presence of F. lutescens during 14–42 days of a 56-day incubation period at both 7 and 13°C, with these higher populations again being attributed to proteolysis of milk proteins by F. lutescens. However, Flavobacterium sp. ATCC 21429 failed to impact growth of L. monocytogenes at 7°C and proved to be slightly inhibitory to the same three Listeria strains when samples were held at 13°C. One strain of Bacillus spp. [170] also prevented Listeria growth in raw milk. These results dispel the previous theory and indicate that L. monocytogenes can readily compete with P. fragi, P. fluorescens, and certain Flavobacterium spp. for nutrients in milk and at the same time can outgrow these organisms at refrigeration temperatures even if the ratio of L. monocytogenes to pseudomonads or flavobacteria is on the order of 1:100,000. Enhanced growth of microorganisms, including L. monocytogenes, in the presence of these psychrotrophs is related to increased levels of nutrients that occur in milk as a result of proteolytic enzymes produced by these organisms [119,174]. Because many of these enzymes are heat-stable and able to survive pasteurization, raw milk must be handled properly and pasteurized within a reasonable time (i.e., 3–4 days) to prevent conditions that may favor growth of listeriae. As previously noted [187], enhanced growth of listeriae in intensively pasteurized as compared to HTST-pasteurized and raw milk also might be related to this phenomenon.

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8

7

L. monocytogenes log10 CFU/mL

6

5 L. monocytogenes 4 L.monocytogenes + P. fragi

3

2

L. monocytogenes + P. fluorescens

1

0 0

1

2

3

4 Days

5

6

7

8

FIGURE 11.12 Growth of L. monocytogenes at 10°C in whole milk preincubated for 3 days with selected Pseudomonas spp. (Adapted from Marshall, D.L. and R.H. Schmidt. 1988. Growth of Listeria monocytogenes at 10°C in milk preincubated with selected pseudomonads. J. Food Prot. 51: 277–282.)

17 16

L. monocytogenes

15

L. monocytogenes P. fragi

14 Generation Time (hours)

L. monocytogenes P. fluorescens

13 12 11 10 9 8 7 6 Whole

Skim

Nonfat Milk Solids

Product

FIGURE 11.13 Generation times of L. monocytogenes at 10°C in various milks preincubated for 3 days with P. fragi or P. fluorescens spp. (Adapted from Marshall, D.L. and R.H. Schmidt. 1988. Growth of Listeria monocytogenes at 10°C in milk preincubated with selected pseudomonads. J. Food Prot. 51: 277–282.)

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NONFLUID DAIRY PRODUCTS Although the aforementioned studies demonstrate the ability of L. monocytogenes to grow to potentially hazardous levels in fluid dairy products held at refrigeration temperatures, concern about the behavior of this organism in dairy products extends well beyond fluid milks and cream. As you will recall from Table 10.5, nearly 50 recalls have been issued in the United States for Listeriacontaminated ice cream. These recalls, along with FDA reports suggesting that about 3.5% of the ice cream and 8.5% of the ice cream novelties produced in the United States may be contaminated with presumably low levels of L. monocytogenes, prompted research on the extent of Listeria survival in frozen dairy products. Additionally, behavior of Listeria during manufacture and storage of nonfat dry milk and butter also has been investigated in the event that these products are inadvertently prepared from contaminated skim milk and cream, respectively.

ICE CREAM Pasteurized milk that has not been sold in retail stores is sometimes returned to dairy factories and reprocessed into ice cream. Because large commercial refrigeration units often fail to maintain a constant temperature of 4°C, virtually all reclaimed milk has undergone some degree of temperature abuse during the period in which the product was on sale. In addition to possible growth of L. monocytogenes during this 2-week period of “cold enrichment,” pseudomonads also can grow in milk and produce an environment that is more favorable for growth of Listeria. However, proper pasteurization of the ice cream mix would be expected to eliminate L. monocytogenes, with any listeriae in the final product most likely coming from the surrounding environment during subsequent product handling and packaging. As indicated from the studies summarized in the following text and the lack of listeriosis outbreaks involving commercial ice cream, Listeria is unable to grow in the final product during frozen storage, with similar lack of growth and partial inactivation reported for ewe’s milk during frozen storage [192]. The numerous Class I recalls issued since the late 1980s for Listeria-contaminated ice cream prompted Berang et al. [80] to investigate the behavior of L. monocytogenes in inoculated samples of chocolate ice cream (and chocolate milk as discussed earlier) prepared from fresh skim milk and commercial skim milk that was held beyond the expiration date. Although growth of listeriae was certainly not expected in ice cream held at −18 to −24°C, the pathogen survived equally well in both types of chocolate ice cream. Hence, use of returned milk in chocolate ice cream did not appear to enhance Listeria survival. Long-term survival of L. monocytogenes was also confirmed in a later study [191] in which the pathogen persisted for 14 weeks in ice cream stored at −18°C with no apparent cell death or injury. In 1996, Dean and Zottola [94] assessed the fate of L. monocytogenes V7 in full-fat (10%) and reduced-fat (3%) soft-serve ice cream prepared with and without 14-ppm nisin. Regardless of fat content, L. monocytogenes populations remained constant in ice cream during freezing and 3 months of storage at −18°C. However, nisin effectively reduced Listeria survival in both full- and reduced-fat ice cream during manufacture, with Listeria populations generally decreasing 2 and 3 orders of magnitude in full- and low-fat ice cream, respectively, following 1 month of frozen storage at −18°C. Although not currently approved in the United States as an ice cream ingredient, incorporation of nisin into ice cream formulations appears to be an effective, albeit costly, means of inactivating listeriae in this product during frozen storage. In 1989, Amelang and Doores [4,5] determined generation times for L. monocytogenes in nine formulations of commercially produced ice cream mix that varied in type and level of fat (cream, butter), sugar (cane sugar, corn sweetener), and milk solids (condensed milk, skim milk, whey powder). To simulate postprocessing contamination, all samples were inoculated to contain 103 L. monocytogenes strain Scott A or V7 CFU/mL and incubated at 4, 21, and 35°C. Overall, L. monocytogenes had average generation times of 21.6, 1.08, and 0.79 h in ice cream mixes incubated at 4, 21, and 35°C, respectively, with similar growth rates occurring in mixes containing

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10, 14, and 15% fat and held at the same temperature. It is noteworthy that these generation times are markedly shorter than those calculated by Rosenow and Marth [213] for growth of the same strains in whole milk, skim milk, chocolate milk, and whipping cream (see Table 11.5). Although L. monocytogenes generally behaved similarly in all ice cream mixes incubated at 4 and 21°C, differences in generation times were noted at 35°C when the pathogen was cultured in ice cream mixes made with alternative fat and milk solids. At 35°C, growth of listeriae was somewhat enhanced in mixes containing butter rather than cream, skim milk powder, or whey powder rather than condensed skim milk, and egg yolk as additional sources of solids. Although the pathogen grew most rapidly in ice cream mix containing a 50:50 ratio of cream to butter, partial replacement of cane sugar (sucrose: glucose + fructose) with corn sweetener (glucose and maltose) or high fructose corn syrup failed to significantly shorten generation times.

BUTTER In 1988, Olsen et al. [189] examined the fate of L. monocytogenes during manufacture and storage of butter in the event that the product is prepared from contaminated cream. According to their report, pasteurized cream was inoculated to contain 104–105 L. monocytogenes CFU/g and churned into butter. After removing the buttermilk, washed butter grains were salted to a level of 1.2% and resultant butter was analyzed weekly for listeriae during 10 weeks of storage at −18, 4–6, and 13°C. During manufacture 95% of the L. monocytogenes population was lost in buttermilk, with the remaining 5% of the population appearing in butter. The pathogen was present at levels of 1.7 × 104 to 1.8 × 105 CFU/g in cream as compared with 1.5 × 103 to 1.6 × 104 CFU/g in butter, indicating that similar to Staphylococcus aureus [183], L. monocytogenes also favors the water rather than lipid phase during butter making. As shown in Figure 10.14, Listeria populations increased 1.9 and 2.7 orders of magnitude in butter stored at 4–6 and 13°C, with maximum numbers being

7.00

L. monocytogenes log

10 CFU/g

6.00 13 °C 5.00 4 to 6 °C 4.00

3.00

–18 °C

2.00 0

20

60

40

80

Days

FIGURE 11.14 Survival of L. monocytogenes in butter manufactured from artificially contaminated cream and stored at 12, 4–6, and −18°C. Each line represents the average of 4 trials. (Adapted from Olsen, J.A., A.E. Yousef, and E.H. Marth. 1988. Growth and survival of Listeria monocytogenes during making and storage of butter. Milchwissenschaft 43: 487–489.)

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observed after 49 and 42 days of storage, respectively. These findings along with similar results by Lanciotti et al. [163] for commercially prepared light butter stored at 4 and 20°C indicate that enough milk solids were trapped in the water phase (containing 6% salt) to support growth of listeriae during storage. Numbers of listeriae then began to decrease; however, the organism was still present at levels >104 CFU/g following 70 days of refrigerated storage. Although freezing the contaminated butter prevented growth of L. monocytogenes, the organism was still present at levels of 103 CFU/g after 70 days of storage at −18°C, as was also reported by Slavchev et al. [224]. Following a 2000 report from Finland linking consumption of butter to 25 listeriosis cases including 6 fatalities [172], researchers at the University of Georgia [152,153] assessed the fate of L. monocytogenes in butter and other related fat spreads as a postmanufacturing contaminant. In the first of these studies [153], sweet cream whipped unsalted butter (pH 4.51), salted light butter (pH 4.58), two yellow fat spreads (pH 4.05 and 5.37), and a light margarine (pH 5.34) were either surface-inoculated with a 6-strain cocktail of L. monocytogenes and then stored at 4.4 or 21°C under high relative humidity for 21 days, or uniformly contaminated by mixing the inoculum into the product followed by storage at 4.4 or 21°C for 14 days. For surface-inoculated samples, growth of L. monocytogenes was only seen on sweet cream whipped salted butter (pH 6.40) with populations increasing < 1 log during 21 days of storage. On all other surface-inoculated samples, numbers of Listeria decreased 1.7 to > 5 logs and > 5 logs during storage at 4.4 and 21°C, respectively. In uniformly contaminated samples of sweet cream whipped salted butter (pH 6.40), Listeria populations remained relatively constant during 14 and 7 days of storage at 4.4 and 21°C, respectively, with decreases of 1.5 to > 2.0 logs reported for the remaining products. Holliday and Beuchat [152] later assessed the impact of storage temperature on L. monocytogenes growth in seven different yellow fat spreads. In this study, one margarine, one butter–margarine blend, and five dairy/nondairy spreads and toppings were inoculated with a 6-strain cocktail of L. monocytogenes at ~106 CFU per 3.5 mL or 4.0 g and then stored at 4.4, 10 and 21°C for up to 94 days. Overall, L. monocytogenes persisted 10–14 to >94 days in the seven fat spreads examined, with longer survival seen in samples stored at 4.4 as opposed to 10 and 21°C. The only growth of Listeria was seen when the butter–margarine blend (pH 6.66) was stored at 21°C with numbers of Listeria increasing ~1.5 logs after 2 weeks and then slowly decreasing to populations near the original inoculation level after 94 days of storage. These findings are also consistent with those of Cirigliano and Keller [87] who reported that L. monocytogenes failed to grow in margarine, yellow fat spreads, or topping held at 5, 10, or 23°C. Lack of Listeria growth in these non-butter-containing yellow fat spreads has been attributed to acidity (pH < 5.60) along with the presence of preservatives (potassium sorbate, sodium benzoate, salt) and the unfavorable growth environment provided by some emulsions. Addition of garlic to butter is also reportedly somewhat effective in reducing Listeria survival when the product is stored at 10 and 37 but not at refrigeration temperatures [3]. Thus far L. monocytogenes has not been isolated from pasteurized cream manufactured in the United States; however, given the massive Listeria recall of Texas-produced fluid dairy products, including half-and-half and whipping cream, in May 1986 (see Table 11.5), one cannot assume that all pasteurized cream and butter manufactured in the United States and elsewhere will be universally free of listeriae. Hence, because at least six Class I recalls have been issued for L. monocytogenes-contaminated butter, and also because growth of L. monocytogenes has been demonstrated experimentally in both cream and butter during refrigerated storage, it is necessary to ensure that cream is pasteurized and that recontamination of pasteurized cream is prevented before and during its churning into butter. However, margarine and other non-dairy-based fat spreads are generally unable to support growth of Listeria and therefore pose a lesser risk for consumers.

NONFAT DRY MILK Dried dairy products, including nonfat dry milk, whey, and casein, also may become contaminated with pathogenic microorganisms both before and after drying. Such concerns have been raised

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recently in Australia and New Zealand [157]. Although all dry dairy products examined thus far have been Listeria-free, methods used to detect listeriae in these surveys were generally unable to recover cells that may have been injured during the drying process. Two factors, namely, the unusual thermal resistance of L. monocytogenes and the report of a milkborne listeriosis outbreak in Massachusetts during 1983, prompted Doyle et al. [104] to examine behavior of L. monocytogenes during manufacture and storage of nonfat dry milk. Samples of concentrated (30% solids) and unconcentrated (10% solids) skim milk were inoculated to contain 105–106 L. monocytogenes (strain Scott A or V7) CFU/mL and dried to moisture contents of 3.6–6.4% in a gas-fired pilot-plant-sized spray dryer with inlet and outlet air temperatures of 165 ± 2 and 67 ± 2°C, respectively. All samples of nonfat dry milk were stored at 25°C for up to 16 weeks and periodically analyzed for listeriae, using both direct plating on McBride Listeria Agar (detects uninjured cells) and cold enrichment in Tryptose Broth (detects injured and uninjured cells). Listeria populations decreased approximately 1.0–1.5 orders of magnitude during spray drying regardless of whether or not nonfat dry milk was prepared from concentrated or unconcentrated skim milk. Strain V7 was generally hardier than strain Scott A during both spray drying and storage of nonfat dry milk. Twelve to 16 weeks of storage at room temperature were required to decrease populations of strain V7 >1000-fold in nonfat dry milk, whereas only 6 weeks of storage were necessary to obtain similar decreases in numbers of strain Scott A. Overall, strains Scott A and V7 survived a maximum of 8 and 12 weeks in nonfat dry milk, respectively. Although strain Scott A generally survived equally well in nonfat dry milk prepared from concentrated and unconcentrated skim milk, strain V7 survived 2 weeks longer in nonfat dry milk manufactured from concentrated rather than unconcentrated skim milk. The higher moisture content of nonfat dry milk (i.e., 5.7 and 6.4%) prepared from concentrated skim milk may have enhanced survival of listeriae in this product during extended storage. Overall, populations of L. monocytogenes decreased >10,000-fold in nonfat dry milk during 16 weeks of storage at room temperature. Hence, if commercially produced nonfat dry milk is ever found to contain L. monocytogenes, presumably at very low levels, it may be possible to eliminate this pathogen by holding the product at room temperature for several months.

REFERENCES 1. Abou-Donia, S.A. and A.K. Al-Medhagi. 1992. Detection and survival of Listeria monocytogenes in Egyptian dairy products. J. Dairy Sci. 75(Suppl. 1): 138. 2. Abou-Eleinin, A.A.M., E.T. Ryser, and C.W. Donnelly. 2000. Incidence and seasonal variation of Listeria species in bulk tank goat’s milk. J. Food Prot. 63: 1208–1213. 3. Adler, B.B. and L.R. Beuchat. 2002. Death of Salmonella, Escherichia coli O157: H7, and Listeria monocytogenes in garlic butter as affected by storage temperature. J. Food. Prot. 65: 1976–1980. 4. Amelang, J. and S. Doores. 1989. The effect of ingredients in ice cream formulations on the growth of Listeria monocytogenes. Annual Meeting of the Institute of Food Technologists, Chicago, IL, June 25–29, Abstr. 468. 5. Amelang, J. and S. Doores. 1989. The effect of medium, growth phase and temperature on the growth of Listeria monocytogenes in ice cream mix. Annual Meeting of the Institute for Food Technologists, Chicago, IL, June 25–29, Abstr. 469. 6. Andre, P., H. Roose, R. Van Noyen, L. Dejaegher, I. Vyttendaele, and K. De Schrijver. 1990. Neuromeningeal listeriosis associated with consumption of an ice cream. Med. Mal. Infect. 20: 570–572. 7 Anonymous. 1986. Ice Cream Bars Recalled. FDA Enforcement Report, July 16. 8. Anonymous. 1986. Ice Cream Recalled. FDA Enforcement Report, October 22. 9. Anonymous. 1986. Ice Cream Recalled. FDA Enforcement Report, October 29. 10. Anonymous. 1986. Ice Cream, Sherbet and Glacee Recalled. FDA Enforcement Report, September 3. 11. Anonymous. 1986. Ice Milk Mix Recalled. FDA Enforcement Report, June 25. 12. Anonymous. 1986. Large class I recall made of ice cream because of Listeria. Food Chem. News 28(24): 11–12.

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164. Legnani, P., E. Leoni, F. Soppelsa, and P. Bisbini. 1995. Prevalence of Listeria spp. in food products in the province of Belluno (Italy). L’Igiene Moderna 103: 143–155. 164a. Leuchner, L., T. Annamalai, T. Hoagland, Y. Zhao, and K. Venkitanarayanan. 2002. Behavior of Listeria monocytogenes in pasteurized and unpasteurized goat milk at different storage temperatures. Abstract 61C-34. Annual Meeting of the Institute of Food Technologists, Anaheim, CA. 165. Liewen, M.B. and M.W. Plautz. 1988. Occurrence of Listeria monocytogenes in raw milk in Nebraska. J. Food Prot. 51: 840–841. 166. Liewen, M.B., D.L. Peters, and M.W. Plautz. 1987. Incidence of L. monocytogenes in raw milk in Nebraska. Annual Meeting of the Institute of Food Technologists, Las Vegas, NV, June 16–19, Abstr. 118. 167. Little, C.L. and J. de Louvois. 1999. Health risks associated with unpasteurized goats’ and ewes’ milk on retail sale in England and Wales. A PHLS Dairy Products Working Group study. Epidemiol. Infect. 122: 403–408. 168. Lovett, J., D.W. Francis, and J.M. Hunt. 1987. Listeria monocytogenes in raw milk: detection, incidence, and pathogenicity. J. Food Prot. 50: 188–192. 169. Luisjuan-Morales, A., R. Alaniz-de la O, M.E. Vazquez-Sandoval, and B.T. Rosas-Barbosa. 1995. Prevalence of Listeria monocytogenes in raw milk in Guadalajara, Mexico. J. Food Prot. 58: 1139–1141. 170. Lund, A.M. and E.A. Zottola. 1990. Inhibition of Listeria species by Bacillus in raw milk. J. Food Prot. 59: 903 (abstr.). 171. Lund, A.M., E.A. Zottola, and D.J. Pusch. 1991. Comparison of methods for isolation of Listeria from raw milk. J. Food Prot. 54: 602–606. 172. Lyytikainen, O., T. Autio, R. Maijala, P. Ruutu, T. Honkaanen-Buzalski, M. Miettinen, M. Hatakka, J. Mikkola, V.J. Anttila, T. Johansson, L. Rantala, T. Aalto, H. Korkeala, and A. Siltonen. 2000. An outbreak of Listeria monocytogenes serotype 3a infections from butter in Finland. J. Infect. Dis. 181: 1838–1841. 173. Marshall, D.L. and R.H. Schmidt. 1988. Growth of Listeria monocytogenes at 10°C in milk preincubated with selected pseudomonads. J. Food Prot. 51: 277–282. 174. Marshall, D.L. and R.H. Schmidt. 1991. Physiological evaluation of stimulated growth of Listeria monocytogenes by Pseudomonas species in milk. Can. J. Microbiol. 37: 594–599. 175. Massa, S., D. Cesaroni, G. Poda, and L.D. Trovatelli. 1990. The incidence of Listeria spp. in soft cheeses, butter, and raw milk in the province of Bologna. J. Appl. Bacteriol. 68: 153–156. 176. Mathew, F.P. and E.T. Ryser. 2002. Competition of thermally injured Listeria monocytogenes with a mesophilic lactic acid starter culture in milk of various heat treatments. J. Food Prot. 65: 643–650. 177. Mattingly, J.A., B.T. Butman, M.C. Plank, R.J. Durham, and B.J. Robison. 1988. Rapid monoclonal antibody-based enzyme-linked immunosorbant assay for detection of Listeria in food products. J. Assoc. Off. Anal. Chem. 71: 679–681. 178. McBean, L.D. 1988. A perspective on food safety concerns. Dairy Food Sanit. 8: 112–118. 179. Mena, C., G. Almeida, L. Carneiro, P. Teixeira, T. Hogg, and P.A. Gibbs. 2004. Incidence of Listeria monocytogenes in different food products commercialized in Portugal. Food Microbiol. 21: 213–216. 180. Meyer-Broseta, S., A. Diot, S. Bastian, J. Riviere, and O. Cerf. 2003. Estimation of low bacterial concentration: Listeria monocytogenes in raw milk. Int. J. Food Microbiol. 80: 1–15. 181. Mickova, V. 1991. Listeria monocytogenes in foods. Vet. Med. (Praha) 36: 745–750. 182. Mickova, V. and S. Konecny. 1990. Listeria monocytogenes in foods. Veterinarstyi 40: 327–328. 183. Minor, T.E. and E.H. Marth. 1972. Staphylococcus aureus and enterotoxin A in cream and butter. J. Dairy Sci. 55: 1410–1414. 184. Monge, R., D. Utzinger, and L. Arias. 1994. Incidence of Listeria in pasteurized ice cream and soft cheese in Costa Rica, 1992. Rev. Biol. Trop. 43: 327–328. 185. Moura, S.M., M.T. Destro, and B.D. Franco. 1993. Incidence of Listeria species in raw and pasteurized milk produced in Sao Paulo, Brazil. Int. J. Food Microbiol. 19: 229–237. 186. Muraoka, W., C. Gay, D. Knowles, and M. Borucki. 2003. Prevalence of Listeria monocytogenes subtypes in bulk milk of the Pacific Northwest. J. Food Prot. 66: 1413–1419. 187. Northolt, M.D., H.J. Beckers, U. Vecht, L. Toepoel, P.S.S. Soentoro, and H.J. Wisselink. 1988. Listeria monocytogenes: heat resistance and behavior during storage of milk and whey and making of Dutch types of cheese. Neth. Milk Dairy J. 42: 207–219.

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188. O’Donnell, E.T. 1995. The incidence of Salmonella and Listeria in raw milk from farm bulk tanks in England and Wales. J. Soc. Dairy Technol. 48: 25–29. 189. Olsen, J.A., A.E. Yousef, and E.H. Marth. 1988. Growth and survival of Listeria monocytogenes during making and storage of butter. Milchwissenschaft 43: 487–489. 190. Oz, H.H. and R.J. Farnsworth. 1985. Laboratory simulation of fluctuating temperature of farm bulk tank milk. J. Food Prot. 48: 303–305. 191. Palumbo, S.A. and A.C. Williams. 1991. Resistance of Listeria monocytogenes to freezing in foods. Food Microbiol. 8: 63–68. 192. Papageorgiou, D.K., M. Bori, and A. Mantis. 1997. Survival of Listeria monocytogenes in frozen ewe’s milk and Feta cheese curd. J. Food Prot. 60: 1041–1045. 193. Patterson, R.L., D.J. Pusch, and E.A. Zottola. 1989. The isolation and identification of Listeria spp. from raw milk. J. Food Prot. 52: 745. 194. Pearson, L.J. and E.H. Marth. 1990. Behavior of Listeria monocytogenes in the presence of cocoa, carrageenan, and sugar in a milk medium incubated with and without agitation. J. Food Prot. 53: 30–37. 195. Pearson, L.J. and E.H. Marth. 1990. Behavior of Listeria monocytogenes in the presence of methylxanthines—caffeine and theobromine. J. Food Prot. 53: 47–50, 55. 196. Pearson, L.J. and E.H. Marth. 1990. Inhibition of Listeria monocytogenes by cocoa in a broth medium and neutralization of this effect by casein. J. Food Prot. 53: 38–46. 197. Peters, D.L. and M.B. Liewen. 1988. Growth and survival of Listeria monocytogenes in unfiltered milk. Annual Meeting of the Institute of Food Technologists, New Orleans, June 19–22, Abstr. 326. 198. Pine, L., G. B. Malcolm, J.B. Brooks, and M.I. Daneshvar. 1989. Physiological studies on the growth and utilization of sugars by Listeria species. Can. J. Microbiol. 35: 245–254. 199. Pitt, W.M., T.J. Harden, and R.R. Hull. 1999. Antibacterial activity of raw milk against Listeria monocytogenes. Aust. J. Dairy Technol. 54: 90–93. 200. Prentice, G.A. 1994. Listeria monocytogenes. In The Significance of Pathogenic Microorganisms in Raw Milk. Brussels, International Dairy Federation Ref. S.I. 9405, pp. 101–115. 201. Proctor, M.E., R. Brosch, J.W. Mellen, L.A. Garrett, C.W. Kasper, and J.B. Luchansky. 1995. Use of pulsed-field gel electrophoresis to link sporadic cases of invasive listeriosis with recalled chocolate milk. Appl. Environ. Microbiol. 61: 3177–3179. 202. Quagilo, G., C. Casolari, G. Menziani, and A. Fabio. 1992. The incidence of Listeria monocytogenes in milk and milk products. L’Igiene Moderna 97: 565–579. 203. Quinto, E.J., C.M. Franco, C.A. Fente, B.I. Vazquez, and A. Cepeda. 1996. Effects of Pseudomonas fluorescens on the growth of Listeria monocytogenes and Listeria innocua in skimmed milk. Arch. Lebensmittelhygiene 47: 107–110. 204. Rajikowski, K.T., S.M. Calderone, and E. Jones. 1994. Effect of polyphosphate and sodium chloride on the growth of Listeria monocytogenes and Staphylococcus aureus in ultra-high temperature milk. J. Dairy Sci. 77: 1503–1508. 205. Razavi-Rohani, M. and Y. Hedaiatinia. 1990. A study of the contamination of milk to Listeria in Urmia, Iran. In Posters and Brief Communications of the XXIII International Dairy Congress, Montreal, October 8–12. Abst. 364. 206. Rea, M.C., T.M. Cogan, and S. Tobin. 1992. Incidence of pathogenic bacteria in raw milk in Ireland. J. Appl. Bacteriol. 73: 331–336. 207. Rodler, M. and W. Körbler. 1989. Examination of Listeria monocytogenes in dairy products. Acta Microbiol. Hung. 36: 259–261. 208. Rodriguez, J.L., P. Gaya, M. Medina, and M. Nunez. 1994. Incidence of Listeria monocytogenes and other Listeria spp. in ewe’s raw milk. J. Food Prot. 57: 571–575. 209. Rodriguez, L.D., J.F.F. Garayzabal, J.A.V. Boland, E.R. Ferri, and G.S. Fernandez. 1985. Isolation de micro-organismes du genre listeria à partir de lait cru destiné à la consommation humaine. Can. J. Microbiol. 31: 938–941. 210. Rohrbach, B.W., F.A. Draughon, P.M. Davidson, and S.P. Oliver. 1992. Prevalence of Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica and Salmonella in bulk tank milk: risk factors and risk of human exposure. J. Food Prot. 55: 93–97. 211. Rola, J., K. Kwiatek, B. Wojton, and M.M. Michalski. 1994. Incidence of Listeria monocytogenes in raw milk and dairy products. Medycyna Wet. 50: 323–325. 212. Rosenow, E.M. and E.H. Marth. 1987. Addition of cocoa powder, cane sugar, and carrageenan to milk enhances growth of Listeria monocytogenes. J. Food Prot. 50: 726–729, 732.

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and Behavior 12 Incidence of Listeria monocytogenes in Cheese and Other Fermented Dairy Products Elliot T. Ryser CONTENTS Introduction ....................................................................................................................................406 U.S. Surveillance Programs and Recalls for L. monocytogenes in Domestic and Imported Cheese ................................................................................................407 Domestic Cheese ..................................................................................................................407 Imported Cheese...................................................................................................................414 France .......................................................................................................................414 Other Western European Countries..........................................................................418 Surveys and Monitoring Programs for Listeria spp. in Cheese Produced outside the United States ............................................................................................................................419 Canada ..................................................................................................................................419 France ...................................................................................................................................421 Germany ...............................................................................................................................428 Italy .......................................................................................................................................430 Switzerland ...........................................................................................................................430 Other European Countries....................................................................................................432 Other Countries ....................................................................................................................435 Behavior of L. monocytogenes in Fermented Milks .....................................................................436 Starter Cultures, Cultured Milks, and Cream ......................................................................436 Mesophilic Starter Cultures......................................................................................436 Cultured Buttermilk..................................................................................................439 Cultured Cream ........................................................................................................441 Thermophilic Starter Cultures..................................................................................441 Yogurt .......................................................................................................................443 Kefir ..........................................................................................................................445 Traditional Fermented Milk Products ..................................................................................445 Behavior of L. monocytogenes in Cheese .....................................................................................446 Coagulants ............................................................................................................................447 Coloring Agents and Starter Distillates ...............................................................................448 Mold-Ripened Cheeses.........................................................................................................449 Camembert Cheese...................................................................................................449 Blue Cheese ..............................................................................................................453

405

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Bacterial Surface–Ripened Cheeses.....................................................................................454 Brick Cheese.............................................................................................................455 Taleggio Cheese........................................................................................................456 Tilsiter Cheese ..........................................................................................................456 Trappist Cheese ........................................................................................................456 Soft Italian Cheeses..............................................................................................................457 Mozzarella ................................................................................................................457 Semisoft and Hard Cheeses .................................................................................................457 Gouda and Maasdam Cheeses .................................................................................458 Colby Cheese............................................................................................................458 Cheddar Cheese ........................................................................................................459 Swiss Cheese ............................................................................................................462 Parmesan Cheese ......................................................................................................462 Hard Italian-Type Cheese.........................................................................................463 Hispanic Cheeses..................................................................................................................464 Queso Blanco Cheese...............................................................................................464 Queso de los Ibores Cheese .....................................................................................464 Mexican Manchego ..................................................................................................464 Chihuahua .................................................................................................................465 Pickled Cheeses ....................................................................................................................465 Feta ...........................................................................................................................465 Turkish White-Brined Cheese ..................................................................................467 Domiati Cheese ........................................................................................................467 Bulgarian White-Pickled Cheese..............................................................................467 Sudanese White-Pickled Cheese ..............................................................................467 Yugoslavian White-Brined Cheese...........................................................................468 Ewe’s and Goat’s Milk Cheese ............................................................................................468 Kachkaval Cheese.....................................................................................................468 Manchego Cheese.....................................................................................................469 Goat’s Milk Cheese ..................................................................................................469 Soft Unripened Cheese.........................................................................................................471 Cottage Cheese .........................................................................................................471 Cream Cheese...........................................................................................................474 Whey Cheeses ......................................................................................................................474 Cold-Pack Cheese Food ...........................................................................................476 Pasteurized Processed Cheese..............................................................................................477 Behavior of L. monocytogenes in Cheese as Affected by Cheese Composition ................478 Feasibility of Preparing Cheese from Raw Milk.................................................................481 Whey.....................................................................................................................................482 Brine Solutions .....................................................................................................................483 References ......................................................................................................................................486

INTRODUCTION On June 14, 1985, Listeria monocytogenes emerged from relative obscurity to the front page of many American newspapers because of a large listeriosis outbreak in California that was directly linked to consumption of Mexican-style cheese manufactured in metropolitan Los Angeles. By the time this outbreak subsided in August 1985, as many as 300 cases of listeriosis had been reported, including 85 deaths—at least 40 of which were traced to the tainted cheese. In response

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to this foodborne outbreak of listeriosis, U.S. Food and Drug Administration (FDA) officials added L. monocytogenes to their list of pathogenic organisms that should be of concern to cheesemakers and began surveying various soft domestic cheeses for listeriae. Approximately 6 months later, isolation of L. monocytogenes from several imported Brie cheeses purchased at a supermarket led to the eventual recall of approximately 300,000 tons of Brie cheese imported from France and to a real concern about the incidence of this pathogen in other European cheeses. Recall of this cheese prompted two corrective measures: (1) adoption of a cheese certification program by the United States and France to prevent importation of Listeriacontaminated cheese and (2) initiation of numerous large-scale surveys to determine the extent of Listeria contamination in virtually all types of cheese manufactured in the United States, Canada, and Western Europe. Throughout 1986 and most of 1987, the impact of Listeria on European cheesemakers was primarily in the form of economic losses from destruction of contaminated product. However, L. monocytogenes struck again late in 1987 with the report of a large listeriosis outbreak in Switzerland (see Chapter 11) in which Vacherin Mont d’Or soft-ripened cheese was incriminated as the vehicle of infection. This pathogen has continued to plague consumers of cheese with a series of smaller outbreaks traced to commercially produced European soft cheeses and, most recently, several small outbreaks in the United States that involved soft homemade Mexican-style cheese prepared from raw milk. These cheeseborne listeriosis outbreaks have prompted ongoing worldwide efforts to better understand the incidence of and behavior of L. monocytogenes during manufacture and storage of numerous cheese varieties as well as other fermented dairy products. The first half of this chapter summarizes Listeria-related recalls of cheese in the United States and results from surveys dealing with the incidence of listeriae in domestic and imported fermented dairy products. The second half of this chapter addresses the fate of L. monocytogenes during manufacture and storage of buttermilk, yogurt, and various cheeses (including whey) and the potential for cheese ingredients, such as rennet, salt brine, and coloring agents, to serve as vehicles of contamination during cheesemaking.

U.S. SURVEILLANCE PROGRAMS AND RECALLS FOR L. MONOCYTOGENES IN DOMESTIC AND IMPORTED CHEESE DOMESTIC CHEESE The concept of listeriosis as a foodborne illness is not new. Consumption of contaminated raw milk was believed to have caused several cases of listeriosis in post–World War II Germany. In 1961, Seeliger [358] also suggested sour milk, cream, and cottage cheese as possible vehicles of infection in this outbreak. Although results from two Yugoslavian studies concerned with behavior of L. monocytogenes in various fermented dairy products (i.e., cultured cream, unsalted skim milk cheese, Kachkaval cheese, and yogurt) were published in 1964 [246] and 1981 [367], no surveys dealing with the incidence of listeriae in fermented dairy products were made before contaminated Mexicanstyle cheese was linked to the California listeriosis outbreak in June 1985. Public health concerns about presence of L. monocytogenes in domestic and imported fermented dairy products as well as other foods, such as meat, poultry, seafood, fruits, and vegetables, can be traced either directly or indirectly to the 1985 listeriosis outbreak in California. Less than 1 month after the first nationwide Class I Listeria-associated recall was issued for 22 varieties (∼500,000 lb) of Mexican-style cheese contaminated with L. monocytogenes (Table 12.1), the FDA developed a series of programs designed to prevent the recurrence of such an outbreak [365] (Figure 12.1). The Domestic Soft Cheese Surveillance Program—the first of the dairy factory initiative programs—was instituted by the FDA in July 1985 and involved on-site inspection of firms manufacturing soft cheese [15]. Priority was given to manufacturers of Mexican-style soft cheese, followed by firms producing other ethnic-type soft cheeses, such as Edam, Gouda, Liederkranz,

California

11/6/1990 2/1/1991 2/14/1991

7/11/1991 10/28/1991 3/10/1992

10/14/1992

Cheese spread Mozzarella

Ricotta Jack Cold-pack cheese food

Queso fresco

Washington

New York Wisconsin Wisconsin

Arizona, Arkansas, California, Colorado, Georgia, Guam, Hawaii, Idaho, Illinois, Kansas, Louisiana, Marshall Islands, Massachusetts, Nevada, New Jersey, New Mexico, New York, Oklahoma, Oregon, Rhode Island, Samoa, Texas, Utah, Washington State Nationwide, Puerto Rico Arizona, California, Oregon, Texas Virginia, Washington, DC Illinois, North Carolina, Ohio, Pennsylvania Nationwide California, Washington State Arizona, California, Florida, Texas, Washington State Arizona, California, Idaho, Nevada, Oregon, Washington State Southeastern United States Connecticut, Georgia, Illinois, Michigan, New York, Ohio, Pennsylvania, Texas, West Virginia, Wisconsin Florida, New York Iowa, Minnesota, Wisconsin Arizona, California, Colorado, Florida, Georgia, Illinois, Indiana, Maryland, Michigan, Minnesota, New York, Ohio, Pennsylvania, Tennessee, Texas, Vermont, Virginia, Wisconsin Oregon, Washington State

Distribution

Unknown

1109 12,500 Unknown

~1362 >89,000

500,000

~10,000 127,607 10,850 1150 ~13,800 ~1400 Unknown

~500,000

Quantity (lb)

87

84 81 83 85

83 82

80

9,10,13 21,39 43,55 53 52 54,365 65

11,12

Reference FDA Enforcement Report

408

Florida Wisconsin

Ohio California Virginia Illinois Kentucky Wisconsin California

8/14/1985 3/5/1986 9/11/1986 4/17/1987 5/6/1987 8/21/1987 1/29/1988

Liederkranz (Brie,a Camembert) Soft Mexican-style: Queso fresco and 5 other varieties Semisoft Salvador-style white Soft-ripened: Old Heidelberg Soft-ripened: Bonbel and Gouda Raw milk sharp Cheddar Soft Mexican-style: Cotija, Queso fresco, and 8 other varieties; Baby Jack and Monterey Jack Mexican-style soft cheese

California

Origin

6/13/1985

Date Recall Initiated

Jalisco-brand soft Mexican-style: Cotija, Queso fresco, and 20 other varieties

Type of Cheese

TABLE 12.1 Chronological List of Class I Recalls in the United States for Domestic Cheese Contaminated with L. monocytogenes

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12/18/1992 3/4/1993 10/19/1993

4/15/1994 5/11/1994 5/20/1994 5/21/1994 5/23/1994 5/24/1994 6/15/1994

8/11/1994 8/11/1994 10/28/1994 2/2/1996

10/30/1997

11/14/1997

2/4/1998

Limburger Cheese spread

Cream cheese

Queso Prensado Cream cheese and lox Mexican-style soft white Mexican-style soft white Mexican-style soft white Queso blanco Goat milk cheese

Torte loaf cheese

Swiss cold-pack cheese food Swiss Gorgonzola

Cream cheese with vegetables

Cream cheese

Queso fresco

Wisconsin

Massachusetts

Massachusetts

Wisconsin Ohio Wisconsin

Missouri

Wisconsin Massachusetts Texas Texas Texas Wisconsin California

Wisconsin

Wisconsin Tennessee Wisconsin Alabama, Illinois, Indiana, Kentucky, Mississippi, Tennessee California, Florida, Georgia, Illinois, Indiana, Iowa, Minnesota, Nebraska, North Carolina, Ohio, South Carolina, South Dakota, Tennessee, Wisconsin Florida, New Jersey, Wisconsin Connecticut, Georgia, Massachusetts Texas Texas Texas New Jersey California, Colorado, Georgia, Illinois, Massachusetts, Michigan, New York, Oregon, Texas Illinois, Indiana, Kansas, Louisiana, Missouri, Texas Missouri, Ohio Pennsylvania California, Colorado, Florida, Georgia, Illinois, Minnesota, New Jersey, New York, North Carolina, Pennsylvania, Tennessee, Washington State, Wisconsin Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Pennsylvania, Vermont Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont Nationwide 248,938

Unknown

7340

510 2270 4500

301

1429 20 Unknown Unknown Unknown 1220 ~5682

3075

1500 11,789

(continued)

107,108

104

103

97 99 100

98

96 91 94 93 93 95 92

90

86,88 86

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Incidence and Behavior of Listeria monocytogenes 409

Date Recall Initiated 3/20/1998 3/23/1998 3/26/1998 3/27/1998 4/10/1998 4/11/1998 5/1/1998 5/26/1998 5/26/1998 7/27/1998 3/29/1999 5/22/1999

6/1/1999 6/11/1999

7/24/2000 10/17/2000 10/27/2000 11/1/2000 11/1/2000 11/2/2000 11/4/2000 11/6/2000

Type of Cheese

Queso blanco Queso fresco

Queso blanco Blue cheese Mozzarella Blue cheese Blue cheese salad dressing Queso fresco Spanish white Mexican-style fresh white Shredded Monterey Jack Raw milk Colby

Queso fresco Raw milk mild Cheddar

Cheddar Cheddar, Monterey Jack, and Muenster (19 varieties) Shredded Colby and Monterey Jack Cheddar Cheddar Jack, cheese sticks Cheddar Colby and Monterey Jack Oregon Wisconsin Wisconsin Wisconsin Wisconsin Wisconsin Mississippi Wisconsin

Nationwide Alabama, Florida, Georgia, North Carolina, South Carolina, Tennessee, Virginia Florida Nationwide California, Colorado, Nevada Nationwide Nationwide New York New York New York Texas Arizona, California, Colorado, Georgia, Illinois, Maryland, Missouri, New York, Pennsylvania, South Carolina, Tennessee, Utah New Jersey, New York Alabama, Arkansas, Florida Georgia, Michigan, Missouri, North Carolina, Pennsylvania, Tennessee, Washington State Alaska, California, Oregon, Washington State Nationwide Nationwide Nationwide Georgia, Oklahoma, Wisconsin States east of the Rocky Mountains Alabama, Florida, Louisiana, Mississippi Nationwide (except far western United States)

Distribution

1260 1,325,000 62,180 33,963 159,850 74,302 17,362 70,216

500 228

Unknown 123,000 Unknown Unknown Unknown Unknown Unknown Unknown 6000 135

248,938 Unknown

Quantity (lb)

389 389 389 389 389 389 389 389

389 389

389 389 389 103 106 389 389 389 389 389

389 109

Reference FDA Enforcement Report

410

New York Missouri

Wisconsin Wisconsin California Wisconsin Louisiana New York New York New York Iowa Missouri

Wisconsin Domestic

Origin

TABLE 12.1 (CONTINUED) Chronological List of Class I Recalls in the United States for Domestic Cheese Contaminated with L. monocytogenes

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Listeria, Listeriosis, and Food Safety

12/8/2000 5/1/2001 12/3/2001 2/27/2002 4/25/2003 9/12/2003 10/3/2003 10/16/2003 10/22/2003 11/9/2003 12/12/2003 12/17/2004 8/22/05 12/10/05 12/14/05

Mozzarella string cheese Cheddar, Jack, and spread cheese Cheddar, Colby, and American cheese cubes Ackawi Queso fresco Blue cheese Queijo Minerio Queso fresco Raw milk Cheddar Sharp Cheddar, cold-pack cheese food Queso fresco Manouri Greek cheese

Queso seco American pasteurized processed cheese

Bleu cheese Later found to contain only L. innocua.

a

11/6/2000 11/6/2000 11/8/2000

Cheddar Colby and Monterey Jack Sharp Cheddar

Massachusetts

Florida Wisconsin

Wisconsin California Ohio Michigan New York Colorado Pennsylvania North Carolina Oregon Wisconsin New York New Jersey

Wisconsin Wisconsin Wisconsin Alabama Nationwide California, Florida, Illinois, Maryland, North Carolina, Tennessee, Virginia Minnesota, Ohio, Oklahoma, Wisconsin Nationwide Maryland, Pennsylvania Michigan New York, New Jersey Nationwide New Jersey, New York Illinois Oregon Illinois, Minnesota, Wisconsin New Jersey, New York California, Illinois, Michigan, New Jersey, New York, Pennsylvania, Virginia Florida Alabama, Illinois, Minnesota, Missouri, Texas, Washington State Massachusetts 36

2000 550,254

8688 Unknown 6200 Unknown 350 360 4758 4266 46 2736 Unknown 32

18,000 1,431,539 17,712

389

389 389

389 389 389 389 389 389 389 389 389 389 389 389

389 389 389

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Incidence and Behavior of Listeria monocytogenes 411

DK3089_C012.fm Page 412 Wednesday, February 21, 2007 6:56 PM

412

Listeria, Listeriosis, and Food Safety

DOMESTIC

California listeriosis outbreak — January–August 1985

Domestic soft cheese surveillance program — begun July 1985 L. monocytogenes accidentally isolated from French Brie cheese — January 1986

IMPORTED Imported soft cheese surveillance program — begun March 1986

Aged and ripened cheese survey — begun January 1986

Temporary FDA/French soft-ripened cheese testing program — begun April 1986

Continuation of soft cheese survey — March 1987

French certification program — begun April 1987 Survey of cheese manufactured from raw milk — begun April 1987

Cheese under general pathogen surveillance program — 1988

L. monocytogenes isolated from Italian Romano cheese — June 1987

Italian cheese surveillance program — begun July 1987 Survey of import cheese in domestic status — begun December 1987

FIGURE 12.1 Surveillance programs for Listeria spp. in domestic and imported cheese. (Adapted from Archer, D. L. 1988. Review of the latest FDA information on the presence of Listeria in foods. WHO Working Group on Foodborne Listeriosis. Geneva, Switzerland, February 15–19.)

Limburger, Monterey Jack, Muenster, and Port du Salut, made from raw, heat-treated (<71.7°C [161°F]/15 sec) or pasteurized (≥71.7°C [161°F]/15 sec) milk. In addition to determining the firm’s compliance with good manufacturing practices (i.e., use of proper pasteurization, cleaning, and sanitizing procedures), FDA inspectors collected and analyzed cheese samples for L. monocytogenes using the original FDA procedure. Cheese samples also were tested for the presence of enteropathogenic strains of Escherichia coli and for phosphatase activity, which, if present, generally indicates improper pasteurization of cheesemilk and/or subsequent contamination with raw milk. However, suitability of the phosphatase test for cheese has since been questioned. Less than 2 months into this program, FDA officials isolated a pathogenic strain of L. monocytogenes from one sample of domestically produced Liederkranz cheese (see Table 12.1). The manufacturer subsequently recalled the product nationwide. Following preliminary FDA reports of further Listeria contamination, this recall was extended to include all lots of Brie and Camembert cheese manufactured at the same facility [9,13]. However, final laboratory reports indicated that both Brie and Camembert cheese were contaminated with Listeria innocua, which is nonpathogenic, rather than L. monocytogenes. Although the Domestic Soft Cheese Surveillance Program also was responsible for temporarily closing two soft cheese factories in California that produced phosphatase-positive cheese [10], it must be stressed that L. monocytogenes was never isolated from cheeses produced at either facility. In general, FDA inspections of other soft cheese factories uncovered problems similar to those encountered during inspections of Grade A fluid milk factories: (1) potential bypasses of the pasteurizer, (2) postpasteurization blending of product, and (3) a general lack of education and training of plant personnel [282]. Items of particular concern to cheesemakers and that were not generally found during visits to Grade A milk factories included defects in the pasteurization process, discrepancies in pasteurization/production records, and a higher incidence (than in Grade A milk factories) of pathogenic microorganisms (including L. monocytogenes) on environmental surfaces in production and storage areas. Inspections of domestic cheese factories continued throughout 1986, 1987, and 1988 under four separate programs (see Figure 12.1), with FDA officials reaching nearly half of the 400 soft cheese factories in the United States by April 1986 and the remaining factories (including follow-up

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inspections of problem factories) by late 1987 [47]. According to FDA records [113], L. monocytogenes was confirmed in 12 of 658 (1.82%) domestic cheese samples analyzed during 1986. During these inspection programs, six Class I recalls were issued for various ethnic-type soft and semisoft cheeses containing L. monocytogenes. In response to (1) a 1987 report of a woman who developed listeriosis in San Bernadino, CA, after consuming illegally produced Mexican-style cheese and (2) the widespread availability of uninspected, unbranded Mexican-style cheese illegally produced from raw milk in metropolitan Los Angeles [73], Genigeorgis et al. [216], in conjunction with the California Department of Food and Agriculture, U.S. Department of Agriculture’s Food Safety and Inspection Service (USDA–FSIS), the Immigration and Naturalization Service, and the Los Angeles District Attorney’s Office, surveyed 100 California-produced soft Hispanic-style cheeses that were either seized or purchased undercover between June and November of 1988. Overall, two samples each were positive for L. monocytogenes and L. innocua. These four Listeria-contaminated cheeses had a pH of 6.2–6.5 and were presumably prepared from raw milk, as evidenced by a positive alkaline phosphatase test. Given the ability of L. monocytogenes to grow in such cheeses during refrigerated storage and marketing, Hispanic-style cheeses continue to constitute a significant public health threat. Hispanic-style cheeses manufactured commercially in the United States accounted for 23 of 68 (33.8%) recalls issued through 2005 including one large recall in June 1990 involving approximately 500,000 lb of product. However, of greatest concern are those Hispanic-style cheeses that have been illegally manufactured from raw milk, as evidenced by continued sporadic cases of listeriosis among the Hispanic population, with Gombas et al. [230] recovering L. monocytogenes from only 5 of 2,931 (0.17%) commercially produced soft Hispanicstyle cheeses recently purchased in Maryland and California over a 50-week period. As part of this same survey, L. monocytogenes was also isolated from 23 of 1,623 (1.42%) and 14 of 1,347 (1.04%) retail samples of blue-veined and mold-ripened cheese, respectively, with some of these cheeses also having been imported. As previously mentioned, although all products containing L. monocytogenes must be retrieved from the marketplace, formal Class I recalls do not have to be issued for contaminated products that have not yet reached retail stores. Because such situations typically lead to nonpublished “internal recalls” issued by the manufacturer, far more cheese was likely destroyed during this 20-year period than has actually been reported. Several such informal recalls involved a part-skim milk cheese manufactured in California [58] as well as ricotta, Parmesan, and mozzarella cheese of uncertain origin [297]. Following a report by Ryser and Marth [340] that L. monocytogenes can survive more than 1 year in Cheddar cheese (i.e., well beyond the mandatory 60-day aging period for Cheddar cheese manufactured from raw milk), the FDA modified its Domestic Cheese Program in August 1987 to include cheese prepared from unpasteurized milk [47]. Between April and October of 1987, 181 samples of domestic aged (held a minimum of 60 days at ≥1.7°C [35°F]) natural cheese manufactured from raw milk, as well as similar imported cheeses in domestic status, were collected from retail stores by FDA field personnel and analyzed for L. monocytogenes (Table 12.2). These efforts uncovered one positive sample—a sharp Cheddar cheese manufactured in Wisconsin, which was subsequently recalled from the market in July 1987 (see Table 12.1). Thus far, 17 of the 68 (25%) Listeria-related cheese recalls have involved Cheddar or Colby, including four cheeses prepared from raw milk, with 14 of these recalls issued since 2000. Late in 1987, the FDA announced plans for a 2-year pathogen surveillance program [44] that was designed to examine domestic and imported cheese as well as other high-risk foods (i.e., milk, vegetables, and seafood) for the presence of L. monocytogenes and other selected pathogens, including Vibrio cholerae, V. parahaemolyticus, E. coli, enteropathogenic E. coli, Staphylococcus aureus, Salmonella spp., Yersinia enterocolitica, Campylobacter jejuni, and C. coli. Under this program, samples of soft-ripened and raw milk cheese as well as imported hard and artificial blended cheese were examined for all of the aforementioned organisms except Vibrio spp. Domestic cheeses were collected at the wholesale level, whereas samples of imported cheese were obtained

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TABLE 12.2 Incidence of L. monocytogenes in “Domestic” Cheese Manufactured from Raw Milk—FDA 1987a Type of Cheese Blue Brick Cheddar Colby Edam Goat Gouda Monterey Jack Swiss Other Total

Number of Samples Analyzed

Number of Positive Samples (%)

18 5 71 8 4 6 1 9 42 17 181

0 0 1b (1.4) 0 0 0 0 0 0 0 1 (0.55)

a

Includes imported cheese in domestic status. Two samples with L. innocua.

b

Source: Adapted from Archer, D. L. 1988. Review of the latest FDA information on the presence of Listeria in foods. WHO Working Group on Foodborne Listeriosis. Geneva, Switzerland, February 15–19.

from retail stores. Although this program prompted only one Listeria-related recall of domestic cheese during 1989 and 1990, five separate recalls of Anari and Halloumi cheese imported from Cyprus were reported during this same 2-year period along with one additional recall of Italian soft-ripened/semisoft cheese. However, because additional cheese-related recalls after 1990 have been limited to eight imported cheeses, the present FDA inspection program in combination with increased vigilance by commercial cheesemakers appears to be highly effective in limiting consumer exposure to both domestic and imported Listeria-contaminated cheese. Several other fermented dairy products also were examined for L. monocytogenes in conjunction with the FDA Dairy Initiative Program [113] (see Chapter 11). In 1986, 10 samples of cottage cheese were found to be free of listeriae. Other than cheese and cheese food, 1% fat cultured buttermilk [34,36] and frozen yogurt [72] are the only other domestically produced, fermented dairy products known to have been contaminated with L. monocytogenes. The first of these products was included in a 1986 Class I recall involving approximately 1 million gallons of dairy products (fluid milk, chocolate milk, half-and-half, whipping cream, ice milk, ice milk mix, ice milk shake mix, ice cream, and ice cream mix), all of which presumably contained L. monocytogenes. Three years later, officials from the Wisconsin Department of Agriculture, Trade and Consumer Protection issued a statewide recall for one particular brand of frozen yogurt after routine testing revealed the presence of L. monocytogenes in one sample of mandarin orange frozen yogurt [75]. Both these products were retrieved from the marketplace without incident.

IMPORTED CHEESE France International concern over the potential health hazard of consuming Listeria-contaminated cheese also stems from the California listeriosis outbreak of 1985. This outbreak and an earlier link between consumption of French Brie and/or Camembert cheese and several outbreaks of foodborne illness in

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415

the United States and Europe caused by enterotoxigenic and/or enteropathogenic E. coli [275,298,385] prompted a meeting in September 1985 between FDA officials and representatives of the French Embassy/French Delegation on Food Safety and Food Distribution to discuss the FDA’s plans for inspecting imported soft cheese [14]. Late in September, representatives from the Codex Committee on Food Hygiene and the International Dairy Federation agreed with FDA officials that a “Code of Hygienic Practices” should be developed for manufacturing fresh and soft cheese [8]. Use of raw milk (a known source of L. monocytogenes) to improve organoleptic properties of certain cheeses was cited as a particular area of concern. Although a basic certification program for soft cheese produced in France was in operation for some time, agreement on a general Code of Hygienic Practices for manufacture of soft cheese was not reached during the remainder of 1985. In January 1986, as part of a research effort to enhance recovery of listeriae from cheese, FDA officials inadvertently isolated a pathogenic strain of L. monocytogenes from two uninoculated control samples of French Brie cheese purchased at a local supermarket [20,113]. Ironically, both cheeses were prepared from pasteurized milk in a cheese factory certified by the French government under the existing soft-ripened cheese agreement. In response to these findings, the first in a series of nationwide recalls was issued in February 1986 for Listeria-contaminated French Brie cheese (Table 12.3). The following week, the French firm that manufactured the tainted cheese agreed to halt all production [20]. Shipment of additional cheese that was previously certified “Listeria free” by an independent French laboratory (certification by the French Ministry of Agriculture began on January 1, 1986) was also stopped pending identification of the contamination source. In addition, all suspect lots of French Brie cheese en route to the United States were detained on entry and tested for Listeria by the FDA before being released. Shortly thereafter, sampling was extended to include virtually all lots of French Brie cheese produced by this manufacturer. The French cheese industry was dealt its most serious blow in March 1986, when approximately 660 million pounds of Brie cheese produced by five different manufacturers were recalled in the United States (Table 12.3). This recall, which involved nearly 60% of all Brie cheese marketed in the United States, immediately raised the possibility of block-listing French firms that produced contaminated cheese [22]. Consequently, FDA officials immediately began testing all shipments of French soft-ripened cheese as well as 20% of French soft/semisoft cheese and 20% of all other imported soft cheeses for Listeria, E. coli, and phosphatase (see Figure 12.1) [25]. Recognizing the danger of contracting listeriosis from consuming contaminated French softripened cheese, FDA officials drafted the following proposal to detain, test, and certify all French soft-ripened cheeses exported to the United States [74]: FDA intends to detain any entry that is found to be Listeria-positive, regardless of the species of Listeria found. French soft-ripened cheese without a certificate indicating negative results for the Listeria analysis or with a positive analysis for Listeria will be detained. … In addition, all French soft-ripened cheeses are to be sampled and analyzed for the presence of Listeria. [Under the previous agreement, cheeses were shipped with a certificate of analysis that only indicated the level of E. coli and the absence of phosphatase.] The analysis may be carried out by a private laboratory after the cheese has arrived in the United States, or alternatively the analysis may be conducted so that the availability of the results will coincide with the arrival of the shipment in the United States. The timing of the analysis is important to ensure that the nature of the cheese tested, particularly the pH, is identical to that examined by the FDA, if we decide to perform an audit on the entry. Thus, importers of French soft-ripened cheese should provide FDA with certificates indicating the dates testing was initiated and completed. Cheese shipments will not be released without a certificate indicating negative results for Listeria analysis. …

In this proposal, FDA officials stressed that other methods used to detect Listeria in cheese should conform to the 7-day FDA method, and also suggested that 1/16-in.-thick slices from the cheese surface (sample with highest pH) be analyzed for listeriae rather than cross-sectional plugs of cheese. Following FDA threats to halt importation of soft-ripened cheese, the French Ministry of Agriculture agreed to begin lot-by-lot testing in April 1986, as outlined in the March FDA proposal [26].

France France France France Italy Denmark Denmark Denmark Denmark

2/12/1986 2/14/1986 2/14/1986 2/14/1986 2/14/1986 2/21/1986 2/24/1986 3/14/1986 4/1/1986 6/23/1986 8/13/1986 8/18/1986 4/16/1987 1/27/1988 2/11/1988 2/12/1988 2/18/1988 4/6/1988 5/26/1989 7/27/1989 8/9/1989 9/15/1989 7/27/1990

Brie

Brie Brie

Brie Brie Brie Brie Brie Brie

Soft-ripened: Tourre de l’Aubier and Fromage des Burons Soft-ripened: Tourte de l’Aubier Semisoft: Morbier Rippoz Soft-unripened, full fat Semisoft Semisoft: L’Amulette Danish Estron Semisoft: L’Amulette Danish Estron Semisoft: L’Amulette Danish Estron Blue

Anari Anari Anari Halloumi Italian soft-ripened and semisoft

California, Illinois, Maine, Massachusetts, New Jersey, New York, Oregon Illinois, Massachusetts, Michigan New Jersey, New York, Texas Nationwide California East, Midwest, North, South Florida, New Jersey, New York, Massachusetts California, Florida, Illinois, Massachusetts, Michigan, Minnesota, New Jersey, New York, North Carolina, Oregon, Pennsylvania, Texas New York Illinois, Texas New York Florida, New Jersey, New York California, Connecticut, New Jersey, New York, Pennsylvania

Georgia, New Jersey Colorado, Connecticut, Florida, Louisiana, Maryland, Massachusetts, New Jersey, New York, Ohio, Washington, DC Florida, New York, Washington, DC Nationwide Oregon, Washington State Illinois, Minnesota, New Jersey Nationwide Colorado, Connecticut, Georgia, New Jersey, New York, North Carolina, Texas, Washington, DC New York, Ohio, Pennsylvania

Bermuda, Nationwide

Distribution

50 cases 79 cases 80 cases 14,400 Unknown

1056 ~1600 15 wheels 410 cartons Unknown ~11,500 ~1,150 ~5,000

Unknown

10 cases Unknown Unknown 909 cases ~660 million ~230

57,000 2–6 lb wheels 40 cases 100 cases

Quantity

69 70 71 77 78

33,29,31,35 29,31,35 48, 49 60 57,58,61 57,58,62 57–59,63 56, 57

29,32

17 17,38 16,38 18,40 37,41 28,37

17 17

18,20,27,30

Ref.

416

Cyprus Cyprus Cyprus Cyprus Italy

France France France France France France

France France

France

Date Recall Initiated

Type of Cheese

Country of Manufacture

TABLE 12.3 Chronological List of Class I Recalls in the United States for Imported Cheese Contaminated with L. monocytogenes

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Listeria, Listeriosis, and Food Safety

4/6/1993

2/29/1996 6/7/1996 5/25/1999

6/20/2002 11/21/2002 11/26/2002 12/9/2004

Fontina

Limburger

Jarlsberg

Ricotta

St. Nectaire Havarti Ricotta Manouri France Unspecified Italy Greece

Italy

Norway

Germany

Sweden California, Connecticut, Maryland, Massachusetts, Minnesota, New Hampshire, New York, North Carolina, Pennsylvania, Rhode Island, Washington State Florida, Indiana, Maine, Massachusetts, New Jersey, New York, Ohio, Utah, Virginia Alaska, California, Guam, Hawaii, Idaho, Montana, Nevada, Oregon, Utah, Washington State Connecticut, Florida, Delaware, Georgia, Indiana, Maryland, Massachusetts, Nebraska, New Hampshire, New Jersey, New York, North Carolina, Pennsylvania, South Carolina, Tennessee, Virginia California, Illinois, Ohio, New York Nationwide Nationwide California, Illinois, Michigan, New Jersey, New York, Pennsylvania, Virginia 236 52,998 6702 1–7 cases

164 cases

30,727

813

85,080

389 389 389 389

389

101

102

89

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Incidence and Behavior of Listeria monocytogenes 417

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418

Listeria, Listeriosis, and Food Safety

However, French authorities stressed that such a program would not be practical on a long-term basis and hoped that the FDA would accept an expansion of the existing factory/product certification program to include Listeria testing in the near future. Beginning in May 1986, FDA officials announced that all shipments of French soft-ripened cheese lacking certification of analysis for Listeria would be detained [24]. Inspections during the next 2 months uncovered L. monocytogenes in two French cheeses—a noncertified Brie and a 6-lb certified lot of Muenster [24]—both of which were presumably recalled internally. Continued problems with Listeria-contaminated French soft-ripened cheeses prompted FDA officials to revise the imported cheese surveillance program in August 1986 [19]. These changes allowed immediate detention of French cheeses that were (1) manufactured at a non-governmentcertified factory, (2) unaccompanied by a Listeria-free government certificate, (3) positive for phosphatase, or (4) manufactured by one of several firms that were block-listed for Listeria. Although these changes made importation of French cheeses more difficult, sampling of cheeses that were manufactured at certified factories and accompanied by Listeria-free certificates was decreased to the 20% level. Between June and August 1986, three additional Class I recalls were issued for French semisoft/ soft-ripened cheese contaminated with L. monocytogenes (see Table 12.3). Two of three firms involved in these recalls were previously block-listed by the FDA [19]. An additional Class I recall issued for semisoft Morbier Rippoz cheese (see Table 12.3) was accompanied by the following press release [35]: Although Listeria is a rare cause of human illness, it can be life-threatening to pregnant women and their fetuses, frail elderly persons, or other persons with weakened immune systems. In healthy adults, it is a transient illness with such mild-to-moderate flu like symptoms as fever, headaches, and/or gastrointestinal tract distress.

The language used in such press releases also has received considerable attention. These messages to the public must be firm enough to accomplish the goals of the recall but not so alarming as to create undue panic. After considerable consultation, the governments of France and the United States reached agreement on a French certification program for soft cheese [50,113]. Under this program, which began on February 15, 1987, cheeses were tested before shipping using methods that were mutually acceptable by both governments. French cheeses manufactured at certified factories would be sampled at the 5% level, whereas other French cheeses (and cheeses manufactured in other countries without certification programs) would be analyzed at the 20% level. In the event of a Listeriapositive shipment, personnel at the French cheese factory would be required to investigate the potential source of contamination and analyze every lot of cheese for listeriae in at least the next 20 consecutive shipments destined for the United States. Although a positive finding would not automatically result in suspension of the certified status for a cheese factory under this program, FDA officials reserved the right to initiate detentions and recalls if a product was found to contain L. monocytogenes. After this certification program was accepted, only two additional recalls involving a French soft-ripened full-fat cheese have been reported (see Table 12.3). Other Western European Countries Despite the adverse publicity that the French cheese industry received throughout 1986 and 1987, it must be recognized that the problem of Listeria-contaminated cheese was not limited to France. Between October and December 1986, FDA inspectors isolated Listeria spp. from 4 of 74 (5.4%) cheeses imported from Italy, 2 cheeses of which also contained high levels of phosphatase [46]. After finding similar percentages of positive samples during January, February, and March 1987, FDA officials told representatives of the Italian government to either submit a draft for a certification

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419

program (or recommend an alternate solution) or face a ban on importation of potentially hazardous cheeses into the United States. As of April 30, 1987, only 13 of all Italian cheese samples analyzed complied with current FDA safety standards: free of Listeria, phosphatase, and enteropathogenic strains of E. coli. Additionally, 144 cheese samples examined as part of an import alert were suspected to contain L. monocytogenes [45]. After isolating listeriae from Italian Pecorino Romano cheese prepared from goat’s milk (see Figure 12.1) in June 1987 [365], the previous import alert was extended to include both soft and hard varieties of Italian cheese [51]. (This was the first instance in which L. monocytogenes was isolated from hard cheese.) Subsequently, the FDA ordered intensified sampling of soft and hard cheese for the next 2 months [45]. Late in 1987, FDA officials also increased the number of cheeses sampled from Austria, Denmark, Germany, Italy, and Switzerland as part of the agency’s ongoing imported cheese surveillance program (see Figure 12.1) [58]. Although this action prompted the recall of several Danish cheeses in early 1988 (see Table 12.3), no additional Class I recalls were issued during the remainder of 1988 for imported cheese contaminated with L. monocytogenes. Heightened concern over the presence of this pathogen in European cheeses (which stems from the 1987 cheeseborne outbreak of listeriosis in Switzerland) and subsequent initiation of corrective action are probably both responsible for the lack of Class I recalls issued during the remainder of 1988 and early 1989. However, during the latter half of 1989, FDA officials issued (1) four separate Class I recalls for Listeria-contaminated soft cheeses manufactured in Cyprus (see Table 12.3) and (2) an import alert for contaminated soft and hard cheeses produced by two Italian firms [202]. The overall situation regarding presence of L. monocytogenes in imported cheese has greatly improved since 1986 [79], with only nine additional recalls of imported cheese issued during the 15-year period since 1990. However, continued detection of listeriae in imported cheeses, as evidenced by the importation refusal of 24 cheeses (23 from France and 1 from Colombia) from July 2004 to May 2005 [389], confirms that ongoing surveillance of the soft and semisoft surface-ripened varieties, in particular, is still necessary to safeguard public health, with these cheese categories having been documented as important sources of L. monocytogenes in recent risk assessments.

SURVEYS AND MONITORING PROGRAMS FOR LISTERIA SPP. IN CHEESE PRODUCED OUTSIDE THE UNITED STATES In response to the 1985 cheeseborne listeriosis outbreak in California, scientists worldwide began testing many types of cheese for Listeria spp. Given the possible ramifications of selling Listeriacontaminated cheese and the fact that increasingly large quantities of European specialty cheeses are exported to the United States and Canada, high priority was given to determining the incidence of listeriae in Western European cheeses. Since 1986, over 100 surveys have dealt with the incidence of Listeria spp. in various cheeses. However, because these surveys now span a 20-year period and differ in design (i.e., number and size of sample, site of sample collection [cheese factory or retail store], age of sample, portion of cheese analyzed [surface, interior, or both]), and methods for detecting and identifying listeriae, many of the Western European studies and those conducted elsewhere need to be interpreted with some caution.

CANADA Reports from federal monitoring programs in both Canada and the United States [58,247] indicate that the incidence of listeriae in Canadian cheese (and nonfermented dairy products as described in Chapter 11) is relatively low. However, one outbreak was reported in Quebec during 2002 in which 17 cases of listeriosis were traced to four types of soft or semihard raw milk cheese that were aged at least 60 days before consumption [149].

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In the only Canadian survey thus far reported, Farber et al. [203] examined 182 samples of soft and semisoft cheese for listeriae using the original FDA method. The cheeses analyzed in this survey were produced at 61 different factories, most of which were located in the provinces of Ontario and Quebec. Although all cheeses examined were Listeria-free, 19 of 79 samples (24%) were positive for phosphatase, which suggests that these cheeses may have been prepared, at least in part, from raw milk. In addition to this survey, the literature also contains one report of the unconfirmed isolation of L. monocytogenes from Cheddar and Colby cheeses [260], both of which were manufactured from raw milk and held a minimum of 60 days at ≥1.7°C (≥35°F) as required by the Canadian government. Although the incidence of L. monocytogenes in Canadian-produced cheese appears to be low, the pathogen has been detected in cheese exported to Canada from several Western European countries, including Denmark, France, Switzerland, and Germany [67,247]. During April 1997 to June 2005, the Canadian Food Inspection Service issued health hazard alerts for eight primary soft or semisoft cheeses, of which four were domestic, with the remainder imported from France, Greece, Italy, and Portugal [150]. In conjunction with the Canadian survey just discussed, Farber et al. [203] also examined 187 samples of Western European soft and semisoft cheese (98 different brands from 12 different countries) that were regularly exported to Canada. Three soft and semisoft cheeses produced by the same manufacturer in France were positive for Listeria spp. (Table 12.4). Two cheeses contained L. monocytogenes alone, whereas the third contained both L. monocytogenes and L. innocua. In keeping with the previously described 1988 policy regarding foods that have been directly linked to major listeriosis outbreaks, Canadian officials immediately recalled the contaminated cheese. Although some of the tainted cheese was likely consumed before the recall, no cases of listeriosis linked to consumption of this cheese were reported. Despite being labeled as “manufactured from pasteurized milk,” the three French cheeses from which listeriae were isolated yielded positive results with the phosphatase test, as did other cheeses imported from Denmark, Finland, and Switzerland. Such findings, along with unpublished reports of phosphatase in pasteurized dairy products, have raised serious questions as to the validity of the phosphatase test. Results from one study [318] demonstrated that certain heat-labile, microbially produced

TABLE 12.4 Incidence of Listeria spp. in Soft/Semisoft European Cheeses Exported to Canada between October 1985 and March 1987 Country of Origin Austria Denmark Finland France Germany Greece Italy The Netherlands Norway Portugal Sweden Switzerland Total

Number of Positive Samples (%)

Number of Samples Analyzed

L. monocytogenes

L. innocua

Other Listeria spp.

2 43 1 104 16 2 3 2 7 2 2 3 187

0 0 0 3 (2.9) 0 0 0 0 0 0 0 0 3 (1.6)

0 0 0 1 (1.0) 0 0 0 0 0 0 0 0 1 (0.5)

0 0 0 0 0 0 0 0 0 0 0 0 0

Source: Adapted from Farber, J.M., M.A. Johnston, U. Purvis, and A. Loit. 1987. Surveillance of soft and semi-soft cheeses for the presence of Listeria spp. Int. J. Food Microbiol. 5: 157–163.

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TABLE 12.5 Incidence of Listeria spp. in French Cheese Destined for Domestic (France) and Foreign Markets during 1986 and 1987 Number of Positive Samples (%) Market Domestica Foreignb Foreignb

Type of Cheese

Number of Samples Analyzed

L. monocytogenes

Soft Other Soft (pasteurized milk) Soft (pasteurized milk) Soft (raw milk)

192 135 736 736 355 1418

2 (1.0) 0 11 (1.5) 11 (1.5) 6 (1.7) 19 (1.34)

Total

Other Listeria spp. 3 1 10 10 5 19

(1.6) (0.7) (1.4) (1.4) (1.4) (1.34)

a

Results from January 1987 to November 1987. Results from January 1986 to September 1987.

b

Source: Adapted from Bockemuhl, J., G. Schulze, G. Marcy, and H.P.R. Seeliger. 1992. Number and distribution of Listeria monocytogenes in soft cheese: how relevant is the natural contamination for causing disease? In Proceedings of the 3rd World Congress on Foodborne Infections and Intoxications, Berlin, Germany, June 16–19, pp. 496–500.

alkaline phosphatases can mimic the natural phosphatase found in milk and produce false-positive results in the Scharer test. Hence, the ability of the phosphatase test to determine whether or not a dairy product such as cheese was made from pasteurized milk (or from pasteurized milk contaminated with raw milk) needs to be reexamined.

FRANCE Beginning in early 1986, sporadic Listeria-contamination problems have been associated with French soft cheeses exported to the United States and Canada as well as England [247], Germany [76], The Netherlands [128], Norway [247,402], Sweden [247], and Australia [241]. Hence, in an effort to bolster public confidence in the safety of cheeses produced in France, the French government, in cooperation with the Veterinary Service for Food Hygiene in France, conducted a series of systematic surveys to determine the incidence of Listeria spp. in French cheeses destined for domestic and foreign markets (Table 12.5) [136,225]. Overall, 1.34% of predominantly soft, 30-day-old French cheeses examined during 1986 and 1987 contained detectable levels of both L. monocytogenes and other Listeria spp., with few differences observed between cheeses destined for domestic or foreign consumption. It also is noteworthy that comparable levels of contamination were seen in soft cheeses prepared from raw and pasteurized milk. These findings agree with those of most other surveys and suggest that soft cheeses are most likely to become contaminated with L. monocytogenes during the latter stages of manufacture and ripening. Although L. monocytogenes also was recovered from 10.3% of soft/semisoft French cheeses marketed in Sweden from 1989 to 1993 [268], most surveys have suggested contamination rates of <10%, with 108 of 2425 (4.5%) French cheeses surveyed (Table 12.6) reportedly harboring L. monocytogenes. Based on the six independent surveys in Table 12.6, 4.7% of soft/semisoft and 4.5% of other cheese types yielded L. monocytogenes with contamination rates of 9.7 and 1.7% for soft cheese prepared from raw/heat-treated and pasteurized milk, respectively. Thus, soft cheeses prepared from raw milk were 5.7 times more likely to be contaminated as the same cheeses prepared from pasteurized milk. Contamination rates of 46.9 and 87.0% have been reported for soft surface-ripened cheeses prepared from raw milk [364], with L. monocytogenes populations as high as 106 CFU/g detected on the cheese surface.

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TABLE 12.6 Incidence of Listeria spp. in Cheeses Manufactured outside the United States

Country Europe Austria

Belgium

Croatia Czechoslovakia

Denmark

France

Germany

Type of Cheese

Soft red smear Semisoft red smear Hard red smear Soft Unspecified Unspecified Raw milk Unspecified Hard sheep’s milk Soft-ripened Sheep’s milk Hard Unspecified Soft/semisoft Soft red smear Semisoft red smear Unspecified Soft-ripened (raw milk) Soft, surface-ripened (raw milk Soft (raw milk) Soft (heat-treated milk) Soft-ripened (pasteurized milk) Soft (pasteurized milk) Soft red smear Semisoft red smear Semihard Hard red smear Blue Cottage Unspecified Soft Soft Soft Soft (raw milk) Soft (unripened) Soft (mold-ripened) Soft red smear Soft (smear-ripened)

Number of Samples Analyzed

L. monocytogenes

4 4 2 886 929 262 71 37 38 77 10 33 24 46 3 1 25 330

1 (25.0) 0 0 62 (6.9) 214 (23.0) 35 (13.4) 2 (2.8) 0 0 6 (7.8) 0 0 2 (8.3) 0 0 0 8 (32.0) 3 (0.9)

23

L. innocua

ND ND ND ND ND ND ND

Other Listeria spp.

Ref.

ND ND ND ND ND ND ND ND 6 (1.8)

ND ND ND ND ND 68 (25.9) ND 0 0 ND ND ND ND ND ND ND 8 (32.0) 1 (0.3)

339 339 339 268 182 393 328 139 349 291 291 291 291 268 339 339 136 224

20 (87.0)

ND

ND

290

32 5

15 (46.9) 0

13 (40.6) 2 (40.0)

1 (3.1)a 0

198 198

873

12 (1.4)

5 (0.6)

3 (0.3)

224

32

3 (9.4)

5 (15.6)

0

198

124 25 289 1 126 149 242 712 248 166 22 8 117 52 41

5 (4.0) 0 10 (3.5) 0 0 2 (1.3) 20 (8.3) 33 (4.6) 3 (1.2) 7 (4.2) 2 (9.1) 0 4 (3.4) 6 (11.5) 3 (7.3)

ND ND 1 (0.3) ND 0 0 ND 58 (8.1) 16 (6.4) ND 2 (9.1) 0 5 (4.3) ND 5 (12.2)

0 0

ND ND 1 (0.3) ND 0 0 61 (25.2) 4 (0.6)a 0 ND 0 0 0 ND 0

339 339 224 339 224 224 336 374 356 377 198 396 396 339 396

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TABLE 12.6 (CONTINUED) Incidence of Listeria spp. in Cheeses Manufactured outside the United States

Country

Greece/Crete Hungary

Ireland Italy

Type of Cheese Soft/semisoft Soft/semisoft Semisoft Semisoft Semisoft (unripened) Semisoft (moldripened) Semisoft red smear Semisoft (smearripened) Semihard Semihard Hard red smear Hard Acid curd Acid Acid Fresh Processed Unspecified Pichtogalo Chanion Soft Soft Semisoft Molded Mold-ripened Hard Hard Curd Processed Processed Soft Soft/semisoft Soft Soft Soft Soft Soft Soft Soft Soft-unripened Soft-unripened Soft-ripened Soft surface-ripened (mold)

Number of Samples Analyzed

L. monocytogenes

256 31 268 45 89

23 1 9 0 4

12

(9.0) (3.2) (3.4)

L. innocua

Other Listeria spp.

Ref.

ND ND 7 (2.6) 1 (2.2) 1 (1.1)

7 (2.7) ND 8 (3.0)a 0 3 (3.4)a

136 268 374 356 396

0

0

0

396

42 7

4 (9.5) 2 (28.6)

0

237 108 26 42 41 61 48 149 21 89 62 25 15 25 10 10 15 10 15 20 15 40 17 1,284 400 54 29 21 30 12 69 18 136 16

7 (3.0) 6 (5.6) 1 (3.8) 2 (4.8)b 2 (4.8) 2 (3.3) 1 (2.1) 0 0 8 (9.0) 4 (6.5) 0 0 0 2 (20.0) 0 1 (6.7) 0 0 0 0 1 (2.5) 0 65 (5.1) 8 (2.0) 2 (3.7) 0 2 (1.6) 0 1 (8.3) 6 (8.7) 0 9 (6.6) 0

(4.5)

ND

14 (5.9) ND ND 0 8 (19.5) 22 (36.1) ND 0 0 17 (19.0) ND ND ND ND ND ND ND ND 0 ND 0 ND ND 140 (10.9) ND 4 (7.4) ND 2 (1.6) 1 (3.3) ND ND ND ND ND

ND 0 0 ND ND 1 (2.4)a 0 1 (1.6)a ND 0 0 0 ND ND 4 (26.7)c ND ND ND 4 (26.7)c ND 0 ND 0 ND ND 218 (17.0)d ND 0 ND 0 0 ND ND ND ND ND

339 396 374 377 339 376 338 376 377 376 376 357 310 331 245 331 331 331 245 331 245 331 245 247 169 163 160 176 215 280 381 316 326 162 326 176

(continued)

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TABLE 12.6 (CONTINUED) Incidence of Listeria spp. in Cheeses Manufactured outside the United States

Country

Type of Cheese Soft surface-ripened Soft red smear Fresh Fresh Fresh Soft/semisoft Soft/semisoft Semisoft red smear Semisoft Ripened Hard red smear Hard Hard Hard Goat’s milk Goat’s milk Sheep’s milk Unspecified Unspecified Unspecified Unspecified Unspecified Unspecified Asiago Caciocavallo Corleonese Crescenza Crescenza Gorgonzola Gorgonzola Gorgonzola Gorgonzola Gorgonzola Gorgonzola Mozzarella Mozzarella— buffalo’s milk Mozzarella Mozzarella Mozzarella Mozzarella Mozzarella Mozzarella Mozzarella Pecorino

Number of Samples Analyzed

L. monocytogenes

L. innocua

Other Listeria spp.

Ref.

90 5 239 38 17 64 36 14 118 50 4 99 40 10 24 21 40 1,846 373 115 75 62 52 12 12

1 (1.1) 0 0 2 (5.3) 0 0 1 (2.8) 3 (21.4) 0 1 (2.0) 1 (25.0) 0 0 0 0 1 (4.8) 0 24 (1.3) 22 (5.9) 6 (5.2) 0 3 (4.8) 4 (7.7) 0 0

10 (11.1) ND 0 2 (5.3) ND ND ND ND 5 (4.2) 0 ND 0 0 ND ND ND ND ND 50 (13.4) 3 (2.6) ND ND 0 ND 0

1 (1.1)c ND 0 0 ND ND ND ND 0 0 ND 0 0 ND ND ND ND ND 70 (18.8)b 0 ND ND 0 ND 0

381 339 163 176 215 162 268 339 163 335 339 163 176 162 143 162 143 123 164 320 303 147 136 143 386

212 15 67 58 44 40 40 25 94 94

0 0 6 (9.0) 3 (5.2) 4 (9.1) 2 (5.0) 2 (5.0) 2 (8.0) 15 (16.0) 0

ND 0 28 (41.8) 21 (36.2) ND 4 (10.0) 2 (5.0) ND 10 (10.6) ND

ND 0 1 (1.1)a 0 ND 0 0 ND 1 (1.1)a ND

143 139 320 315 316 176 139 215 320 219

74 50 30 29 24 20 14 8

0 0 0 4 (13.8) 0 2 (10.0) 0 0

2 (2.7) 0 0 2 (6.9) ND ND ND ND

2 (2.7)e 0 0 1 (3.4)e ND ND ND ND

176 116 198 315 143 316 215 143

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TABLE 12.6 (CONTINUED) Incidence of Listeria spp. in Cheeses Manufactured outside the United States

Country

The Netherlands

Norway Portugal

Spain

Sweden

Type of Cheese Ricotta Ricotta Ricotta Ricotta—buffalo’s milk Talleggio Talleggio Taleggio Taleggio Tosone—raw milk Soft (raw milk) Soft (pasteurized milk) Soft (domestic) Soft (imported) Semihard raw sheep’s milk Hard pasteurized sheep/goat’s milk Hard pasteurized sheep/cow’s milk Hard raw sheep/cow’s milk Hard/semihard Fresh Soft Soft Semihard/hard Blue (raw milk) Unspecified Unspecified (raw milk) Unspecified (raw milk) Unspecified (pasteurized milk) Carneros—soft goat’s milk Cebrero Queso fresco/cottage Soft/semisoft Soft Soft Soft (mold-ripened)

Number of Samples Analyzed

L. monocytogenes

L. innocua

50 32 11 18

2 (4.0) 0 0 0

45 38 15 12 45 938 484

0 0 0 0 0 43 (4.6) 10 (2.0)

7 (15.6) ND 0 ND 2 (4.4) ND ND

850 90 9

0 10 (11.0) 4 (44.4)

12 11

Other Listeria spp.

ND

ND ND

144 167 153 219

2 (4.4) ND 0 ND ND ND ND

315 316 139 143 266 382 382

ND ND ND

NDg ND ND

333 333 237

2 (16.7)

ND

ND

237

0

ND

ND

237

2

2 (100)

ND

ND

237

68 23 99 14 20 11 ~100 49

8 (11.8) ND 1 (1.0) ND 0 0 0 1 (2.0)

5 (7.4) ND 6 (6.1) ND 0 ND ND 0

4 (5.9) 1 (4.3)b 1 (1.0) 1 (4.3)g 0 ND ND 1 (2.0)a

236 278 392 278 278 269 247 321

42

10 (23.8)

ND

ND

156

0

ND

Ref.

0 ND ND

21

0

ND

ND

156

18

1 (5.6)

ND

ND

300

49 91

1 (2.0) 7 (7.7)

ND 4 (4.4)

ND 2 (2.2)a

322 137

27 24,954 604 54

0 324 (1.3) 40 (6.6) 0

ND ND 38 (6.3) 0

ND ND

268 304 142 42

0 ND

(continued)

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TABLE 12.6 (CONTINUED) Incidence of Listeria spp. in Cheeses Manufactured outside the United States

Country

Switzerland

Turkey

Yugoslavia United Kingdom England England/Wales

Northern Ireland Scotland Elsewhere Australia

Brazil

Type of Cheese Soft (smear-ripened) Semisoft Semisoft (moldripened) Semisoft (smearripened) Semisoft (smearripened) Semisoft red smear Hard/semihard Hard Hard red smear Unspecified Kashor White White White Unspecified Unspecified (raw milk) White-brined Soft Soft/semisoft Soft Soft Soft Soft Soft-ripened Soft-unripened Hard Ewe’s milk Goat’s milk Soft Soft Unspecified Soft Soft Unspecified Unspecified Minas Frescal Coalho Gorgonzola, Brie, Roquefort

Number of Samples Analyzed 18 205 261

L. monocytogenes 4 (22.2) 4 (1.9) 7 (2.7)

L. innocua 3 (16.7) 0 ND

Other Listeria spp. ND

Ref.

ND

42 142 142

0

343

33 (9.6)

ND

ND

142

69

6 (8.7)

13 (18.8)

ND

42

ND ND

ND ND

6 36,379 88 12 17 30 82 50 30 224 40 170 251 12 1,437 251 222 131 769 366 66 141 476 33 27 305 437 28 338 126 20 43 53

0 2,692 (7.4) 0 0 0 0 11 (13.4) 2 (4.0) 0 4 (1.8) 2 (6.1)

0

0

ND ND ND ND 6 (12.0) ND 7 (3.1) ND

ND 1 (5.9) ND ND 0 ND 0 ND

339 304 142 339 136 232 231 114 232 238 232

ND

182

ND ND ND ND ND ND ND ND ND ND 1 (3.3)a ND 1 (3.3)a

271 268 218 271 314 135 234 234 234 234 234 241 337 124

24 (5.5)c 0 ND 0 0 ND 1 (1.9)

115 115 390 233 155 205 172

9 (5.3)

ND

1 (0.4) 0 16 (1.1) 10 (4.0) 23 (10.4) 0 63 (8.2) 4 (1.1) 1 (1.5) 1 (0.7) 22 (4.6) 0 3 (11.1) 3 (1.0)c

9 (3.6) ND ND ND ND ND ND ND ND ND ND 0 ND 0

15 (3.4) 1 (0.2) 6 (1.8) 1 (0.8) 0 1 (2.3) 3 (5.7)

ND 0 ND 1 (0.8) 0 ND 7 (13.1)

0

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TABLE 12.6 (CONTINUED) Incidence of Listeria spp. in Cheeses Manufactured outside the United States

Country

Chile Costa Rica Egypt Japan

Jordan Korea Mexico

Morocco

United Arab Emirates

Type of Cheese Minas Frescal Homemade Minas Frescal Commercial Minas Frescal and Ricotta Soft Hard Soft Damietta Kareish Domestic fresh Domestic soft/semisoft Domestic semihard/hard Imported fresh Imported soft/semisoft Imported semihard/hard Imported other White brined Pasteurized processed Chihuahua— pasteurized milk Fresh Panela—raw and pasteurized milk Manchego— pasteurized milk Domestic fresh Imported moldripened Domestic Imported

Number of Samples Analyzed

Other Listeria spp.

Ref.

0 7 (41.2)

1 (8.3) 3 (17.7)

0 0

362 172

33

1 (3.0)

3 (9.0)

6 (18.2)

172

256 155 20 50 100 92 94

2 (0.8) 0 9 (45.0) 1 (2.0) 1 (1.0) 0 0

ND ND ND 1 (2.0) 3 (3.0) ND ND

105

0

94 418

0 16 (3.8)

ND ND ND 0 1 (1.0)h ND ND

167 167 293 204 204 296 296

ND

ND

296

ND ND

ND ND

296 296

403

0

ND

ND

296

55 67 45

0 0 0

ND ND

ND ND

0

0

296 192 372

40

0

0

0

372

40

6 (15.0)

6 (15.0)

40

0

0

20 45

0 0

53

0

0

0

228

2 (1.0)

2 (1.0)

0

228

196

L. seeligeri. Isolated from sheep’s milk cheese. cNon–L. monocytogenes. d185 L. welshimeri, 18 L. ivanovii, 15 L. murrayi. eL. grayi f63 L. welshimeri, 6 L. ivanovii, 1 L. murrayi. gListeria sp. hL. welshimeri. b

L. innocua

12 17

Note: ND = Not determined. a

L. monocytogenes

14 (35.0) 0

ND ND

348 372

ND ND

190 190

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In another French survey [224], workers at the Veterinary Service for Food Hygiene recovered L. monocytogenes as well as L. innocua and other Listeria spp. from 0.3 to 3.5% of cottage, softripened, and semihard cheeses examined (Table 12.6). However, in contrast to other surveys, comparable contamination rates were observed for soft-ripened cheese prepared from raw and pasteurized milk. Additional efforts in France have focused on characterizing listeriae isolates from cheese and other milk products. Listeria spp. recovered from French dairy products during 1986 included L. monocytogenes (370 strains), L. innocua (134 strains), L. seeligeri (17 strains), and L. ivanovii (1 strain), with 299 of 370 (80%) and 48 of 370 (13%) L. monocytogenes strains belonging to serovars 1/2 and 4b, respectively. Additional surveys conducted in France [136,225] and Belgium [116] from 1985 to 1990 indicated that a disproportionately large number of L. monocytogenes strains isolated from cheese and other dairy products were serovar 1/2. This situation appears to be reversed in the United States, with isolates of serovar 4b typically outnumbering those of serovar 1/2. In one of the earlier studies, phage typing was used to characterize particular L. monocytogenes strains isolated from dairy products. Although only 33.8% of all L. monocytogenes strains isolated from French dairy products during 1986 and 1987 were typeable using the available set of phages, some phage types were unique to particular regions within France [136]. In some instances, excellent correlations were observed between specific phage types and certain cheese varieties, with some phage types even being specific to a particular dairy. Such findings have led to better control of the listeriosis problem within the dairy industry. The inadvertent isolation of L. monocytogenes from French soft-ripened cheese by FDA officials in January 1986 prompted several additional surveys of French cheese exported to other Western European countries. Working in The Netherlands, Beckers et al. [128,129] examined 69 samples of French soft cheese (i.e., Brie and Camembert) for L. monocytogenes using both direct plating and cold enrichment. The pathogen was recovered from 7 of 69 (10.1%) cheeses at levels ranging between 103 and 106 CFU/g. Cold enrichment uncovered three additional cheeses with L. monocytogenes for a total of 10 positive samples. Although all 10 Listeria-positive cheeses were prepared from raw milk, comparable rates of contamination have been reported for cheese manufactured from raw and pasteurized milk [136,224].

GERMANY Germany experienced a major outbreak of listeriosis shortly after World War II. This outbreak, which may have resulted from consumption of contaminated raw milk, led to an increased interest in listeriosis research, and this in turn prompted Professor H. P. R. Seeliger to publish his timehonored monograph Listeriosis in 1961. During the last 40 years, the late Professor Seeliger emerged as one of the world’s leading authorities on listeriosis. In addition, he operated a listeriosis research center at the Institut für Hygiene und Mikrobiologie der Universität Würzburg to which Listeria isolates could be sent for biochemical and serological confirmation. Hence, it is not surprising that the incidence of listeriae in cheese has received considerable attention in Germany. Since 1990, German officials have been enforcing a policy similar to that adopted in Canada in which only contaminated foods previously associated with foodborne listeriosis outbreaks are recalled from the marketplace. Although spared the heavy economic losses experienced by the United States and France, Germany and most other European countries have not escaped the Listeria problem completely unscathed. Despite rigorous testing, Listeria-laden German blueveined cheese was recalled from France [247], with a similar recall being issued for sour milk cheese exported to Canada [67,247] and The Netherlands [247]. In 2000, Germany also recalled one hard and one unspecified cheese because of L. monocytogenes contamination. Consequently, a series of Listeria-monitoring programs now continues for German soft, semisoft, semihard, and hard cheeses as well as cultures, cheese byproducts, and the general environment within cheese factories.

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Results from various surveys made since 1986 (see Table 12.6) indicate that 0–11% (average of 4.4%) and 0–28.6% (average of 2.9%) of the soft and semisoft cheeses marketed in Germany contained L. monocytogenes, respectively, with the highest incidence of listeriae generally occurring in smear-ripened varieties. With few exceptions, L. innocua was isolated more frequently from soft and semisoft cheese than was L. monocytogenes. Although somewhat similar average percentages were reported for the incidence of L. monocytogenes in semihard (3.8%) and hard cheese (4.8%), two of the three hard cheeses that contained L. monocytogenes were reportedly manufactured from ewe’s rather than cow’s milk. Overall, it appears that the Listeria contamination rate for hard cheeses prepared from cow’s milk may still be relatively low, as also was observed in Switzerland (see Table 12.6). Hence, these results from Germany generally agree with those from other surveys in that L. monocytogenes was found more frequently in high- rather than low-moisture cheese, particularly smear- and surface-ripened varieties. Of the three remaining categories of German cheese shown in Table 12.6, only acid curd cheese was positive for listeriae. The apparent absence of Listeria spp. from samples of fresh (i.e., cottage) and processed cheese is expected, because relatively severe heat treatments are used in their manufacture. Even if a few listeriae survived cheesemaking, most, if not all, of the survivors would have been sublethally injured during exposure to heat and/or acid and would therefore be unable to grow in most selective enrichment broths that are commonly used for examining cheese. In the only other study thus far reported, Weber et al. [396] examined various German cheeses, including 11 types manufactured from ewe’s and goat’s milk, for listeriae (Table 12.7). Although all cheeses prepared from ewe’s or goat’s milk were free of L. monocytogenes, L. innocua was detected in one sample of fresh goat’s milk cheese. In addition to cows, lactating sheep and goats can also shed L. monocytogenes in their milk. However, information concerning the incidence of L. monocytogenes in cheese prepared from nonbovine milk is far less plentiful. From the survey data presented in Table 12.6, 29 of 575 (5.0%) samples of goat’s milk cheese from Italy and the United Kingdom yielded L. monocytogenes, as did 5 of 228 (2.2%) sheep’s milk cheeses analyzed in Italy, the United Kingdom, and Croatia, with 112 samples of Italian buffalo milk Mozzarella testing negative. In addition to the aforementioned survey of German hard cheese produced from ewe’s milk [374], Tham [378] also reported isolating

TABLE 12.7 Incidence of Listeria spp. in Domestic and Imported Cheese Analyzed in Germany between October 1987 and June 1988 Type of Cheese/Milka Fresh/cow Fresh/goat Soft/cow Soft/goat Soft/ewe Semisoft/cow Semisoft/ewe Semihard/cow Hard/cow a

Number of Samples Analyzed 21 1 307 1 3 144 6 22 4

Number of Positive Samples (%) L. monocytogenes

L. innocua

2 (9.5) 0 8 (2.6) 0 0 19 (13.2) 0 0 0

0 1 (100.0) 11 (3.6) 0 0 8 (5.6) 0 11 (50.0) 0

Milk from which cheese was manufactured.

Source: Adapted from Weber, A. von, C. Baumann, J. Potel, and H. Friess. 1988. Nachweis von Listeria monocytogenes und Listeria innocua in Käse. Berl. Münch. Tierärztl. Wochenshr. 101: 373–375.

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L. monocytogenes from one sample of 8-week-old goat cheese marketed in Sweden. Given similar L. monocytogenes contamination rates for cheeses prepared from cow’s milk, the cheesemaking procedure as well as the moisture content and other aspects of the cheese that can enhance growth and/or survival of Listeria are of greater importance than the source of milk. The incidence of L. monocytogenes in the remaining cheeses prepared from cow’s milk was similar to those values obtained in other studies (see Table 12.6), with the pathogen being detected in 2.6 and 13.2% of the soft and semisoft (presumably mold- and smear-ripened varieties) cheeses examined, respectively. The unusually high incidence of L. monocytogenes in fresh curd cheese (9.5%) is probably the result of contamination during later stages of manufacture or packaging as well as the small number of samples examined. As was true for surveys of French soft-ripened cheese discussed earlier, most L. monocytogenes isolates were serovar 1/2 (22 strains), as also reported by Schönberg et al. [357], with the remaining seven isolates being classified as 4ab or 4b. However, because most clinical L. monocytogenes isolates from Germany are serovar 4b, it appears that some questions remain concerning the ability of present isolation methods to recover L. monocytogenes serovar 4b as compared with other serovars from various foods, including cheese.

ITALY Public health concerns raised in the United States and elsewhere following the isolation of L. monocytogenes from various cheeses have now prompted nearly 30 surveys of cheeses manufactured in Italy (see Table 12.6). Overall, L. monocytogenes was recovered from 204 of 6655 (3%) Italian cheeses surveyed, with this pathogen being most prevalent in Gorgonzola (6.9%), followed by mozzarella (4.9%) and various soft cheeses (4.3%). In another study, Cantoni et al. [151] reportedly isolated L. monocytogenes from 14 of 375 (3.7%), 14 of 216 (6.5%), and 5 of 95 (5.3%) samples of Gorgonzola (blue-veined), Tallegio (soft, surface-ripened), and other Italian cheeses, respectively. Although a follow-up study demonstrated L. monocytogenes at levels of <100 to 12,000 CFU/g in Gorgonzola and Taleggio cheese, respectively, the pathogen was never recovered from 1150 samples of soft, semisoft, semihard, or hard cheese or from 72 samples of pasta filata-type cheese such as provolone and mozzarella. When present, however, L. monocytogenes serovar 1 typically predominated [351], as has been reported for other European cheeses. Between January 1987 and September 1988, Massa et al. [280] also examined 54 soft rindless (i.e., Mascarpone, mozzarella, Crescenza) and 67 soft thin-rind (i.e., Italico, Caciotta) cheeses produced by both large and small northern Italian factories for listeriae and E. coli. Listeria monocytogenes was detected in only 2 of 47 (4.2%) thin-rind cheeses manufactured by one small factory, with core samples from these two positive cheeses being negative for the pathogen. Although all other thin-rind and rindless soft cheeses were free of L. monocytogenes, two mozzarella cheeses contained detectable levels of L. innocua. According to these investigators, E. coli populations in these cheeses ranged from <10 to 8 × 105 CFU/g, with the two L. monocytogenes-positive cheeses containing 104 E. coli CFU/g. However, because 14 similar Listeria-free cheeses also contained 103 E. coli CFU/g, E. coli is clearly a poor indicator organism for possible presence of listeriae.

SWITZERLAND Most Western European countries have experienced various degrees of economic loss from Listeriacontaminated cheese; however, thus far only Switzerland, France, and the United States have been forced to deal with major outbreaks of cheeseborne listeriosis. Well before the 1987 listeriosis outbreak in Switzerland (linked to consumption of Vacherin Mont d’Or soft-ripened cheese), Swiss officials began examining various cheeses for listeriae. Although these surveys apparently were prompted by the 1985 listeriosis outbreak in California, an unusually high incidence of unexplainable listeriosis cases in certain areas of Switzerland may have provided added incentive to initiate these surveys. A two-stage Listeria-monitoring program was later established for cheese and other

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dairy products with random testing of 10-g samples obtained at both the factory and retail levels [247]. According to the Federal Bureau of Health, such samples must be completely free of L. monocytogenes before the product is deemed acceptable. Based on results from a 10-year-long survey of more than 60,000 cheeses conducted by the Swiss Dairy Research Station during 1990–1999, 7.4 and 1.3% of all hard/semihard and soft cheeses yielded L. monocytogenes, respectively. In this study, serotype 1/2b was more frequently isolated from hard/semihard cheese, whereas serotype 1/2a predominated in soft cheese. Working in Switzerland, Breer [142] examined 799 domestic and imported cheese samples for listeriae during the winters of 1985 and 1986. Various Listeria spp. were detected in 19.2% of the soft surface-ripened cheeses, all of which were traced to 10 Swiss and a few foreign manufacturers. During follow-up investigations of these 10 cheese factories in Switzerland, Listeria spp. were isolated from surfaces of various cheeses and also from curing and smearing brines, wastewater sinks, and surfaces of wooden boards used in cheese ripening. In addition, identical serovars of L. monocytogenes (1/2b and/or 4b) and L. innocua were isolated repeatedly from the same cheese factories. These findings demonstrate that ample opportunity existed for cheese to become contaminated with listeriae during the later stages of manufacture and ripening. Subsequently, Breer [142] reported that 4.9 and 4.7% of all cheeses sold in Switzerland contained L. monocytogenes and L. innocua, respectively (see Table 12.6). Of equal importance is the fact that both Listeria spp. were isolated more frequently from soft (6.6, 6.3%) than semisoft cheese (1.9, 0%) and that neither organism was detected in 88 samples of hard cheese. During 1986, Breer [141] also found that 12.9 and 10.0% of soft surface-ripened cheeses manufactured in Switzerland were contaminated with L. monocytogenes and L. innocua, respectively (Table 12.8). The incidence of both Listeria spp. was generally twice as high in smear- rather than mold-ripened cheese. As in previous studies [339], the rate of Listeria contamination was typically independent of the type of milk (raw or pasteurized) from which smear-ripened cheeses were manufactured. These Swiss studies, along with several of the aforementioned German surveys, indicate a greater likelihood of isolating L. monocytogenes and L. innocua from high- rather than low-moisture cheese, with special emphasis on mold- and smear-ripened varieties. In support of this observation, Bannerman and Bille [125] found that 110 of 449 (24.5%) rinds from soft cheese produced in

TABLE 12.8 Incidence of Listeria spp. in Soft Mold- and Smear-Ripened Cheeses Manufactured in Switzerland during 1986 from Pasteurized and Raw Milk Type of Cheese Mold-ripened Raw milk Pasteurized milk Smear-ripened Raw milk Pasteurized milk Total

Number of Samples Analyzed

Number of Positive Samples (%) L. monocytogenes

L. innocua

22 9

2 (9.1) 0

0 1 (11.1)

17 22 70

3 (17.6) 4 (18.2) 9 (12.9)

4 (23.5) 2 (9.1) 7 (10.0)

Source: Adapted from Breer, C. 1986. The occurrence of Listeria spp. in cheese. In Proceedings of 2nd World Congress on Foodborne Infections and Intoxications, Institute of Veterinary Medicine, Robert von Ostertag Institute, Berlin, pp. 230–233.

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Switzerland were contaminated with Listeria spp., including a high percentage of samples with L. monocytogenes. During an additional survey made between October 1986 and September 1987 [42], L. monocytogenes was detected in 4 of 18 (22.2%) and 6 of 67 (8.7%) smear-ripened soft (i.e., Limburger, Romadur, Muenster, Reblochon) and semisoft (i.e., St. Paulin, Tilsiter, Mutschli, Raclette) cheeses, respectively, with many cheeses also containing L. innocua. From the apparent widespread distribution of Listeria within some cheese factory environments, it follows that cheeses prepared from raw and pasteurized milk are equally likely to contain listeriae. Additional information concerning the incidence and control of listeriae in dairy factories and other food processing facilities is given in Chapter 18.

OTHER EUROPEAN COUNTRIES As already implied, ongoing Listeria problems that have affected the European cheese industry are not limited to France, Germany, Italy, and Switzerland. Other European nations, including Austria, Belgium, Denmark, England, Finland, Greece, Hungary, The Netherlands, Spain, and Sweden, also expressed concern in a March 1989 poll conducted by the International Dairy Federation [247]. According to the survey, L. monocytogenes was isolated from domestic cheese sold in Austria (soft cheese), Belgium (rind-type cheese), Denmark (various soft cheeses), England (various cheeses), Ireland (soft farm-house cheese), The Netherlands (young farm-house Gouda), and Sweden (goat’s milk cheese), with this pathogen later being identified in cheeses from Czechoslovakia (soft-ripened) [291], Greece (Feta, Pichtogalo Chanion) [182,310], Hungary (mold-ripened) [245], Ireland (soft) [247], Norway (soft imported) [333], Portugal (hard/semihard) [237], Turkey (soft, white) [232,238], and Yugoslavia (white brined) [182] (see Table 12.6). Denmark, England, and Sweden have also experienced problems with soft or semisoft cheeses imported from Denmark, France, Germany, and Italy (Table 12.9). In contrast, Norway [124,399] and Spain [247] have been primarily

TABLE 12.9 Incidence of L. monocytogenes in Cheeses Marketed in Sweden from 1989 to 1993 Type of Cheese

Type of Cheese Milk

Country of Origin

White Mold

Green/Blue Mold

Smear-Ripened

Other

Heat-Treated

Raw

Austria Denmark England France Germany Greece Italy The Netherlands Norway Romania Spain Sweden

— 0/19 — 15/119 (12.6) 0/8 — 0/5 — — — — 0/3

0/1a 0/27 0/12 0/23 1/19 (5.3) — 0/25 — 0/1 — 0/1 0/13

— — — 2/18 (16.7) 0/1 — 1/2 (50.0) 0/2 — — — 0/3

— — — 0/14 0/3 0/1 0/4 — — 0/1 — 0/8

0/1 0/46 0/12 5/144 (3.5) 1/31 (3.2) 0/1 1/36 (2.8) 0/2 0/1 0/1 0/1 0/26

— — — 13/30 (43.3) — — — — — — — 0/1

Total

15/154 (9.7)

1/122 (0.8)

4/26 (15.4)

0/31

7/302 (0.4)

13/31 (41.9)

a

Number of positive samples/number of samples analyzed (%).

Source: Adapted from Loncarevic, S., M.-L. Danielsson-Tham, and W. Tham. 1995. Occurrence of Listeria monocytogenes in soft and semi-soft cheeses in retail outlets in Sweden. Int. J. Food Microbiol. 26: 245–250.

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affected by imported soft and blue-veined cheeses. Thus, in addition to France, Italy, Switzerland, and Germany, other EU member countries including Austria, Belgium, Denmark [68], England, Finland, Greece, Hungary, The Netherlands, and Sweden also have developed programs to actively monitor the incidence of listeriae in domestic/imported cheese (especially soft surface-ripened varieties) and manufacturing environments within cheese factories. As of March 1989 [247], Austria, Denmark, France, Germany, Greece, Hungary, Italy, Sweden, and Switzerland had regulations regarding the sale of cheese and other foods contaminated with L. monocytogenes, with most European countries now attempting to prevent distribution and sale of cheese (and in some instances other ready-to-eat foods) containing ≥100 L. monocytogenes CFU/g. Because these observations along with the 1987 listeriosis outbreak in Switzerland involving Vacherin Mont d’Or cheese both support the widespread notion that L. monocytogenes poses a significant health threat to certain segments of the population, the European Economic Community Consumers Association published a list of soft and semisoft cheeses manufactured in France and Switzerland that should be avoided by susceptible individuals—pregnant women, immunocompromised adults, and the elderly. This highly controversial list of cheeses (and brands when applicable) included Brie (La Renommee), Muenster (Ermitage), Crème de Bleu (Diapason), Lys Bleu, Camembert (I Signy), Tilsit, Fourme de Bresse, Bleu de Bresse, Reblochon, Pont L’Eveque, Gruyère, and Vacherin Mont d’Or. In February 1988, a case of cheeseborne listeriosis was reported in England in which a 40-year-old woman contracted meningitis shortly after consuming Anari-type soft goat’s milk cheese that contained L. monocytogenes at levels >107 CFU/g. During follow-up investigations at the factory [224], the same L. monocytogenes strain also was isolated from 8 of 11 and 4 of 8 factory and retail samples of Halloumi and Cheddar cheese, respectively, as well as single samples of Gjestost and soft chive cheese. In addition, L. innocua also was recovered from several samples of Halloumi and Cheddar cheese. As in the previously described studies by Pini and Gilbert [283] and Massa et al. [280], no clear relationship was observed between the presence of L. monocytogenes/ L. innocua and coliforms/E. coli. According to several additional surveys, some Costa Rican [293] and Turkish cheeses [179] contained 104 L. monocytogenes and ≥102 coliforms CFU/g. Hence, coliforms appear to be relatively poor indicators of Listeria contamination. The Public Health Laboratory Service in London coordinated a large-scale survey in which various dairy products marketed in England and Wales were sent to 46 laboratories throughout the country for Listeria testing. Results from this comprehensive survey (see Table 12.6) indicated that 8.2, 1.1, 1.5, and 4.1% of the soft-ripened, soft-unripened, hard, and goat’s milk cheese manufactured in England and Wales contained L. monocytogenes; 75, 42, and 7 isolates classified as serovar 1/2, 4b, and 4, respectively. Among the soft-ripened varieties, 13 cheeses harbored >103 L. monocytogenes CFU/g with 3 samples exceeding 105 CFU/g. Of these 13 cheeses, 7 were prepared from raw milk, with only 1 being manufactured in the United Kingdom. In contrast, only 2 of 33 cheeses prepared from ewe’s or goat’s milk contained >500 L. monocytogenes CFU/g. Overall, the incidence of this pathogen was similar in imported (7.4%) and U.K.-produced cheese. These incidence rates and serovar distribution patterns for L. monocytogenes in soft-ripened and unripened cheese are generally similar to those observed in most other European studies [265,268]. As just suggested, numerous surveys for incidence of listeriae in cheese also have been completed in many of these aforementioned countries which, with the exception of Denmark and France, have not experienced major economic problems associated with Listeria-contaminated cheese. Following the 1986 report of an English woman who contracted listeriosis after consuming French soft cheese [126], two English researchers [314] examined 45 domestic soft cheeses as well as 177 soft cheeses imported from France, Italy, Cyprus, Germany, Denmark, and Lebanon for Listeria spp. and E. coli. (Table 12.10). Overall, L. monocytogenes was isolated from 2 of 45 (4.4%) English cheeses and 21 of 177 (11.9%) soft cheeses imported from France, Italy, and Cyprus. Populations of L. monocytogenes in contaminated cheese ranged from <102 to 105 CFU/g, with 9 of 12 French cheeses containing ≥104 CFU/g. Despite differences in media and methods used in

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TABLE 12.10 Incidence of L. monocytogenes and E. coli in Soft Cheese Sampled in England during 1987

Country of Origin

Number of Samples Analyzed

L. monocytogenes Number of Positive Samples (%)

Level/g

Number of Samples with >10 E. coli CFU/g (%)

England France Italy Cyprus West Germany Denmark Lebanon

85 45 44 20 17 6 5

12 2 7 2 0 0 0

(14.1) (4.4) (15.9) (10.0)

<102–105 <102 <102–104 <102 ND ND ND

32 14 12 3 9 2 1

Total

222

23 (10.4)

ND–105

73 (32.9)

(37.6) (31.1) (27.3) (15.0) (52.9) (33.3) (20.0)

Note: ND = not detected. Source: Adapted from Pini, P.N. and R.J. Gilbert. 1988. The occurrence in the U.K. of Listeria species in raw chickens and soft cheese. Int. J. Food Microbiol. 6: 317–326.

various surveys, the contamination rate of 14.1% for soft French cheeses calculated in this study was close to the 14.5% previously observed for French soft cheese exported to The Netherlands. As was true of previous surveys of French dairy products, all strains of L. monocytogenes (except one nontypeable strain) were of serovar 1/2 or 4b, with the former predominating. Listeria innocua, the only other Listeria sp. detected during this survey, was isolated from 9 of 85 (10.6%), 7 of 44 (15.9%), 2 of 45 (4.4%), and 1 of 6 (16.7%) soft cheeses produced in France, Italy, England, and Denmark, respectively, with 6 of 222 (2.7%) cheeses containing both Listeria spp. Although E. coli populations exceeded 10 CFU/g in 73 of 222 (32.9%) cheeses examined, no correlation was again observed between the presence of L. monocytogenes or L. innocua and contamination with E. coli. In fact, E. coli was detected at >10 CFU/g in only 10 of 23 (43.5%) cheeses that contained the pathogen. In this study, 10 of 23 (43.5%) cheeses contaminated with L. monocytogenes were prepared from pasteurized milk, whereas 2 and 11 of the remaining positive cheeses were manufactured from raw milk and milk of undetermined processing, respectively. Thus, as in previous studies, the type of milk (i.e., raw or pasteurized) from which cheese is made appears to be a poor indicator of possible Listeria contamination. Problems regarding the occasional presence of listeriae in soft cheese also have surfaced in the Scandinavian countries, with L. monocytogenes being recovered from 0.3% of Norwegian cheeses [151] and also identified in Danish Esrom and Blue Costello cheese that was exported to Norway [196,308], Sweden [196], and the United States. Although four Class I recalls were issued for Danish Esrom and Blue cheese in the United States (see Table 12.3), both of these cheeses (~20% of which were contaminated) were on sale for up to 2 months in Norway before being removed from the market, apparently without incident [399]. Danish officials also took steps to prevent unsold cheese from reaching consumers and have since developed a Listeria surveillance program [68] similar to that instituted in the United States, with routine testing of various cheeses as well as cheesemaking facilities. Additional concern over the microbiological safety of various cheeses also led to isolation of L. monocytogenes serovar 1/2b from two presumably French soft-ripened cheeses (one prepared from raw milk and the other from pasteurized milk) that were exported to Norway and Sweden [379]. Surface and interior samples from the raw milk cheese contained 7.5 × 105 and 1.0 × 102

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L. monocytogenes CFU/g, respectively, whereas corresponding samples from the pasteurized milk cheese contained 4.0 × 106 and 1.0 × 106 L. monocytogenes CFU/g. The reasons for nonuniform distribution of listeriae in soft-ripened cheese will be explored in the second half of this chapter. Although cheese prepared from raw milk contained 3 × 106 to 7 × 106 coliforms CFU/g, coliform tests indicated that the remaining cheese manufactured from pasteurized milk was fit for consumption. These findings reinforce the fact that coliform-free cheese may not necessarily be free of L. monocytogenes.

OTHER COUNTRIES Reports of Listeria-contaminated cheese in countries beyond North America and Europe also are beginning to surface (see Table 12.6). In 1987, L. monocytogenes was recovered from ricotta cheese manufactured in Melbourne, Australia [390], and again in 2002 in ricotta cheese from New Zealand [111]. This earlier event, along with identification of L. monocytogenes in the same imported brands of Danish blue and French brie cheese that were recalled in the United States [241], prompted Venables [390] to determine the incidence of listeriae in Camembert, blue vein, ricotta, cottage, pasta filata, high-moisture, low-acid, and other cheese varieties manufactured in and around Melbourne. Overall, L. monocytogenes was recovered from 6 of 338 (1.8%) cheeses produced by five different manufacturers, with the pathogen identified as being present in pasta filata (three samples), ricotta (two samples), and shredded (one sample) cheese. One cheese also contained L. seeligeri. Simultaneous identification of L. monocytogenes in environmental samples from all factories producing Listeria-positive cheese strongly suggests that these cheeses were contaminated during manufacture or ripening. In keeping with U.S. policies, attempts were made to remove tainted cheese from the marketplace. Furthermore, after thoroughly cleaning and sanitizing the factory, government officials required that Listeria-free cheese be produced for 12 consecutive days before being released to the public. Recent discovery of nonpathogenic listeriae in raw milk from neighboring New Zealand and later L. monocytogenes in several cheeses manufactured in New Zealand [110] also has prompted the New Zealand government to develop the Food Safety Authority Certificate of Listeria Clearance for shipment of products to Australia. Information regarding the presence of listeriae in dairy products produced elsewhere is scarcer still, with results from a March 1989 IDF Survey [247] indicating that L. monocytogenes had not yet been isolated from any dairy products manufactured in South Africa, Israel, or the former Soviet Union. Most cheese-related surveys from other countries have yielded negative results, with detection of L. monocytogenes largely being limited to a few high-moisture domestic cheeses produced in Egypt [204] as well as Chile [166], Costa Rica [293], and Venezuela [177]. However, notably higher isolation rates have been reported for soft and Hispanic-style cheeses from Brazil and Mexico—particularly for those cheeses that were homemade or prepared from raw milk. In the Brazilian survey by DaSilva et al. [172], homemade Minas Frescal (a fresh white soft cheese) had an L. monocytogenes contamination rate nearly 14 times greater than commercially produced cheese of the same type, with isolates belonging to serotype 1/2a being most common followed by serotype 4b—the serotype most often associated with outbreaks of foodborne listeriosis. Similar findings were reported in the Mexican survey by Saltijeral et al. [348], with L. monocytogenes being more prevalent in raw milk Fresh Panela cheese sold by street vendors as compared to the same cheese manufactured commercially from pasteurized milk. Such homemade cheeses clearly pose a greater public health risk than do commercially produced cheeses, as has been reported in regard to several illegally produced Hispanic-style cheeses that were linked to cases of listeriosis in the United States. Given the enormous volume of dairy products exported to other countries and the fact that L. monocytogenes has been isolated from the natural environment of all seven continents except Antarctica, it appears that developing countries are unlikely to remain completely untouched by the problems associated with Listeria-contaminated foods. Consequently, interest in the incidence of listeriae in dairy products and other ready-to-eat foods will likely continue in the years ahead.

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BEHAVIOR OF L. MONOCYTOGENES IN FERMENTED MILKS Before the well-known 1985 outbreak of cheeseborne listeriosis occurred in California, very little information was available about the behavior of L. monocytogenes in fermented milks and cheese. In fact, at the time of this outbreak, a search of the scientific literature uncovered only four such studies, which were reported from Bulgaria [246,383] and Yugoslavia [363,367] between 1965 and 1979. Hence, in addition to prompting numerous surveys for Listeria spp. in cheese, confirmation of cheese as an important vehicle in foodborne listeriosis has led to several hundred publications addressing the fate of L. monocytogenes in fermented dairy products during manufacture and storage. As described elsewhere in this book, cows, sheep, and goats can shed L. monocytogenes naturally in their milk during lactation. According to results from recent environmental surveys of dairy processing facilities, ample opportunity exists for this pathogen to enter pasteurized milk as a postpasteurization contaminant before the fermentation process begins as well as afterward as a contaminant of the finished product. Thus far most studies have dealt with behavior of L. monocytogenes in fermented dairy products inoculated with the pathogen either before or after fermentation, with relatively few studies addressing the fate of listeriae in fermented dairy products manufactured from naturally contaminated raw milk. Although the extent to which L. monocytogenes survives in cultured dairy products is partly dictated by whether or not the pathogen enters the product before or after fermentation, viability of Listeria in fermented dairy products, particularly cheese, depends on the type of product in which the pathogen is found as well as the degree of acid tolerance possessed by the contaminating strain [172]. Hence, to better understand the complex interactions between the various factors that affect viability of listeriae in cheese (i.e., amount, activity and type of starter culture, aw, pH, salt content, temperature during manufacture and storage), it is appropriate to begin this section by first discussing the behavior of L. monocytogenes in milk fermented with mesophilic and thermophilic lactic starter cultures. Commingled with this information will be data concerning the fate of this foodborne pathogen during manufacture and storage of cultured buttermilk, cream, and yogurt. The viability of L. monocytogenes in coagulants (e.g., calf rennet, microbial rennet, and bovine-pepsin rennet extract), coloring agents (e.g., annatto), and starter distillates (e.g., natural flavor compounds derived from cultured milk) used in cheesemaking also will be considered before our discussion of the many cheese varieties. Two additional areas of concern to cheesemakers, namely, the fate of L. monocytogenes in whey and salt brine solutions, will be examined at the end of this chapter.

STARTER CULTURES, CULTURED MILKS,

AND

CREAM

Fermented or cultured buttermilk, cream, and yogurt were among the first dairy products to be massproduced commercially using pure bacterial starter cultures. Today, two mesophilic (optimal growth at 30°C) lactic acid bacteria starter cultures, namely, Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris (formerly Streptococcus lactis and Streptococcus cremoris, respectively), are commonly used either alone or in combination to manufacture cultured buttermilk and cream, whereas a mixture of two thermophilic (optimal growth at or above 37°C) lactic acid bacteria, namely, Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrückii subsp. bulgaricus (formerly Streptococcus thermophilus and Lactobacillus bulgaricus, respectively), is used to produce yogurt. These same mesophilic and thermophilic starter cultures also are used to produce over 400 varieties of cheese that were recognized by the U.S. Department of Agriculture [387] in 1978, with over 1200 cheese varieties now manufactured worldwide. The variety of cheese to be produced depends, in part, on which of the lactic acid bacteria are used, either alone or in combination, as the starter culture. Mesophilic Starter Cultures Viability of L. monocytogenes in the presence of mesophilic lactic acid bacteria was first examined by Schaack and Marth [353]. Samples of autoclaved skim milk were inoculated to contain approximately

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=0.1%

=1.0%

=0.5%

=5.0%

1.50 1.25 1.0 0.75 0.50 0.25 0 21°C

30°C

S. cremoris

21°C

30°C

S. lactis

FIGURE 12.2 Changes in populations of L. monocytogenes in skim milk following a 15-h fermentation at 21 and 30°C with 0.1, 0.5, 1.0, or 5.0% (v/v) added S. cremoris or S. lactis. (Adapted from Schaack, M.M. and E.H. Marth. 1988. Behavior of Listeria monocytogenes in skim milk during fermentation with mesophilic lactic starter cultures. J. Food Prot. 51: 600–606.)

103 L. monocytogenes CFU/mL along with 0.1, 0.5, 1.0, or 5.0% of a S. cremoris or S. lactis milk culture and then fermented at 21 or 30°C for 15 h to simulate preparation of a conventional bulk starter culture for cheesemaking. As shown in Figure 12.2, L. monocytogenes grew to some extent in all samples during fermentation regardless of the species of lactic acid bacterium, inoculum level, or incubation temperature. Maximum Listeria populations after 15 h of incubation at 21°C were ~1.0–2.3 orders of magnitude lower in skim milk containing 0.1–5.0% of either starter culture than in control samples without starter culture. Increasing the incubation temperature to 30°C led to final Listeria populations that were ~2.5–4.0 orders of magnitude lower than in controls. As expected, growth of L. monocytogenes increased as the starter culture inoculum level decreased from 5.0 to 0.1%. Acid production by the starter culture played a major role in limiting multiplication of Listeria, with the pathogen never growing at pH <5.0. However, in two instances, the organism was almost completely inhibited at pH 5.6–6.0—well above the minimum pH value generally required for growth. Twelve years later Pitt et al. [317] conducted an abbreviated study in which samples of pasteurized rather than autoclaved milk were inoculated to contain approximately 103 L. monocytogenes CFU/mL followed by Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, or Lactobacillus plantarum at a level of approximately 106 CFU/mL. After 72 h of incubation at 30°C, L. monocytogenes populations decreased 89, 98, and 100% using L. lactis, L. cremoris, and L. plantarum, respectively; these findings confirm the earlier work by Schaack and Marth [353]. Buttermilk, yogurt, sour cream, and other similar fermented milk products must be prepared from pasteurized milk, whereas raw and/or heat-treated milk can be used in place of pasteurized milk for the manufacture of many aged cheeses including Cheddar, Colby, and brick provided that the cheese is aged for at least 60 days at ≥1.7°C before consumption. Recognizing the ability of Listeria to become sublethally injured in heat-treated milk processed at <71.6°C for 15 sec and survive in certain aged cheeses well beyond this 60-day ripening period, Mathew and Ryser [285] assessed the competition of thermally injured L. monocytogenes with a mesophilic lactic acid bacteria starter culture in milks exposed to various heat treatments. The study design was similar to that of

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TABLE 12.11 Percentage Increase in Injury of Healthy, Low-Heat-Injured, and HighHeat-Injured L. monocytogenes Cells in Different Milks Following a 6-h/31.1°C Fermentation with a 1.0% (v/v) Commercial L. lactis subsp. lactis/L. lactis subsp. cremoris Starter Culture Percentage Increase in L. monocytogenes Injury Type of Milk Raw Low-heat-treated High-heat-treated Pasteurized Ultrapasteurized

Uninjured

Low-Heat Injured

High-Heat Injured

60.58a 56.38b 55.31b 53.82b 48.00c

46.76a 46.32a 46.10a 45.92a 45.01a

52.54a 50.00b 48.09c 47.39c 45.72d

Means (n = 3) in the same column with different superscripts are significantly different ( p < 0.05). Source: Adapted from Mathew, F.P. and E.T. Ryser. 2002. Competition of thermally injured Listeria monocytogenes with a mesophilic lactic acid starter culture in milk of various heat treatments. J. Food Prot. 65: 643–650.

Schaack and Marth [353] except for the use of (1) a commercial starter culture comprising L. lactis subsp. Lactis/L. lactis subsp. cremoris, (2) a three-strain cocktail that was sublethally injured by heating in Tryptose Phosphate Broth at 56°C for 20 min (low-heat-injured) or at 64°C for 2 min (high-heat-injured) before inoculating the milk, and (3) milk that was raw, low-heat-treated (56°C/20 min), high-heat-treated (64°C/2 min), pasteurized (72°C/25 sec), or commercially sterile ultra-high-temperature–pasteurized milk. In the starter-free controls, 76 to 81% and 59 to 69% of the low- and high-heat–injured cells, respectively, repaired after 8 h of incubation at 31.1°C. Increased injury was observed for previously healthy L. monocytogenes cells using starter culture inoculum levels of 1 and 2% as opposed to 0.5%, with the extent of injury related to acid production. However, less injury was seen for the low- and high-heat–injured cells, with sublethal injury affording some protection against the drop in pH, as was previously discussed in Chapter 6. Furthermore, raw and subpasteurized milk were less conducive to recovery of uninjured and high-heat–injured cells of L. monocytogenes as compared to pasteurized and ultrapasteurized milk (Table 12.11). Given that most fermentation studies have used pasteurized milk inoculated with Listeria, the less favorable environment associated with raw and subpasteurized milk would suggest a decreased likelihood for survival in cheeses prepared from raw or heat-treated as opposed to pasteurized milk. Although still in use today, conventionally prepared bulk starter cultures have one major disadvantage in that excessive acid development from an inherent lack of buffering capacity frequently leads to some cell damage and partial loss of starter culture activity. Consequently, in industrially prepared bulk starter cultures, the final pH is now normally held constant at 5.1–5.2 by either adding (manual or automated) a neutralizer during the fermentation process (i.e., external pH control) or by using a specially prepared growth medium containing chemical buffers that solubilize as the pH decreases (i.e., internal pH control). Given the popularity of internal-pH-controlled media for starter culture preparation, researchers at the University of Wisconsin investigated the ability of L. monocytogenes strains Scott A, V7, and CA to compete with S. lactis [397] and S. cremoris [398] in one commercially available starter culture medium having internal pH control. In both studies, the starter culture medium was inoculated to contain 103 L. monocytogenes CFU/mL together with either a 0.25 or 1.0% inoculum of S. lactis or S. cremoris and incubated at 21 or 30°C for 30 h. Growth of the pathogen was only

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partially inhibited by S. lactis and S. cremoris when compared to starter-free controls, with the greatest inhibition occurring at the higher inoculum level and higher temperature. However, Listeria populations of 105–106 and 104–105 CFU/mL developed in samples fermented with S. lactis and S. cremoris, respectively, when these cultures were ready for use (pH 5.5) after 15 to 18 h of incubation. Because neither conventional bulk starter technology nor internal-pH-controlled media will completely inhibit this pathogen, manufacturers of cheese and other fermented dairy products should not discount the starter culture as a possible source for Listeria but rather should adopt rigorous sanitation standards and Hazard Analysis Critical Control Point (HACCP) programs to minimize Listeria contamination and potential product loss. Ultrafiltered milk, a type of concentrated milk sometimes used for commercial manufacture of certain cheeses including mozzarella, ricotta, cottage, and Cheddar, also possesses a higher buffering capacity than that of unfiltered milk because of higher concentrations of proteins and insoluble salts. Consequently, listeriae may have greater opportunity to grow in ultrafiltered as opposed to unfiltered milk during fermentation. ElGazzar et al. [189] examined this question by inoculating samples of unfiltered skim milk as well as retentate (concentrated twofold and fivefold by volume) and permeate from unfiltered skim milk to contain 103–105 L. monocytogenes CFU/mL together with 107–108 L. lactis subsp. cremoris CFU/mL. In contrast to the aforementioned studies involving skim milk and internal-pH-controlled bulk starter media, Listeria failed to grow in ultrafiltered milk containing starter culture, with populations remaining constant in unfiltered skim milk and decreasing up to 10- and 100-fold in 2-fold retentate and permeate, respectively, after 36 h of incubation at 30°C. Increased inactivation of L. monocytogenes in the permeate as compared to retentate and unfiltered skim milk is again related to the lower buffering capacity of the permeate that results from ultrafiltration. When these samples were refrigerated at 4°C, L. monocytogenes persisted 4–6 weeks in skim milk (pH 4.2), 3–5 weeks in retentate (pH 4.6), and 1 week in permeate (pH 4.1). Thus, fermentation of ultrafiltered milk at 30°C will not guarantee complete inactivation of Listeria even after the finished product is moved to refrigerated storage. Cultured Buttermilk Cultured buttermilk is essentially pasteurized skim milk that has undergone a 12- to 15-h fermentation at 20°C with S. cremoris or S. lactis (0.5% initial inoculum) and certain flavor-enhancing lactic acid bacteria such as Leuconostoc cremoris or L. dextranicum. After fermentation, the final product is packaged and refrigerated until consumed. As part of a follow-up study [354], all 15-h-old fermented milk samples from the aforementioned study by Schaack and Marth [353] were stored at 4°C and examined for L. monocytogenes. Survival of the pathogen ranged from an average of 5 weeks in skim milk fermented at 30°C with a 5.0% inoculum of S. cremoris to 12.5 weeks in skim milk fermented at 21°C with a 0.1% inoculum of S. cremoris (Table 12.12). Similarly, Listeria viability averaged 2.5–13.0 weeks in skim milk previously fermented at 30 and 21°C with 5.0 and 0.1% S. lactis, respectively. Slower inactivation of the organism in skim milks fermented at 21 rather than 30°C may be related to the rate of acid production during fermentation, because pH values for fermented milks ranged between 4.3–6.0 and 4.2–4.6 immediately after 15 h of incubation at 21 and 30°C, respectively. The fact that L. monocytogenes can survive 10.5 weeks in this refrigerated product (fermented 15 h at 21°C with 0.5% S. cremoris) emphasizes the importance of maintaining sanitary conditions during manufacture of cultured buttermilk. In addition to entering pasteurized skim milk as a postpasteurization contaminant, L. monocytogenes also can be introduced into cultured buttermilk as a postfermentation contaminant. Choi et al. [159] studied the second of these scenarios. Samples of commercially produced, cultured buttermilk (pH 4.21) were inoculated separately with each of four strains to contain an average of 3.5 × 103 L. monocytogenes CFU/mL and stored at 4°C. Under these conditions, the pathogen survived an average of 22.8 days (Table 12.13), with populations of three of four Listeria strains decreasing

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TABLE 12.12 Weeks of Survival of L. monocytogenes in Skim Milks Fermented with S. cremoris or S. lactis at 21 and 30°C for 15 h and Then Stored at 4°C S. cremoris Inoculum (%)

S. lactis

21°C

30°C

21°C

30°C

12.5a 10.5 8.0 9.0

6.5 6.0 4.5 5.0

13.0 ND 8.5 6.0

2.0 ND 3.0 2.5

0.1 0.5 1.0 5.0

Note: ND = Not determined. a

Average of two trials.

Source: Adapted from Schaack, M.M. and E.H. Marth. 1988. Survival of Listeria monocytogenes in refrigerated cultured milks and yogurt. J. Food Prot. 51: 848–852.

more than 100-fold during the first 8–12 days of refrigerated storage. It is important to realize that although L. monocytogenes was inactivated faster when added directly to cultured buttermilk than when skim milk was fermented into a “buttermilk-like” product in the previous study [354], the pathogen still survived throughout the normal shelf life of the product. Furthermore, results from an earlier study [226] demonstrating that E. coli and Enterobacter aerogenes are inactivated faster than L. monocytogenes in cultured buttermilk imply that coliform-free buttermilk may not

TABLE 12.13 Survival of L. monocytogenes in Inoculated Samples of Commercially Produced Buttermilk and Yogurt Stored at 4–5°C Product Buttermilk Plain yogurt Brand Da Brand Yb Vanilla-flavored yogurt Brand D Brand Y Plain low-fat yogurt Plain low-fat yogurt

PH

L. monocytogenes Inoculum (log10 CFU/mL)

Initial

Final

Survival (days)

3.55

4.21

4.38

22.8

4.26 4.36

4.02 4.03

4.08 4.09

21.2 24.7

4.21 4.70 2.10 7.10

4.03 4.03 4.10 4.10

4.10 4.10 4.10 4.10

24.7 22.3 3 9

a

Custard-style. Fluid-style.

b

Source: Adapted from Choi, H.K., M.M. Schaack, and E.H. Marth. 1988. Survival of Listeria monocytogenes in cultured buttermilk and yogurt. Milchwissenschaft 43: 790–792; and Siragusa, G.R. and M.G. Johnson. 1989. Persistence of Listeria monocytogenes in yogurt as determined by direct plating and cold enrichment methods. Int. J. Food Microbiol. 7: 147–160.

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necessarily be free of listeriae. These findings again emphasize the importance of good sanitation in producing Listeria-free buttermilk. Cultured Cream Unlike cultured buttermilk, far less is known about the viability of L. monocytogenes in cultured cream. In the only study reported thus far, Stajner et al. [367] manufactured cultured cream from naturally contaminated raw cow’s milk containing approximately 5 × 105 L. monocytogenes CFU/mL. According to these Yugoslavian authors, viable listeriae were detected in the finished product throughout 7 days of storage at 3–5°C. Thermophilic Starter Cultures

Change in Listeria Population (log10 CFU/mL)

Practically speaking, thermophilic fermentations used to produce yogurt and certain cheeses (e.g., Swiss, Parmesan, mozzarella, and Romano) are not normally continued beyond 4–6 h. The only two exceptions are in production of Bulgarian buttermilk and acidophilus milk, which require thermophilic fermentations of 10–12 and 18–24 h, respectively. Therefore, primary emphasis will be placed on behavior of Listeria during the first 6 h of fermentation. In addition to determining the fate of L. monocytogenes in the presence of mesophilic starter cultures [353], Schaack and Marth [352] also investigated the ability of this organism to grow during fermentation of skim milk with thermophilic lactic acid bacteria. As in the previous study, samples of autoclaved skim milk were inoculated to contain 103 L. monocytogenes CFU/mL. After adding 0.1, 1.0, or 5.0% S. thermophilus, L. bulgaricus, or a mixture of the two species, all samples were examined for numbers of listeriae during 15 h of incubation at 37 and 42°C. Limited growth of L. monocytogenes occurred in all samples, with the organism generally attaining maximum populations after 6 h of incubation at either temperature (Figure 12.3). At this

=0.1%

1.75

=1.0%

1.50

=5.0% 1.25 1.0 0.75 0.50 0.25 0 37°C

42°C

S. thermophilus

37°C

42°C

L. bulgaricus

37°C

42°C

L. bulgaricus and S. thermophilus

FIGURE 12.3 Changes in populations of L. monocytogenes in skim milk following a 6-h fermentation at 37 and 42°C with 0.1, 1.0, and 5.0% (v/v) added Lactobacillus bulgaricus, Streptococcus thermopilus, or L. bulgaricus + S. thermopilus. (Adapted from Schaack, M.M. and E.H. Marth. 1988. Behavior of Listeria monocytogenes in skim milk and in yogurt mix during fermentation by thermophilic lactic acid bacteria. J. Food Prot. 51: 607–614.)

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point, Listeria populations were generally 1.0–1.5 orders of magnitude lower in fermented than in unfermented control samples, as was also reported by Lukasova [270], indicating that growth of the pathogen was markedly suppressed by the thermophilic starter culture, particularly when used at inoculum levels of 5%. In addition, greater inhibition of listeriae was consistently observed in milks fermented at 42 rather than 37°C. Listeria monocytogenes behaved similarly in milks fermented with S. thermophilus, L. bulgaricus, and a mixture of both starter cultures during the initial 6 h of incubation; however, viability of the pathogen in milks fermented beyond 6 h varied with the species of lactic acid bacterium used in the fermentation. Although populations of listeriae remained relatively unchanged in all milks fermented 6–15 h with S. thermophilus (final pH of 4.55–4.90), the pathogen frequently survived only 9–15 h in milks fermented with L. bulgaricus alone, with similar findings reported 12 years later by Pitt et al. [317], who used pasteurized rather than autoclaved milk. Rapid inactivation of the pathogen coincided with pH values near 4.0 which developed in milks fermented 9–15 h with L. bulgaricus. The combination of L. bulgaricus and S. thermophilus was more inhibitory to Listeria than was S. thermophilus alone but less inhibitory than L. bulgaricus alone. Although populations of listeriae failed to decrease in milks fermented at 37°C with the mixed starter culture, some inactivation was noted in all corresponding samples incubated at 42°C. As was true when L. bulgaricus was used alone, inactivation of listeriae by the mixed starter culture again was most pronounced in samples having pH values near 4.0. In addition to the mesophilic starter culture work by Pitt et al. [317] discussed earlier, these investigators also assessed the ability of L. monocytogenes to compete with thermophilic starter cultures during the fermentation of pasteurized as opposed to autoclaved milk. Using commercial cultures of L. bulgaricus and S. thermophilus separately at inoculation levels of approximately 106 CFU/mL, populations of L. monocytogenes in the milk increased from 103 to 105 and from 103 to 106 after 12 and 20 h of incubation at 37°C, respectively, and thereafter declined. In the presence of S. thermophilus and L. bulgaricus, Listeria growth was inhibited 93 and 100%, respectively, with Schaack and Marth [352] reporting similar findings 12 years earlier. In the previous study by Schaack and Marth [354], following 15 h of incubation all fermented milk samples were stored at 4°C and monitored for listeriae. Using S. thermophilus alone, L. monocytogenes survived 21–32 and 5–15 weeks in milks fermented at 37 and 42°C, respectively (Table 12.14). Failure of these milks to attain pH values near 4.0 after fermentation with S. thermophilus helps explain the unusually long survival of listeriae. As expected, L. bulgaricus was most detrimental to listeriae, with the pathogen surviving beyond 15 h only in samples fermented with the lowest inoculum. Using a 0.1% L. bulgaricus inoculum the pathogen was eliminated from

TABLE 12.14 Weeks of Survivala of L. monocytogenes in Skim Milks Fermented with S. thermophilus or S. thermophilus + L. bulgaricus (LBST) at 37 and 42°C for 15 h and Then Stored at 4°C S. thermophilus

LBST

Inoculum (%)

37°C

42°C

37°C

42°C

0.1 1.0 5.0

28.5 32.0 21.0

15.0 8.5 5.0

7.5 1.5 1.0

1.5 12 h 15 h

a

Average of two trials. Source: Adapted from Ref. 352.

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milks fermented at 37 and 42°C following 7 and 3 days of refrigerated storage, respectively. Milks cultured with a combination of S. thermophilus and L. bulgaricus yielded results that were between both extremes observed when the two starter cultures were used separately (Table 12.14). Once again, L. monocytogenes survived longer in refrigerated milks fermented at 37 than 42°C, with slower inactivation in the former being attributed to slightly higher pH values. These findings are similar to those of Zuniga-Estrada et al. [403], who found that Listeria persisted for only 8 h when milk containing 103 L. monocytogenes and 106 S. salivarius subsp. thermophilus/L. delbrueckii subsp. bulgaricus CFU/mL was fermented at 42°C to pH 4.4. Yogurt As previously mentioned, L. monocytogenes can enter yogurt either before the fermentation as a milk contaminant or afterward as a contaminant of the finished product. Schaack and Marth [352,354] examined the behavior of Listeria under the first of these conditions by inoculating yogurt mix to contain ~103–104 L. monocytogenes (strains V7, OH, Scott A, or CA) CFU/mL and 2% of a commercial starter culture containing S. thermophilus, L. bulgaricus, and L. acidophilus. Yogurt mix was fermented at 45°C for 5 h and then stored at 4°C. Populations of all four Listeria strains increased an average of 2.5- to 10.0-fold in yogurt mix during the fermentation. After finished yogurt at pH 4.75 was refrigerated at 4°C, numbers of listeriae decreased, with strains V7, OH, Scott A, and CA surviving 7–12, 7–12, 4–12, and 1–5 days, respectively. The pH of yogurts in which listeriae were last detected ranged from 3.88 to 4.11. Subsequent work has shown Listeria survival to be somewhat more variable than first reported, with L. monocytogenes persisting 2–7 days [279], 4 days [5], 3–5 days, 18–24 days [255], 10 days [239], and 14–25 days [329] in yogurts of pH 4.2, 4.0–4.3, 4.1–4.7, 3.8–3.9, 4.1, and 4.5–5.0, respectively, during refrigerated storage, with growth of the pathogen also generally not being observed during fermentation. However, these survival times can reportedly be shortened by fermenting the yogurt mix with a bacteriocinproducing strain of S. thermophilus or by adding purified enterocin—a bacteriocin produced by Enterococcus faecium—to the mix after the start of fermentation [264]. In one additional French study [168] involving highly acidic yogurt (pH 3.5) prepared from yogurt mix inoculated to contain 102–107 L. monocytogenes CFU/mL, the pathogen was eliminated after only 1–2 days of storage at 4°C. Although acid development and differences in acid tolerance/injury during manufacture [211] play major roles in determining the fate of listeriae in yogurt, other factors including various starter culture metabolites and antilisterial compounds (bacteriocins) produced by certain strains of L. acidophilus [323] also may contribute to the rapid death of Listeria in yogurt, provided this lactic acid bacterium is in the product, with addition of 100 mg/L lysozyme to yogurt mix before fermentation also reportedly decreasing Listeria survival in the finished product [329]. Although nearly all yogurt in the United States is now prepared from cow’s milk, this product is sometimes manufactured from ewe’s and goat’s milk, particularly in Europe and the Middle East. In a 1964 Bulgarian study [246], L. monocytogenes viability in yogurt prepared from naturally contaminated ewe’s milk was markedly influenced by the storage temperature, with the pathogen surviving <24–48 h and >6 days in yogurt held at 18–22 and 10°C, respectively. Faster demise of listeriae at the higher rather than lower storage temperature was attributed to increased acid production by S. thermophilus and L. bulgaricus. When Abdel-Gawad et al. [personal communication] prepared yogurt from cow’s, ewe’s, and goat’s milk inoculated to contain 103–104 L. monocytogenes CFU/mL, 97–99.9% of the population was sublethally injured within 24 h of manufacture with no cells repairing or surviving in any yogurt samples (pH 4.0–4.3) beyond 5 days of cold storage. Many dairy industry personnel believe that contamination of yogurt is more likely to occur after rather than before fermentation. Choi et al. [159] simulated postfermentation contamination of yogurt by inoculating two commercial brands of plain and vanilla-flavored custard- and fluidstyle yogurt to contain 104–105 L. monocytogenes CFU/mL. The pathogen survived an average of

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21.2–24.7 days in yogurt held at 4°C (Table 12.13), with most listeriae being inactivated during the first 8–12 days of refrigerated storage. Khattab et al. [255] reported similar findings, with listeriae persisting 18–24 days in experimentally produced Egyptian yogurt (pH 3.8–3.9) inoculated to contain 106 L. monocytogenes CFU/mL. After inoculating various formulations of experimentally produced yogurt to contain 106 L. monocytogenes CFU/mL, Griffith and Deibel [235] also detected the pathogen in yogurt samples having a pH value of 4.3 following 28 days of storage at 4°C. More recently, Tipparaju et al. [380] inoculated retail samples of low-fat and nonfat plain (pH 4.3–4.4) as well as low-fat and nonfat vanilla yogurt (pH 4.1–4.5) to contain approximately 104 L. monocytogenes CFU/mL and stored the products at 8°C. Listeria populations decreased 2.0 to 3.5 logs during 31 days of storage. Greatest reductions were seen after 14 to 21 days with populations also significantly lower in vanilla-flavored yogurt containing vanillin. Based on these studies, the point at which yogurt becomes contaminated greatly influences Listeria survival, with the pathogen more vulnerable to inactivation as a pre- than a postfermentation contaminant. Siragusa and Johnson [364] conducted a similar study in which three different brands of commercial, unflavored, low-fat yogurt were inoculated to contain approximately 102 and 107 L. monocytogenes CFU/g and then stored at 5°C. Listeriae survived <3 days in yogurt inoculated with low levels of the pathogen even though pH values of yogurt were similar to those in the study by Choi et al. [159] (Table 12.13). Using the high inoculum, viable listeriae were found for only 9 days, with populations decreasing approximately 100-fold each after 3 and 6 days of refrigerated storage. Ayre et al. [120] reported similar results using an L. monocytogenes inoculum level of 107 CFU/g. In contrast, Ribeiro and Carminati [329] examined the effect of yogurt pH on Listeria survival by inoculating experimentally produced (pH 4.5), commercial fruit (pH 4.05), and plain yogurt (pH 3.76) to contain ~104 L. monocytogenes CFU/mL. Overall, the pathogen survived 2–3 days, 3–4 days, and 12–14 days in refrigerated yogurts having pH values of 3.76, 4.05, and 4.5, respectively, thereby verifying the impact of pH on Listeria survival. However, Griffith and Deibel [235] reported that L. monocytogenes populations decreased approximately 4 orders of magnitude in artificially acidified (pH 4.2) rather than fermented yogurt during the first 6 days of storage at 4°C. Hence, decreased tenacity of L. monocytogenes in inoculated yogurt samples in these studies as compared with the work by Choi et al. [159] again demonstrates that factors other than pH also are contributing to the death of Listeria. Many yogurts and cultured buttermilks marketed today have pH values of approximately 4.0 and 4.3, respectively. Because large populations of L. monocytogenes were inactivated faster in yogurt than in buttermilk during refrigerated storage, one would expect a lower incidence of listeriae in commercial yogurt. Evidence from FDA surveys discussed earlier in this chapter supports this view, because thus far the pathogen has been detected in commercial buttermilk but not yogurt, with 100 retail and farm-produced samples also negative for L. monocytogenes in the United Kingdom [254]. However, as was true for buttermilk, E. coli and Enterobacter aerogenes are also inactivated faster than L. monocytogenes in yogurt during refrigerated storage [226]. Hence, coliform-free yogurt may not necessarily be free of Listeria. This is important to remember when results of coliform tests on these products are interpreted. It is evident from this discussion that good sanitation practices are of utmost importance in producing Listeria-free yogurt, buttermilk, and other fermented milk products. Because yogurt is occasionally used as an ingredient in other foods, Sikes [361] investigated the fate of L. monocytogenes in low-moisture (1.9% water), medium-acid (pH 4.9) yogurt-based dairy bars supplied to the U.S. military. These bars, which contained ~34% heavy cream, 27.5% yogurt, 27.5% cream cheese, and 11% of other ingredients (i.e., sugar, sunflower oil, whey), were inoculated to contain approximately 1 × 105 L. monocytogenes strain Scott A CFU/g and then periodically examined for numbers of listeriae during extended storage at 25°C. Results indicated that Listeria populations decreased only approximately 100-fold after 40 days of incubation, thus demonstrating the ability of this organism to persist in low-moisture, medium-acid foods. As will soon be discussed, similar behavior has been reported for L. monocytogenes in semihard cheeses, such as Cheddar, Colby, and Gouda, which also have pH values near 5.0.

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Kefir Kefir, an alcoholic milk-based beverage that was originally produced in the Caucasian Mountains of Russia near the Black Sea, is now commercially produced in Europe, Asia, and North America. In the manufacture of kefir, whole cow’s milk is heated to 85°C for 30 min, cooled to 22°C, and then seeded with reusable kefir grains—gelatinous, walnut-sized irregularly shaped granules made up of yeasts (Saccharomyces kefir, Torula spp., or Candida kefir ), lactic acid bacteria (Lacotobacillus kefir, leuconostocs, and lactococci), and polysaccharides. Following overnight fermentation, lactic acid is produced along with ethanol (about 1.0%), diacetyl, acetone, and carbon dioxide. After straining the curdled milk to recover the kefir grains, the product is bottled, with a good kefir foaming much like beer. Working in Turkey, Gulmez and Guven [240] conducted two studies to assess the fate of L. monocytogenes in kefir as both a pre- and postfermentation contaminant. In the first of these studies, milk for kefir manufacture was inoculated to contain about 104 L. monocytogenes CFU/mL and then fermented at 30°C for 24 h. Listeria populations increased about 10-fold during the first 3 h and then slowly decreased during the remainder of the fermentation period as well as during subsequent storage at 4°C, with the pathogen surviving between 5 and 10 days in the final product at pH 4.2. Subsequently, Gulmez and Gowen [240] prepared. Listeriacontaminated kefir as just described, except that the milk was fermented at 30°C for 24 (pH 4.5–4.7) or 48 h (pH 4.2). In addition, Listeria-free kefir that had been similarly prepared was pasteurized at 85°C, cooled, inoculated with L. monocytogenes and then stored at 4°C. Based on their results, the pathogen increased about 100-fold to 106 CFU/mL in samples fermented for 24 h and then decreased about 10-fold after 10 days of refrigerated storage. In contrast, Listeria populations were at or near the original inoculation level after the 48-h fermentation period, with the numbers slowly decreasing to less than 102 CFU/mL at day 10. When L. monocytogenes was introduced into pasteurized kefir that had been previously fermented for 24 and 48 h, populations remained relatively stable and decreased about 100-fold, respectively, after 10 days of storage at 4°C, indicating that this pathogen poses a greater risk in kefir as a prefermentation contaminant.

TRADITIONAL FERMENTED MILK PRODUCTS In certain African and Middle Eastern countries, traditionally fermented ethnic milk products such as ergo (Ethiopia) and labneh (Middle East) comprise a significant portion of the daily diet. Although produced from commercially pasteurized milk under generally adequate hygienic conditions, these products are also frequently prepared at home (particularly in rural areas) from raw milk that is allowed to undergo a natural souring (i.e., fermentation) at ambient temperature without addition of starter cultures. Hence, the likelihood for the presence of Listeria and other bacterial pathogens in raw milk, combined with an often questionable fermentation and a short product shelf life, makes the microbiological safety of such home-fermented milks particularly suspect. Working in Zimbabwe, Dalu and Feresu [142] examined the fate of L. monocytogenes in commercially fermented milk prepared from both raw and pasteurized milk. Commercial fermented milk was prepared from pasteurized milk that was inoculated to contain 104 L. monocytogenes CFU/mL together with an active mesophilic starter culture and then packaged and fermented 24 h at ambient temperature, whereas both traditionally fermented milks were prepared by allowing raw milk to sour naturally in earthenware pots during 24 h of incubation at ambient temperature. Overall, Listeria populations increased ≤10-fold in all three milks during fermentation, with the final product attaining a pH of 4.3–4.6. After 5 days of storage at 4 and 20°C, numbers of listeriae generally decreased about 10- and 100-fold, respectively. Consequently, survival of L. monocytogenes beyond the normal 3–7 day shelf life of these traditionally fermented milks poses a legitimate public health concern.

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In 1994, Ashenafi [117] prepared ergo, a traditional Ethiopian fermented milk, from boiled milk that was inoculated to contain 103 L. monocytogenes CFU/mL along with Lactobacillus and Streptococcus starter organisms from a previous batch of product. After the first 12 h of a 24-h fermentation at 25°C, numbers of listeriae generally increased 10- to 100-fold in ergo (pH 4.5–5.5), with the highest populations being observed in samples fermented in unsmoked rather than olive wood-smoked glass containers. During the next 12 h, the pathogen was slowly inactivated as the pH of the product decreased to ≤4.0. Continued ambient storage led to complete demise of Listeria in ergo fermented in wood-smoked and unsmoked containers within 36–48 and 48–60 h, respectively. Consequently, traditional preparation of ergo in rural areas using wood-smoked fermenting vessels appears to be beneficial in minimizing Listeria survival in this product that is typically prepared from raw milk and consumed immediately after the 24-h fermentation period. Labneh, a Middle Eastern yogurt-like product, is produced using a normal yogurt starter culture of L. delbreuckii subsp. bulgaricus/S. salivarius subsp. thermophilus. Following approximately 5 h of incubation at 42°C, the product is salted (1% NaCl), poured into muslin bags, and hung in a cooler for 48 h. Gentle mixing to obtain a smooth consistency follows, after which the finished product (pH 3.8) is packaged for sale. These manufacturing steps, which afford many opportunities for contamination, prompted Gohil et al. [229] to assess the fate of L. monocytogenes in labneh as a postfermentation contaminant. When commercially produced labneh was inoculated to contain 104 L. monocytogenes CFU/g and stored at 4°C, the pathogen survived 2 and 7 days in products of pH 3.8 and 4.5, respectively, with the addition of 1% NaCl not appreciably altering Listeria survival. Raising the holding temperature to 10, 20, or 30°C led to more rapid demise of listeriae, with samples generally being free of L. monocytogenes after 2–3 days of storage. Several years later, Issa and Ryser [248] examined the behavior of L. monocytogenes in labneh as both a pre-and postfermentation contaminant. In this study, pasteurized milk was heated at 85–87°C for 30 min, cooled to 43°C, and inoculated to contain 103 L. monocytogenes CFU/mL. Following a 4-h fermentation at 43°C with a 2% commercial yogurt starter culture, the fermented milk was stored at 6°C for 12–14 h and then centrifuged to obtain a labneh-like product containing about 22% total solids. Alternatively, traditional labneh containing 0 and 1% salt was prepared using a 3% starter culture, inoculated to contain 103 L. monocytogenes CFU/g, and then stored at 6 and 20°C. During fermentation, growth of Listeria was suppressed as was previously reported by Schaack and Marth [352]. When L. monocytogenes was inoculated into both salted and unsalted labneh as a postfermentation contaminant, the pathogen decreased to nondetectable levels in 9 of 12 lots after 4 days of storage at 4 and 20°C. However, regardless of storage temperature, L. monocytogenes persisted in one lot each of salted and unsalted labneh for at least 15 days. Based on these findings, both labneh and yogurt pose minimal risk for consumers and are unlikely to be associated with cases of listeriosis.

BEHAVIOR OF L. MONOCYTOGENES IN CHEESE Considerable work has been done worldwide to define the behavior of L. monocytogenes during manufacture and ripening of various types of cheese. Most of these studies describe what might happen if cheese is prepared from contaminated milk. Because North American and European surveys have indicated that soft/semisoft cheeses ripened with mold or bacteria are most frequently contaminated with L. monocytogenes, research dealing with such varieties will be discussed first, followed by data on ripened cheeses of progressively lower moisture content, goat’s milk cheese, unripened cheese, whey cheese, and cold-pack cheese food. Whereas this chapter concludes with a discussion of L. monocytogenes behavior in whey and brine solutions, this section begins with an examination of the viability of listeriae in coagulants, coloring agents, and starter distillates, all three of which are commonly used in cheesemaking.

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COAGULANTS To produce cheese curd, milk must first be coagulated or clotted, which can be done either by acidification or addition of a coagulating enzyme. In the first method, an active lactic starter culture is used to lower the pH of the milk to 4.6–4.7 (isoelectric point of casein), at which point the casein micelles in the milk precipitate and form a coagulum. Alternatively, coagulation is occasionally accomplished by adding food-grade acids directly to milk. Coagulation of milk by either means of acidification is primarily confined to the manufacture of cottage cheese and a few ethnic varieties of fresh cheese. The second method, in which a coagulating enzyme is added to destabilize the casein micelles and clot milk at a near-neutral pH, is used to manufacture virtually all other types of cheese. Coagulants presently used in cheesemaking include calf rennet extract, chymosin, bovine pepsinrennet extract, and microbial rennet. Traditionally, calf rennet is extracted from the lining of the abomasum (fourth stomach) of suckling calves and contains two enzymes—pepsin and chymosin (the latter is most important for coagulation of milk). A shortage of calf rennet following World War II led to the use of bovine pepsin-rennet, an extract obtained from the abomasum of somewhat older calves, which can be substituted for calf rennet. Increased production costs of both calf rennet and bovine pepsin-rennet have in turn prompted development of several rennets of microbial origin. Thus far enzyme preparations obtained from molds belonging to the genus Mucor (particularly M. miehei) have proved to be the most satisfactory substitutes for animal rennet. Because two of four coagulants used in cheesemaking are of animal origin and because these animals sometimes carry L. monocytogenes, this pathogen might occasionally appear in both crude enzyme preparations and finished coagulant at the time of shipping. Although microbial rennet should be free of Listeria spp. when manufactured, listeriae within the rennet-manufacturing facility or the cheese factory environment could contaminate any of these products if mishandled. Given the recovery of an apparent sodium benzoate-resistant strain of L. monocytogenes from commercially produced calf rennet extract [188], the possible presence of L. monocytogenes in coagulants should be of concern to cheesemakers, with the International Dairy Federation also having contemplated the addition of rennet to its list of cheesemaking ingredients to be examined for Listeria spp. [375]. During 1988 and 1989, El-Gazzar and Marth published results from three studies examining the viability of listeriae in calf [184], bovine pepsin [185], and microbial rennet [187]. In each of these studies, commercially produced, Listeria-free rennet was inoculated to contain approximately 103, 104, 105, or 106 L. monocytogenes CFU/mL and analyzed for listeriae during 56–70 days of storage at 7°C, using both direct plating and cold enrichment. All samples of calf and bovine pepsin-rennet inoculated with the two lowest levels of Listeria were free of the pathogen after 14–28 days of storage at 7°C (Table 12.15). Even though 42–56 days of storage were required to eliminate the pathogen from samples containing initial inocula of approximately 105 and 106 L. monocytogenes CFU/mL, one must remember that all four inoculum levels used in these studies were many times greater than levels that might occur naturally in commercially produced coagulants. Hence, barring contamination in the cheese factory, these findings suggest that calf and bovine pepsin rennet are normally held long enough in distribution channels to ensure cheesemakers that both coagulants are Listeria-free. Inactivation of L. monocytogenes in calf rennet and pepsin rennet probably results from the combined effects of 5% propylene glycol, 2% sodium propionate, 0.1% (or more) sodium benzoate, 14–21% salt, and a relatively low pH of 5.6. Results from several studies assessing the viability of L. monocytogenes in the presence of benzoic acid and sodium propionate are discussed in Chapter 6. Unlike calf and bovine pepsin-rennet, more than 70 days of storage were required to eliminate L. monocytogenes at even the lowest inoculum level from microbial rennet (see Table 12.15). Enhanced survival of listeriae in microbial rennet may be related to the nature of the coagulant itself as well as the presence of fewer preservatives. Although L. monocytogenes is unlikely to enter

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TABLE 12.15 Survival of L. monocytogenes Strain CA in Three Milk Coagulants Stored at 70°C Number/mL after Days of Storage Product Calf rennet extract

Bovine pepsin-rennet extract

Microbial rennet

a

0

7

2.5 1.1 3.0 2.0 9.5

× × × × ×

103 104 105 106 103

1.5 3.5 6.0 6.0 10

2.0 7.5 1.0 6.0 7.0 2.0 1.0

× × × × × × ×

104 105 106 103 103 105 1.06

30 1.0 2.0 1.5 1.7 1.7 1.5

× × × ×

102 102 104 104

× × × × × ×

103 104 103 103 104 105

(+)a (+) (+) (+)

14

28

42

<10 (−)b <10 (−) 1.2 × 103 (+) 4.5 × 103 (+) <10 (−)

<10 (−) <10 (−) 30 (+) 2.0 × 102 (+) <10 (−)

<10 <10 <10 <10 <10

<10 (−) 1.0 × 102 1.0 × 102 7.6 × 102 1.0 × 103 2.3 × 104 4.0 × 104

<10 (−) <10 (+) <10 (+) 2.8 × 102 2.2 × 102 2.5 × 104 3.6 × 104

<10 (−) <10 (+) <10 (+) 3.3 × 102 4.4 × 102 2.3 × 104 9.3 × 104

(−) (−) (−) (−) (−)

56

70

— — — — <10 (−)

— — — — —

<10 (−) <10 (−) <10 (−) 1.2 × 102 1.4 × 102 1.6 × 104 7.0 × 103

— — — 40 90 8.5 × 103 9.0 × 103

(+) Positive result by cold enrichment. (−) Not detected after 6 weeks of cold enrichment.

b

Source: Adapted from El-Gazzar, F.E. and E.H. Marth. 1988. Loss of viability by Listeria monocytogenes in commercial calf rennet extract. J. Food Prot. 51: 16–18; El-Gazzar, F.E. and E.H. Marth. 1988. Loss of viability of Listeria monocytogenes in commercial bovine pepsin-rennet extract. J. Dairy Sci. 72: 1098–1102; and El-Gazzar, F.E. and E.H. Marth. 1989. Loss of viability by Listeria monocytogenes in commercial microbial rennet. Milchwissenschaft 44: 83–86.

microbial rennet during manufacture, the relatively high incidence of listeriae in cheese factories may lead to inadvertent contamination of the coagulant during cheesemaking. Considering the tenacity of L. monocytogenes in microbial rennet and the long shelf life of this product, it may be prudent for cheesemakers to periodically verify that the microbial rennet they are using is indeed Listeria-free.

COLORING AGENTS

AND

STARTER DISTILLATES

Depending on local preference, various yellow-orange colorants such as annatto (an extract from annatto seed [Bixia orellana]) and turmeric (an extract from the turmeric root [Curcuma longa]) can be added to milk at the beginning of cheesemaking, with annatto most commonly being used in the manufacture of Cheddar, Colby, Muenster, and brick cheese. Although freshly prepared colorants are unlikely to contain microbial pathogens, inadvertent exposure of these coloring agents to L. monocytogenes in the cheese factory could lead to production of a contaminated product. Thus, in addition to the aforementioned coagulants, El-Gazzar and Marth [186] also investigated the fate of listeriae in five commercially available annatto/turmeric extracts that were inoculated to contain approximately 103–107 L. monocytogenes strain CA/mL and stored at 22°C. Regardless of the initial inoculum level, populations of listeriae immediately decreased ≥4 orders of magnitude in all colorants, with the pathogen being completely inactivated immediately after addition to three of five extracts. The almost instantaneous death of Listeria in these three colorants was attributed to the presence of propylene glycol and a pH of 13.3 (one extract). Although Listeria populations of ≤800 CFU/mL were observed in the two remaining colorants immediately after inoculation, with the highest level of listeriae, these samples were free of the pathogen following 7 days of ambient storage.

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Working with a double-strength annatto extract, Galindo-Cuspinera et al. [212] recently reported that a 1:40 dilution of this concentrated extract was inhibitory to L. monocytogenes. Thus, these findings indicate that the length of time that these colorants spend at ambient temperatures during distribution and before use at the cheese factory is more than adequate to inactivate small numbers of listeriae that might enter as chance contaminants. Small levels of starter distillates, that is, mixtures of natural flavor compounds such as diacetyl obtained by distilling specially cultured milks, are frequently used to enhance the flavor of cottage and processed cheese as well as ice cream, margarine, butter, yogurt, snack foods, and certain types of candy. Hence, in connection with the study just described, El-Gazzar and Marth [186] also examined the fate of listeriae in a commercially available starter distillate that was inoculated to contain 102–106 L. monocytogenes strain CA, Scott A, or V7 CFU/mL and held at 7°C. Overall, strain CA decreased to nondetectable levels in all samples after 2–7 days of storage depending on the initial inoculum, whereas 7–28 days of incubation were required to eliminate strains Scott A and V7 from similar samples. Therefore, barring inadvertent contamination in the cheese factory, the time involved in shipping and distributing these starter distillates as well as the aforementioned coagulants and colorants should be more than sufficient to eliminate any inadvertent Listeria contaminants.

MOLD-RIPENED CHEESES Mold-ripened cheeses can be divided into two categories: (a) white mold cheeses, which are surfaceripened by Penicillium camemberti, P. caseicolum, or P. candidum (i.e., Brie and Camembert) and (b) blue-mold or blue-veined cheeses in which ripening results from growth of Penicillium roqueforti or P. glaucum throughout the cheese (i.e., Roquefort, blue, Gorgonzola). The relatively high moisture content of these surface-ripened cheeses, along with a nearly neutral pH in fully ripened cheese, allows rapid growth of L. monocytogenes as well as other foodborne pathogens that would normally be inhibited in more acidic cheeses. Because mold-ripened cheeses also are highly susceptible to surface contamination during ripening, it is not surprising that Brie and Camembert were among the first cheese varieties in which L. monocytogenes was detected and the behavior of the organism studied. Camembert Cheese In one of the first studies to examine behavior of L. monocytogenes during cheese manufacture and storage, Ryser and Marth [341] prepared Camembert cheese from pasteurized milk inoculated to contain approximately 500 L. monocytogenes strains Scott A, V7, CA, or OH CFU/mL. Following 10 days of storage at 15°C/95% relative humidity (RH) to permit proper growth of P. camemberti on the cheese surface, all cheeses were wrapped in foil and ripened at 6°C. Wedge (pie-shaped), surface, and interior samples of cheese were diluted in Tryptose Broth and analyzed for listeriae at appropriate intervals using both direct plating and cold enrichment. Populations of L. monocytogenes increased 5- to 10-fold during the first 24 h of manufacture; however, this increase probably did not result from growth of the organism during cheesemaking. Numerous studies have shown that bacterial populations typically increase 5- to 10-fold during curd formation as a direct result of entrapment of organisms in the curd matrix, with the extent of this increase dependent on the moisture content of the cheese. In all likelihood, L. monocytogenes was similarly concentrated during formation of Camembert cheese curd. Entrapment of L. monocytogenes in the curd is further supported by the fact that in this study only 1.3% of the original Listeria inoculum in the milk was lost in the whey. Yousef et al. [402] later demonstrated that the failure to observe L. monocytogenes population increases of approximately 5- to 10-fold after formation of Camembert as well as Cheddar and cottage cheese curd was probably related to the method of sample preparation. Their improved procedure in which curd samples were homogenized in warm (45°C) Tryptose Broth containing 2% trisodium citrate was subsequently used to examine

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450

FIGURE 12.4 Behavior of L. monocytogenes strain CA (solid symbols) and pH (open symbols) during ripening of Camembert cheese. Solid symbols at <1.0 log10 strain CA/g indicate results for cold enrichment. Numbers indicate the week at which strain CA was found, whereas an “x” signifies that the pathogen was not detected after 8 weeks of cold enrichment. (Adapted from Ryser, E.T. and E.H. Marth. 1987. Fate of Listeria monocytogenes during manufacture and ripening of Camembert cheese. J. Food Prot. 50: 372–378.)

behavior of L. monocytogenes during manufacture and storage of brick [345], Colby [400], feta [307], blue [306], and Parmesan cheese [401]. During the initial 17 days of cheese ripening, the first 10 days of which occurred at ~15°C, populations of three of four L. monocytogenes strains decreased 10- to >1000-fold, with lowest numbers generally being observed in surface samples (Figure 12.4). On further ripening at 6°C, all four Listeria strains grew (particularly between 25 and 30 days of storage) and attained maximum populations of 106–108 CFU/g in wedge and surface samples from fully ripened cheese; however, maximum listeriae populations were generally 10- to 100-fold lower in interior samples from the same cheeses. Although growth of L. monocytogenes clearly paralleled the increase in pH of the cheese during ripening, with growth usually commencing after the cheese attained a pH value of 5.75–6.25, decreased viability of three of four Listeria strains in surface samples having pH values of 6.25–6.50 suggests that factors other than pH, including the presence of potentially inhibitory surface bacteria and yeasts, may also be involved in controlling growth of this pathogen in Camembert cheese. At least six additional studies have addressed the fate of L. monocytogenes during manufacture and ripening of Camembert cheese [122,286,325,368,370,376]. Regardless of the method of inoculation (i.e., milk, surface, brine), the basic conclusions from these reports were similar to those reached by Ryser and Marth [341] years earlier in that (a) L. monocytogenes failed to grow during cheesemaking, (b) low numbers of listeriae were recovered during the period of rapid growth of P. candidum on the cheese surface, (c) populations of listeriae in cheese increased rapidly after 21 to 28 days of ripening, and (d) maximum Listeria populations of >106 CFU/g were detected in surface slices from fully ripened cheese. However, Terplan et al. [376] found that interior samples from 4- to 56-day-old Camembert cheeses ripened at 5°C consistently contained <100 L. monocytogenes CFU/g and never attained pH values >5.6 even after 56 days of ripening. Hence, under

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Strain log10 CFU/g

these conditions, growth of the pathogen was probably suppressed or severely retarded. However, some cells may have been sublethally injured during continuous exposure to this acidic environment, which in turn would have probably decreased the number of Listeria colonies observed on selective plating media. Two of these studies also addressed the influence of cheese ripening temperature on Listeria growth [122,370]. As expected, growth of L. monocytogenes was enhanced as the cheese storage temperature was increased from 3 to 15°C in response to more rapid ripening of the cheese and a concomitant increase in pH. Results from a subsequent study by Ryser and Marth [343] showed greater growth of L. monocytogenes in filter-sterilized Camembert cheese whey previously cultured with P. camemberti than in uncultured whey adjusted to pH values of 5.60–6.80 and thus suggest that P. camemberti is not involved in reducing Listeria populations on the surface of Camembert cheese. In support of these findings, Geisen et al. [214] also failed to observe any antilisterial activity among several strains of P. camemberti that were tested against L. monocytogenes in vitro. Although not yet recognized when this work was published in 1987, a wide range of non-lactic-acid bacteria (i.e., entrococci, staphylococci) [154,195] and yeasts [178] that possess antiliserial activity and colonize the surface of smearripened cheese may also be initially present on the surface of Camembert, with these organisms likely responsible for partial inhibition of Listeria during the initial stages of ripening. To simulate contamination of cheese in the ripening room, Ryser and Marth [341] also inoculated surfaces of 10-day-old wheels of Listeria-free Camembert cheese to contain 2–40 L. monocytogenes (four strains tested separately) CFU/20 cm2. All cheeses were then ripened at 6°C for 60 days, during which time 10-g surface samples were analyzed for listeriae. Three of four L. monocytogenes strains grew on the surface of the cheese and attained maximum populations of ~103–105 CFU/g (Figure 12.5). Although the remaining Listeria strain failed to grow on the cheese surface after 60 days of storage,

FIGURE 12.5 Growth and survival of L. monocytogenes on the surface of Camembert cheese. Half-solid and solid symbols at <1 log10 Listeria/g indicate that the organism was detected in one of two or two of two samples, respectively, using cold enrichment. Numbers indicate the week at which L. monocytogenes was found. (Adapted from Ryser, E.T. and E.H. Marth. 1987. Fate of Listeria monocytogenes during manufacture and ripening of Camembert cheese. J. Food Prot. 50: 372–378.)

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the pathogen was routinely detected throughout the ripening period using cold enrichment. These findings, along with the unfortunate recall of over 300,000 tons of French Brie cheese, stress the importance of manufacturing surface-ripened soft cheeses from high-quality, Listeria-free milk and observing good sanitary practices in the ripening room. Nonetheless, even under ideal manufacturing and ripening conditions, such cheeses may still inadvertently become contaminated with listeriae, as is now well recognized from the high incidence of Listeria in surface-ripened cheese. Because Vacherin Mont d’Or and Brie de Meaux (a raw milk cheese) were directly involved in two major outbreaks of listeriosis in Europe, scientists in both Europe and North America have been exploring various means of eliminating this pathogen from such cheeses that are surfaceripened with mold and bacteria. Not surprisingly, Banks [124] reported that L. monocytogenes grew rapidly in Camembert cheese prepared from raw milk, reaching a population of 106 CFU/g in fully ripened cheeses. Although heat-treating this Listeria-contaminated milk at subpasteurization temperatures (i.e., 62.8 or 65.6°C) led to markedly lower Listeria populations in the cheese immediately after manufacture, the pathogen was not completely inactivated, with some growth being reported during 7 weeks of cheese ripening. Using a different approach, Helloin et al. [242] inoculated a chemically defined medium resembling Camembert cheese (pH 5.0 and 7.0) with healthy and heat-injured (56°C/30 min) cultures of L. monocytogenes and then incubated these samples at 4, 12, or 21°C for up to 14 days. Compared to the uninjured controls, growth of the heat-injured culture was retarded at pH 7.0 during incubations at 4, 10, and 25°C with no growth and faster demise of the heat-injured culture at pH 5.0. Thus, these findings agree with those of Banks [110] and suggest that sublethally heatinjured cells in heat-treated milk are less likely to increase to potentially hazardous levels in Camembert cheese during ripening than are healthy cells. Although initial attempts by Asperger et al. [118] failed, results from several subsequent studies yielded more promising findings concerning the use of nisin and nisin-producing starter cultures to eliminate chance Listeria contaminants from soft surface-ripened cheese. When Maisner-Patin et al. [274] used a nisin-producing strain of L. lactis subsp. lactis to manufacture Camembert cheese from milk inoculated to contain 101, 103, or 105 L. monocytogenes strain V7 CFU/mL, numbers of listeriae decreased dramatically 6–9 h into cheesemaking because of the presence of 700 IU nisin/g of curd. Richard [330] also reported that inactivation of L. monocytogenes can be further enhanced by directly adding as little as 25 IU nisin/mL to the milk at the start of cheesemaking. Inhibition of Listeria continued during the first 2 weeks of ripening; however, regrowth of survivors was then reported in the presence of 250–300 IU nisin/g, first on the cheese surface and later in the interior, with the rate and extent of regrowth again paralleling the increase in cheese pH during ripening. Addition of other inhibitory organisms, including Enterococcus faecalis and Lactobacillus paracasei, to milk failed to arrest L. monocytogenes growth in Camembert cheese during extended ripening. However, a difference of 2.4 log CFU/g between numbers of L. monocytogenes in cheeses made with and without nisin-producing starter cultures was maintained throughout 6 weeks of ripening. Nisin was most effective when the milk for cheesemaking contained 101 L. monocytogenes CFU/mL, with the pathogen being absent in 25-g samples even after 6 weeks of ripening. These findings agree with those of Sulzer and Busse [371], who used a different nisinproducing strain of L. lactis subsp. lactis. In contrast to the previous work, Wan et al. [394] added piscicolin (2048 AU/mL)—a bacteriocin produced by Carnobacterium piscicola—to pasteurized milk containing about 100 L. monocytogenes CFU/mL for manufacture of Camembert cheese. Although piscicolin inhibited growth of Listeria during both manufacture and the first week of cheese ripening, the pathogen was again detected at day 21 and increased to 3.8 log CFU/g as opposed to 6.8 log CFU/g in piscocolin-free cheese after 47 days of ripening. Although both nisin and piscicolin have similar limits to preventing regrowth of Listeria during cheese ripening, these bacteriocins can minimize Listeria growth during Camembert cheese manufacture provided that the cheese milk is of good hygienic quality and contains <103 L. monocytogenes CFU/mL.

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In another report [175], addition of lactoperoxidase system components to the surface of soft bacterial smear-ripened French cheese containing 102–106 L. monocytogenes CFU/g led to complete inactivation of the pathogen following 4 days of storage at 15°C. In 1989, Hughey et al. [244] showed that lysozyme was only bacteriostatic to L. monocytogenes in Camembert cheese. Incorporating 2–10% carrot juice into homogenized Brie cheese was also effective in minimizing growth of L. monocytogenes in refrigerated samples [124]. Although several additional reports also attest to the usefulness of x-ray [140] and gamma irradiation [196] in eliminating high populations of L. monocytogenes from Camembert and other soft surfaceripened cheeses, such treatments do not appear to be very practical, because ripening of the cheese will be adversely affected. Current evidence suggests that L. monocytogenes behaves similarly in naturally contaminated, commercially produced soft and semisoft mold-ripened cheese. While conducting a survey of soft/semisoft cheese sold in Canada, Farber et al. [166] discovered eight 4-month-old French cheeses, presumably of the Brie/Camembert variety, that contained ~104–105 L. monocytogenes CFU/g. Following 1 year of continuous storage at 4°C, Listeria populations remained constant in one cheese and decreased only 10- to 100-fold in the seven remaining cheeses. In view of these results, it is easy to understand why this pathogen has been most frequently detected in soft/semisoft cheeses that have been surface ripened by molds.

Blue Cheese Blue-veined cheeses such as blue, Roquefort, and Gorgonzola that are ripened internally and sometimes externally with P. roqueforti or P. glaucum also have been examined for their ability to support growth and survival of listeriae. Papageorgiou and Marth [306] used the modified Iowa method to manufacture blue cheese from pasteurized milk inoculated to contain approximately 1000 L. monocytogenes (strains Scott A or CA) CFU/mL. All cheeses were ripened 84 days at 9–12°C (90–98% RH) and then held an additional 36 days at 4°C. Numbers of listeriae increased by an average of 1.50 log10 CFU/g during the first 24 h of manufacture, with increases of 0.62 and 0.71 log10 CFU/g being attributed to entrapment of the organism within the curd matrix and growth, respectively. Growth of L. monocytogenes occurred primarily during the first 9 h of manufacture and ceased when the pH of the cheese dropped below 5.0. As expected, somewhat less growth occurred in two lots of cheese with particularly rapid acid production. Unlike the behavior of L. monocytogenes in Camembert cheese, the pathogen not only failed to grow during ripening of blue cheese, but decreased in numbers by 2 to nearly 3 orders of magnitude during the first 56 days of storage at 5°C (Figure 12.6). These decreases, which occurred despite favorable pH values that developed during ripening from growth of P. roqueforti, were most likely caused by formation of free fatty acids [355], with listeriostatic/listeriocidal levels of caproic, caprylic [257], lauric, and other medium-chain fatty acids [258] produced as by-products of P. roqueforti growth during ripening of blue-veined cheeses. However, at least eight different P. roqueforti strains can also produce listeriocins in laboratory media [214]. Nonetheless, combined effects of a relatively high pH and low storage temperature were probably responsible for both Listeria strains surviving at least 120 days in all lots of blue cheese. Although additional tests showed that strain Scott A was evenly distributed throughout blocks of 120-day-old blue cheese, strain CA was far less tolerant to environmental conditions on the cheese surface and was detected in such samples only after cold enrichment. The lengthy survival of L. monocytogenes in blue cheese, coupled with the recall of Danish blue cheese and isolation of L. monocytogenes from Italian Gorgonzola cheese, all stress the importance of preparing blue-mold cheeses from properly pasteurized milk under good hygienic conditions to prevent a possible public health problem involving Listeria.

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FIGURE 12.6 Changes in population of L. monocytogenes strain Scott A and pH during ripening of blue cheese. (Adapted from Papageorgiou, D.K. and E.H. Marth. 1989. Fate of Listeria monocytogenes during the manufacture and ripening of blue cheese. J. Food Prot. 52: 459–465.)

BACTERIAL SURFACE–RIPENED CHEESES This group of cheeses consists of soft and semisoft varieties that are ripened under conditions that induce a progression of microbial growth on the cheese surface. Examples of such cheeses include brick from the United States, Pont l’Evêque and Saint Paulin from France, Tilsiter from Germany, Trappist from Yugoslavia, Havarti from Denmark, Bel Paese and Taleggio from Italy, and Limburger from Belgium. Differences between these varieties result from the shape of the cheese as well as the amount and type of surface growth. Microorganisms are not normally added as pure cultures but rather develop naturally on the cheese surface, because ripening conditions promote growth of organisms that are normally present in the ripening room. Proper aging of these surface- or smear-ripened cheeses results from the sequential growth of halotolerant yeast, lactic-acid-metabolizing bacteria (Micrococcus spp.), and Brevibacterium linens, the last-named organism being essential for proper flavor development. As in mold-ripened cheeses, a pH gradient also develops during aging of bacterial surface-ripened cheeses, which in turn creates a more favorable environment for growth of contaminating microorganisms, including Listeria [119].

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Brick Cheese Using the washed-curd procedure, Ryser and Marth [345] prepared brick cheese from pasteurized milk inoculated to contain ~102–103 L. monocytogenes (strain OH, Scott A, V7, or CA) CFU/mL. Following manufacture, cheeses were smeared with a culture of B. linens and ripened at 15°C/95% RH for 2, 3, and 4 weeks to simulate production of mild, aged, and Limburger-like brick cheese, respectively. Because a natural pH gradient develops as brick cheese ripens, three types of cheese samples—surface, interior, and slice (surface and interior)—were analyzed for numbers of listeriae during 20–22 weeks of additional storage at 10°C. Populations of strains OH, Scott A, CA, and V7 increased approximately 64.6- , 37.2- , 7.4- , and 6.8-fold, respectively, on completion of brining approximately 32 h after the start of cheesemaking. Because a population increase of approximately 10-fold can be attributed to entrapment of listeriae within the curd matrix, with relatively few organisms appearing in the whey, growth of L. monocytogenes during the latter stages of cheesemaking before brining was confined to strains OH and Scott A. Numbers of listeriae remained relatively stable at 103–104 CFU/g of cheese during brining; however, a few organisms leached from the cheese into the 22% NaCl brine solution. Information on behavior of Listeria in salt brine solutions appears at the end of this chapter. Strains OH (isolated from Liederkranz, a bacterial surface-ripened cheese formerly produced in Ohio) and Scott A grew rapidly during the initial 2 weeks of smear development required to manufacture mild brick cheese and generally attained maximum populations of approximately 6.20 and 6.60, 6.90 and 7.0, and 5.10 and 5.60 log10 CFU/g in 4-week-old slice (pH 6.0–6.5), surface (pH 6.5–6.9), and interior (pH 5.6–6.2) samples, respectively. During the remaining 20 weeks of ripening at 10°C, numbers of strains OH and Scott A generally decreased only 1- to 7-fold in mild brick cheese. Both strains also behaved similarly in aged and Limburger-like cheese during smear development and extended storage at 10°C. In contrast, strains CA and V7 failed to grow appreciably during or after smear development, despite favorable pH values of 6.8–7.4 in fully ripened cheese. Although strains CA and V7 were detected only sporadically in 4- to 26-week-old samples of mild, aged, and Limburger-like cheese at levels ranging between 2.7 and 4.6 log10 CFU/g, both strains were routinely recovered from 24- to 26-week-old slice, surface, and interior samples after cold enrichment. Hence, all four L. monocytogenes strains survived beyond the normal shelf life of brick cheese. Subsequent experiments [342] dealing with possible antilisterial effects of several sulfur compounds (i.e., methyl sulfide, dimethyl disulfide, and methyl trisulfide) produced during ripening of brick cheese failed to explain the inability of strains CA and V7 to grow in mild, aged, and Limburger-like brick cheese. Additional possibilities include (a) inhibition of strains CA and V7 by smear-ripening organisms such as Geotrichum candidum [178,273], Lactobacillus plantarum [194,197,267], B. linens [199,276,295], enterococci [195,208,220,221,263,347], coryneform bacteria [302,347], and/or certain staphylococci [154,347], all of which can reportedly produce bacteriocin-like substances active against listeriae or (b) heightened sensitivity of these L. monocytogenes strains to the inhibitory effects of certain listeriocidal fatty acids (i.e., linoleic) and monoglycerides [395] produced during cheese ripening. In conjunction with the previously mentioned European study involving Camembert cheese, Terplan et al. [376] also assessed the behavior of Listeria during manufacture and ripening of red smear-ripened (“bricklike”) cheese. When this cheese was produced from pasteurized milk inoculated to contain 95 L. monocytogenes CFU/mL, numbers of listeriae increased ≤10-fold after the coagulum was cut as a result of entrapment within the curd matrix; however, no growth of the pathogen was detected during the remainder of cheese manufacture. In fact, unlike the study by Ryser and Marth [345], Listeria populations decreased 10-fold by the time the cheese (pH 4.9) was ready for brining, with the cheese containing only 9 L. monocytogenes CFU/g after brining. Following 8 days of smear development at 16.5°C/93% RH, all cheeses were ripened at 5°C for an additional 62 days. Listeria populations close to the cheese surface increased from 2.5 × 101 CFU/g

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immediately after smear development to 1.5 × 104 CFU/g in 14-day-old cheese, during which time the pH increased from 4.9 to 5.1. Continued ripening of bricklike cheese at 5°C led to development of stable L. monocytogenes populations of 2.5 × 105 CFU/g in 1-cm thick surface slices of 42-day-old cheese. However, unlike the study by Ryser and Marth [345], the pathogen was never detected in interior cheese samples that were more than 4 days old, despite pH values of 5.7 in interior samples of 56-day-old cheese. Although results of Ryser and Marth [264] suggest that L. monocytogenes should at least have been isolated occasionally from interior samples of bricklike cheese, the FDA procedure used in this study was unable to detect listeriae in these samples, possibly because of acid injury that may have occurred during exposure to pH values 5.2 for as long as 6 months. Taleggio Cheese First manufactured in the Taleggio Valley in Lombardy, Italy, following World War I, Taleggio is a soft smear-ripened cheese that is particularly prone to contamination during smearing and weekly washing with a brine solution. Hence, several investigators have assessed the fate of Listeria on Taleggo cheese as a postmanufacturing contaminant during cheese ripening. Working in Italy, Marchisio et al. [284] surface-inoculated ripened 35- to 40-day-old Talleggio cheeses with L. monocytogenes and reported that Listeria populations on the cheese surface remained relatively unchanged after 30 to 60 days of additional storage at 6°C. Failure of Listeria to grow on the surface of Taleggio despite the increase in pH during ripening is related to the composition of the surface microflora, with some cultures of Corynebacterium and micrococci [220] as well as bacteriocin-producing strains of Enterococcus faecium [302] being inhibitory to Listeria, with the greatest inhibition coming from well-developed bacterial smears during the later stages of ripening [152]. Tilsiter Cheese In 1995, Bachmann and Spahr [121] manufactured Tilsiter cheese (a semifirm, slightly yellow, smear-ripened cheese similar to brick that originated in the vicinity of Tilsit in East Prussia) from milk inoculated to contain 104 L. monocytogenes CFU/mL. Overall, their findings were similar to those observed for brick cheese containing strains L. monocytogenes strains CA and V7 [345], with Listeria populations varying between 103 and 104 CFU/g in Tilsiter cheese during 90 days of ripening at 10–13°C. Trappist Cheese First manufactured at a monastery in Bosnia during the 1880s, Trappist is another soft surfaceripened cheese similar to Port du Salut in France that undergoes frequent washing to prevent the growth of surface mold. The ability of L. monocytogenes to survive the Trappist cheesemaking process and persist during 90 days of ripening was investigated by Kovincic et al. [259]. Cheeses were prepared from pasteurized milk inoculated to contain 102–105 L. monocytogenes CFU/mL and a 1% starter culture inoculum of L. lactis subsp. lactis and L. lactis subsp. cremoris. After rennet coagulation, the curd was cooked at 39°C for 45 min, hooped, drained, and pressed for 10–12 h. Thereafter, the cheese was brine-salted (18% NaCl, 4 h), dried (5 days at 16–18°C), waxed, and aged at 10°C for up to 90 days. Populations of L. monocytogenes increased ~10-fold in the finished cheese during the first 30 days of ripening, stabilized over the next 30 days, and then gradually decreased to levels approaching the original inoculum after 90 days of storage. Similar results were also obtained when L. monocytogenes was added to the curd/whey mixture rather than the pasteurized milk during cheesemaking. The limited growth and extended survival of L. monocytogenes in Trappist cheese (pH 4.9, 30% moisture, 1.4% NaCl) are generally similar to what has been observed for several common varieties of semisoft/hard cheeses to be discussed shortly.

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SOFT ITALIAN CHEESES Most of the Italian soft cheeses are classified as pasta filata or “plastic curd” cheeses and include such varieties as mozzarella, Provolone, Caciocavallo, and Scamorze, with mozzarella clearly being the most economically important. Traditionally, fresh mozzarella has been produced from the highfat milk of the water buffalo, whereas mozzarella cheese for use on pizza is manufactured from cow’s milk and aged several weeks to develop proper elasticity and meltability. Production of these pasta filata-type cheeses is similar in that the resulting curd is always stretched in hot (65–85°C) water and then kneaded and molded into a characteristically shaped mass of curd which is hardened in cold water, brine-salted, and ripened for various times. Mozzarella The severe heat treatment that the cheese curd receives and the reported thermal tolerance of listeriae led Buazzi et al. [145] to assess the fate of L. monocytogenes during manufacture of mozzarella cheese. When this cheese was prepared from pasteurized milk inoculated to contain 104–105 L. monocytogenes strain OH, CA, or V7 CFU/mL, Listeria populations of 104–105 CFU/mL were reported in the curd after cutting, cooking, and cheddaring. However, immersing and stretching the curd 3 to 4 min in hot water (77°C) led to the complete demise of the pathogen. In subsequent work, Kim et al. [256] reported that L. monocytogenes populations decreased more than 3 logs after mozzarella curd was stretched at 66°C for 3 min or 77°C for 1 min. Because the curd temperature was maintained at 65°C for at least 2 min and L. monocytogenes has a reported D-value of 28.1 sec at 65°C [272], mozzarella cheese should be Listeria-free even if small numbers of the pathogen are present in the curd before stretching. These findings are generally similar to those reported during manufacture of traditional mozzarella cheese from buffalo milk [391], with L. monocytogenes populations decreasing at least 100-fold during brief stretching of the curd at 90–95°C. Although a few survivors remained after curd stretching and molding, all cheeses prepared from milk inoculated to contain 103 and 105 L. monocytogenes CFU/mL were free of this pathogen after 24 and 48 h of refrigerated storage, respectively. The heat treatment given to mozzarella cheese curd is clearly sufficient to inactivate small numbers of listeriae that might be present. However, ample opportunity exists for postprocessing contamination as evidenced by recent surveys and at least three Class I recalls of Listeria-contaminated mozzarella cheese. Stecchini et al. [369] addressed the issue of postprocessing contamination by inoculating the surface and packaging fluid of mozzarella cheese with L. monocytogenes and then storing the product at 5°C for up to 21 days. Under these conditions, numbers of listeriae increased about 10,000-fold during 21 days of storage, with inclusion of a crude heat-treated bacteriocin preparation from L. lactis subsp. lactis yielding final populations only 10-fold lower as compared to untreated controls. Thus, manufacturers of mozzarella cheese must adhere to good manufacturing and sanitary practices to prevent contamination of the curd after stretching and growth of L. monocytogenes to potentially hazardous levels during storage.

SEMISOFT

AND

HARD CHEESES

By definition, hard cheeses are those that contain ≤40% moisture. Cheeses in this category include such varieties as Edam and Gouda (which can also be classified as semisoft cheeses) as well as Colby, Cheddar, Swiss, Emmentaler, Gruyère, Romano, and Parmesan, the last two of which are very hard grating cheeses. Transformation of chalky, acid-tasting curd into a ductile, full-flavored cheese is accomplished during ripening through the action of milk enzymes, rennet, and various microorganisms in the cheese, including the starter culture. The biochemical changes that occur during cheese ripening are complex and involve hydrolysis of fats and proteins with subsequent decarboxylation, deamination, and dehydrogenation as well as production of carbonyls, nitrogenous compounds, fatty acids, and sulfur compounds, all of which contribute to the overall flavor of the

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final product. The amount of aging needed to obtain a fully ripened cheese is directly related to moisture content, with a minimum of 2 and 10 months of ripening being required for Edam (~40% moisture) and Parmesan cheese (~30% moisture), respectively. The current popularity of many of these cheeses, along with the ability of L. monocytogenes to survive in acidic environments during refrigerated storage, has prompted a series of studies examining the behavior of L. monocytogenes during manufacture and storage of at least seven semisoft and hard cheese varieties. Gouda and Maasdam Cheeses Beginning with semisoft/hard cheeses, Northolt et al. [299] prepared Gouda and Maasdam cheese in The Netherlands from pasteurized milk inoculated to contain approximately 500 L. monocytogenes CFU/mL. Gouda cheese was manufactured according to standard procedures, with the exception that one lot was prepared using 0.3 rather than 0.6% starter culture to obtain a cheese with an unusually high moisture content of ~45%. Maasdam cheese was prepared using a culture of propionic acid bacteria in combination with 0.6% mesophilic lactic starter. After brine salting, Gouda cheese was ripened 6 weeks at 13°C, whereas Maasdam cheese was ripened 2 weeks at 13°C, 2 weeks at 18°C, and then stored at 4°C for an additional 2 weeks. As in previous studies, entrapment of listeriae within the curd matrix during cheesemaking resulted in population increases of approximately 10-fold as compared to the original level in pasteurized milk. However, some Listeria growth was noted during manufacture, with populations increasing an additional fourfold in normal Gouda and Maasdam cheese before brining. Six hours after manufacture, slightly higher Listeria populations were detected in Gouda cheese of high rather than normal moisture. Although numbers of listeriae in interior samples from both Gouda and Maasdam cheese remained relatively constant at ~104 CFU/g during the first 2 weeks of ripening, the pathogen was not detected in cheese samples taken at or near the surface. After 6 weeks of ripening, L. monocytogenes reappeared in surface samples from all cheeses at levels between 102 and 104 CFU/g. In contrast, numbers of listeriae in interior samples from 6-week-old cheese were only four- to eightfold lower than populations in the same cheeses immediately after brining. Although L. monocytogenes survived best in high-moisture Gouda cheese that had a pH of 6.0, the selective plating medium used in this study, Trypaflavine Nalidixic Acid Serum Agar, was suboptimal for recovery of stressed or acid-injured listeriae that probably were present in fully ripened Gouda and Maasdam cheese having pH values of 5.48 and 5.44, respectively. Colby Cheese Yousef and Marth [400] prepared Colby cheese from pasteurized milk inoculated to contain 102–103 L. monocytogenes (strain V7 or CA) CFU/mL. Following manufacture, all blocks of cheese were held at 4°C for 140 days. During cheesemaking, most Listeria cells were trapped in the curd matrix, with an average of only 2.4% of the original inoculum escaping in whey. Populations of L. monocytogenes in cheese increased an average of 1.27 orders of magnitude after pressing—about 29 h after the start of manufacture (Figure 12.7). Because an increase of no more than one order of magnitude can be attributed to entrapment of listeriae within the curd matrix after cutting, these findings suggest that slight growth of the organism did occur, particularly during the later stages of cheesemaking and pressing. Numbers of both Listeria strains remained relatively constant in cheese during the first 40 days of ripening, after which populations decreased almost linearly (Figure 12.7). Viability of L. monocytogenes was strongly influenced by moisture content, with strain V7 decreasing more than twice as fast in cheese containing 38.5% (D-value of 54 days) rather than 42.3% (D-value of 124 days) moisture, which is well above the maximum allowable moisture content of 40.0% for Colby cheese.

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L. monocytogenes log10 CFU/g

5

4

3 V7, 42.3% moisture V7, 38.5% moisture CA, 39.5% moisture 2 0 1 2 3 4 0

50

Hours

100

150

Days Time

FIGURE 12.7 Behavior of L. monocytogenes during manufacture and ripening of Colby cheese. (Adapted from Yousef, A.E. and E.H. Marth. 1988. Behavior of Listeria monocytogenes during the manufacture and storage of Colby cheese. J. Food Prot. 51: 12–15.)

Behavior of L. monocytogenes in cheese of normal moisture content also was strain dependent, with strain CA being less stable than strain V7. However, strains V7 and CA were still detected in 140-day-old Colby cheese by direct plating, with cold enrichment results from a follow-up study [402] indicating that both strains were still viable in 5- to 8-month-old Colby cheese stored at 4°C. According to the FDA, Colby and other selected cheeses can be manufactured from raw or heattreated (subpasteurization) milk provided that the finished cheese is held a minimum of 60 days at or above 1.7°C (35°F) before sale in an attempt to eliminate pathogenic microorganisms. These results (and those for Cheddar cheese to follow) have prompted the FDA to reconsider the adequacy of this aging requirement for cheeses prepared from raw milk. Cheddar Cheese Normal stirred-curd Cheddar cheese, which has a moisture content only slightly less than Colby cheese (i.e., 36–38%), was manufactured by Ryser and Marth [340] from pasteurized whole milk inoculated to contain approximately 5 × 102 L. monocytogenes (strain Scott A, V7, or CA) CFU/mL. The resulting 10-lb blocks of cheese were ripened at 6 and 13°C and assayed for numbers of listeriae at appropriate intervals. All curd samples examined during manufacture contained approximately 5 × 102 L. monocytogenes CFU/g, which suggests that the organism was only minimally concentrated in the curd and failed to grow during cheesemaking. However, because only 6.4% of the initial Listeria inoculum was recovered in the whey, the expected 10-fold increase from entrapment of the organism in the curd matrix probably went unnoticed because of inadequate sample preparation methods that have since been improved [402]. Numbers of listeriae increased slightly in cheese during pressing, with all three strains attaining maximum populations of ~3.50–3.75 log10 CFU/g after 14–35 days of ripening at 13 (Figure 12.8) and 6°C (Figure 12.9). This population increase, which was approximately 10-fold higher than that of the original inoculum in milk, probably occurred because of enhanced recovery of the pathogen from older cheese that was easier to homogenize

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4.0 Trial 4 Trial 5

Strain V7 log10 CFU/g

3.0

Trial 6

2.0

1.0 2 0 0

25

50

75

100 125 150 175 200 225 Days

250 275 300 325

FIGURE 12.8 Survival of L. monocytogenes strain V7 in Cheddar cheese ripened at 13°C. Open symbols at <1 log10 Listeria/g indicate that the organism was not detected after 8 weeks of cold enrichment, whereas half-solid or solid symbols at <1 log10 Listeria/g indicate that the pathogen was detected in one of two or two of two samples, respectively, using cold enrichment. Numbers indicate the week at which L. monocytogenes was found. (Adapted from Ryser, E.T. and E.H. Marth. 1987. Behavior of Listeria monocytogenes during the manufacture and ripening of Cheddar cheese. J. Food Prot. 50: 7–13.)

4 Trial 4 Trial 5 Trial 6

Strain V7 log10 CFU/g

3

2

1 4/6 6

6 0 0

50

100

150

200

250

300

350

400

Days

FIGURE 12.9 Survival of L. monocytogenes strain V7 in Cheddar cheese ripened at 6°C. See Figure 12.8 for explanation of symbols. (Adapted from Ryser, E.T. and E.H. Marth. 1987. Behavior of Listeria monocytogenes during the manufacture and ripening of Cheddar cheese. J. Food Prot. 50: 7–13.)

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rather than from actual growth, as shown by Yousef et al. [402]. After 35 days of storage at either temperature, Listeria populations in cheese began to decrease, with all cheeses maintaining pH values of 5.04–5.09 throughout ripening. Strains Scott A, V7, and CA survived 70–224, 126–196, and 70–126 days in cheese ripened at 13°C, respectively, whereas the same strains remained viable for 70–154, 126–434, and 70–154 days in cheese aged at 6°C. Thus, except for strain V7, which was still present in one block of 434-day-old cheese at a level of 30 CFU/g, the remaining two strains survived equally well in cheeses ripened at either temperature. These findings suggest different acid tolerances among the L. monocytogenes strains tested, as has also been reported by Gahan et al. [211]. Additional experiments with strains V7 and CA demonstrated that L. monocytogenes was uniformly distributed in Cheddar cheese during at least the first 98 days of ripening at 6°C. Working in England, Banks [124] also reported that L. monocytogenes persisted 8–9 months and up to 7 months in Cheddar cheese prepared from Listeria-contaminated raw and subpasteurized (i.e., 62.8 or 65.6°C) milk, respectively. Hence, these data provide some of the strongest evidence for the inadequacy of the 60 day/≥1.7°C minimum holding period for cheeses manufactured from raw milk. Several subsequent studies examined the effect of Cheddar cheese compositional changes on Listeria survival during cheese ripening. Mehta and Tatini [287] assessed the behavior of L. monocytogenes strains Scott A and V7 in stirred-curd Cheddar cheese containing 1.3 or 2.5% NaCl or an equal molar mixture of NaCl and KCl. Lowering the level of NaCl enhanced destruction of Listeria, with 1.3% NaCl, 2.5% Na/KCl, and 2.5% NaCl decreasing populations 4.3-, 2.3- and 0.4-orders of magnitude in cheese, respectively, after 10 weeks of aging at 7°C. Thus, in addition to being healthier, low-sodium Cheddar cheese appears to be safer in regard to listeriae. When these same investigators [288] prepared stirred-curd Cheddar cheese from whole milk and reduced-fat milk (1.55 or 2.0% milk fat), L. monocytogenes strains Scott A and V7 persisted in the finished cheese during 20 months of storage at 7°C, with no survival differences being observed between full-fat (28.9% fat) and reduced-fat (20.7%) cheese. However, Listeria survival is strongly influenced by milk fat composition and release of free fatty acids during cheese ripening [312]. Schaffer et al. [355] increased the levels of both long-chain (C18, C18:2) and unsaturated fatty acids in milk by feeding cows a diet of extruded soybeans or sunflower seeds and then used this milk to manufacture stirred-curd Cheddar cheese containing L. monocytogenes strains Scott A and V7 as previously described. During manufacture, numbers of listeriae increased 1.0–1.5 orders of magnitude as previously reported by Ryser and Marth [340]. More important, after 120 days of ripening at 7°C L. monocytogenes populations were 5 to 8 orders of magnitude lower in cheeses prepared from modified-fat milk as compared to unmodified milk. Although some inconsistences were noted in performance of sunflower- and soybean-modified milk, inactivation of L. monocytogenes always occurred most rapidly in cheeses containing the highest levels of free fatty acids, with oleic, linoleic, lauric, and myristic acids shown to be major contributors to Listeria destruction during cheese ripening. In addition to modification in milk and cheese composition, addition of various bacteriocins as well as various bacteriocin-producing starter and nonstarter cultures to milk during cheesemaking has also proved beneficial in reducing survival of Listeria in Cheddar cheese during ripening. Benech et al. [130,131] compared the effectiveness of adding liposome-encapsulated nisin Z (300 IU/g) to milk with the use of a nisin Z L. lactis subsp. diacetylactis starter culture for inactivation of L. innocua during manufacture and ripening of Cheddar cheese. When cheeses were prepared from milk inoculated to contain 105 to 106 L. innocua CFU/mL, the encapsulated form of nisin Z was more effective than the nisin Z-producing starter culture, with Listeria populations decreasing 3 and 1.5 logs immediately after cheesemaking, respectively. Most importantly, L innocua populations in 6-monthold cheeses prepared with encapsulated nisin Z decreased to <10 CFU/g compared to about 104 CFU/g for cheeses using the nisin Z-producing starter culture. The greater effectiveness of encapsulated nisin was related to both nisin stability and location with 90 and only 12% of encapsulated and starterproduced nisin Z remaining active at the fat/casein interface and in the fat of the cheese, respectively.

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In other work, Buyong et al. [148] used a pediocin PA-I-producing strain of L. lactis subsp. lactis in production of Cheddar cheese from pasteurized milk that was inoculated to contain about 103 L. monocytogenes CFU/mL. Overall, pathogen populations were about 3 logs lower in cheese prepared with rather than without the pediocin-producing starter culture. Based on these findings, use of encapsulated nisin and other bacteriocin-producing starter cultures affords an added degree of protection against long-term survival of Listeria and should be considered, particularly if Cheddar and other similar cheeses are to be prepared from raw milk. Swiss Cheese Unlike the aforementioned cheeses, manufacture and ripening of Swiss cheese involves several decidedly different steps, including cooking of the curd at 50–53°C and ripening the finished cheese at an elevated temperature for “eye” development. These observations prompted Buazzi et al. [146] to examine the fate of L. monocytogenes during manufacture and ripening of Swiss cheese. When rindless Swiss cheese was prepared from pasteurized milk inoculated to contain 104–105 L. monocytogenes strain V7, CA, or OH CFU/mL, the pathogen was generally unable to grow during cheesemaking, with populations increasing 43% during the early stages of cooking owing to physical concentration and curd shrinkage. Thereafter, about 57% of the population in the curd was inactivated after 30–40 min of cooking at 50°C. After pressing, the curd contained 50% fewer listeriae, with this population decreasing most sharply after 30 h of brining at 7°C. Storing the finished cheese (pH 5.2–5.4) 10 days at 7°C reduced the Listeria population to very low numbers. Complete inactivation of the pathogen occurred after 66–80 days of ripening at 24°C, with production of propionate by eye-forming bacteria likely contributing to the death of listeriae. Two studies conducted in Switzerland [121,253] demonstrated that the environments within Emmenthaler and Gruyère cheese (i.e., other varieties of Swiss cheese) also are not conducive to Listeria survival, with the pathogen no longer being present in 24-hour-old cheeses (pH 5.2–5.4) prepared from raw milk inoculated to contain 104 L. monocytogenes CFU/mL. Parmesan Cheese Parmesan cheese, a hard grating cheese that undergoes a high-temperature cook resulting in a very low moisture content, was prepared by Yousef and Marth [401] from pasteurized milk inoculated to contain ~104–105 L. monocytogenes (strain V7 or CA) CFU/mL. Unlike the cheeses discussed previously, a lipolytic enzyme (lipase) is often added to cheesemilk to produce the characteristic flavor of fully ripened Parmesan cheese. In addition, the coagulum is cut into very small particles that are cooked at ~52°C (125°F) for 45 to 60 min until the pH decreases to 6.1. This step serves to expel whey, thus producing a dry, ricelike curd that can be pressed to form a very dense, low-moisture cheese. Following manufacture, the cheese produced in this study was brine-salted (22% NaCl) for 7 days at 13°C, dried 4–6 weeks in a humidity-controlled chamber at 13°C, vacuumpackaged, and ripened at 13°C for a minimum of an additional 9 months. During the first 2 h of cheesemaking, populations of both Listeria strains increased approximately 6- to 10-fold, largely from entrapment of the organism within the curd matrix (Figure 12.10). Although Listeria counts remained relatively stable during cooking of the curd at 52°C (125°F) for 45 min, populations decreased appreciably during pressing of the curd. During brining, drying, and ripening at 13°C numbers of both Listeria strains decreased almost linearly, with estimated D-values ranging between 8 and 36 days. Using direct plating, strains V7 and CA were no longer detected in cheese after 21–112 and 14–63 days of ripening at 13°C, respectively. Despite large differences in survival of L. monocytogenes between different batches of cheese, both Listeria strains decreased at a faster rate in Parmesan than in Gouda, Maasdam, Cheddar, and Colby cheese during ripening. Decreased viability of the pathogen in Parmesan cheese is probably related to a combination of factors, including (a) action of lipase added to the milk, (b) heat treatment that the

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107 Manufacture

L. monocytogenes log10 CFU/g

106

105 Ripening 104

Batch #1 Batch #3

103

Batch #6

102

10 0

2

1 Hours

3 0

40

80

120

Days

FIGURE 12.10 Survival of Listeria monocytogenes strain V7 during manufacture and ripening of Parmesan cheese. (Adapted from Yousef, A.E., and E.H. Marth. 1990. Fate of Listeria monocytogenes during the manufacture and ripening of Parmesan cheese. J. Dairy Sci. 73: 3351–3356.)

curd receives during cheesemaking, and (c) lower moisture content (and water activity) of the fully ripened cheese. To decrease the moisture content and develop proper flavor, the present regulation in the United States requires that Parmesan cheese be aged a minimum of 10 months regardless of whether the cheese is prepared from raw or pasteurized milk. According to the results of this study, such an aging process should be sufficient to produce Listeria-free Parmesan cheese. Hard Italian-Type Cheese Working in Italy, Comi and Valenti [161] inoculated the surface and interior of three freshly prepared hard Italian-type cheeses (aw 0.95–0.98, pH 5.2–5.5) to contain 104–105 L. monocytogenes CFU/g. As was true for Parmesan cheese, the pathogen failed to grow in this relatively hard cheese, with Listeria populations decreasing 10- to 100-fold in both surface and interior samples from all three cheeses during the first 28 days of ripening at 4°C. Although the pH of surface and interior samples increased to 5.4–5.5 and 5.8–6.0, respectively, after 35 days ripening, Listeria populations continued to decrease, with the pathogen only being detected by cold enrichment (i.e., populations <102 CFU/g) in samples from 35-day-old cheeses. Compositional analysis of these cheeses suggested that the amount of moisture lost after 35 days of ripening (aw 0.95–0.96) may have offset the benefit for Listeria growth caused by the increase in pH.

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HISPANIC CHEESES Traditional Hispanic-type cheeses comprise a wide range of white cheeses produced in Mexico and in Central and South America. Some of the most popular varieties, including Queso blanco, Queso fresco, and Queso de Puna, are high-moisture fresh cheeses consumed shortly after manufacture, whereas others, such as Queso Anejo, Queso de Bola, Queso de Crema, Queso de los Ibores, and Queso de Prensa, are lower in moisture and undergo various degrees of aging. Although 30 min of heating at 80–85°C is more than adequate to inactivate L. monocytogenes during manufacture of Queso blanco cheese [179], typical production practices for Hispanic-type cheeses involve extensive curd manipulations, including hand stirring, salting, and molding, any of which can easily lead to product contamination. Based on recent risk assessments that are discussed elsewhere and over 20 Class I recalls of Hispanic-style cheeses, Queso blanco, Queso fresco, and other soft varieties pose a greater risk to consumers than most other cheeses, particularly when illegally prepared from raw milk. Queso Blanco Cheese In 1995, Glass et al. [222] reported on the behavior of L. monocytogenes in starter culture-free Queso blanco cheese containing citric, malic, or acetic acid as acidulants and ALTA (a commercial bacteriocin preparation resembling pediocin AcH) as an antilisterial agent. After the finished product (pH 5.2) was inoculated to contain 106 L. monocytogenes CFU/g, populations increased about 10fold in cheeses containing citric or malic acid during 42 days of storage at 4°C, whereas numbers of listeriae decreased slightly in cheeses prepared with acetic acid. These findings are consistent with those of other investigators who used various laboratory media acidified with malic, citric, or acetic acid (see Chapter 6). Addition of 0.6% ALTA to these cheeses yielded slightly lower Listeria counts as compared to cheeses without ALTA. Using an L. monocytogenes inoculum level of 102 CFU/g, these workers concluded that acetic acid was significantly more effective than malic or citric acid in reducing numbers of L. monocytogenes in Queso blanco cheese and that addition of ALTA provided added protection against this pathogen. Similar benefits also were reported when Queso blanco cheese was prepared using a nisin-producing starter culture, with L. monocytogenes populations being about 1000-fold lower in such cheeses (pH 5.3) after 21 days of storage at 4 or 12°C than in nisin-free controls [144]. Although direct addition of Nisaplin (1000 AU/mL) to the cheese milk yielded Listeria populations 100-fold lower in 1-day-old cheeses than in Nisaplin-free controls, the pathogen recovered to control levels within 21 days at 12°C. Hence, incorporating a nisin-producing starter culture was superior to direct addition of Nisaplin for minimizing survival of L. monocytogenes in Queso blanco cheese during storage. Queso de los Ibores Cheese The fate of L. monocytogenes in Queso de los Ibores cheese (a hard, ripened cheese of pH ~5) also was indirectly determined by Mas and Gonzalez-Crespo [277] using commercially available cheeses of various ages. Overall, detecting Listeria spp. in 5 of 10, 2 of 10, and 1 of 10 cheeses that had been aged for 7, 30, and 60 days, respectively, suggests that this hard, low-moisture cheese will not support long-term survival of listeriae. Mexican Manchego Unlike Spanish Manchego cheese, which is a hard aged variety traditionally produced from ewe’s milk (see the section on ewe’s and goat’s milk cheese), Mexican Manchego is a high-moisture cheese that is produced from pasteurized cow’s milk and is ready for consumption within 5 days of manufacture. Solano-Lopez and Hernandez-Sanchez [366] prepared Mexican Manchego cheese from pasteurized cow’s milk inoculated to contain 106 L. monocytogenes CFU/mL along with a 1% mesophilic lactic acid bacteria starter culture. After the curd was cooked at 40°C, salted, and pressed into hoops, numbers

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of listeriae decreased almost 10-fold. An additional 6-fold reduction was seen following 5 days of ripening at 12°C and 85% RH, with the final cheese having a pH of 5.4 and a moisture content of 42.1%. Chihuahua In addition to Mexican Manchego cheese, Solano-Lopez and Hernandez-Sanchez [366] also assessed the fate of L. monocytogenes in Chihuahua. Except for a cheddaring step after curd cooking, manufacture of this semihard aged cheese is similar to Mexican Manchego. When Chihuahua cheese was prepared from pasteurized cow’s milk inoculated to contain 106 L. monocytogenes CFU/mL along with a 1% mesophilic lactic acid bacteria starter culture, numbers of Listeria decreased about 10-fold during cheesemaking and an additional 10-fold in the finished cheese (pH 5.8, 36.4% moisture) after 6 weeks of ripening at 12°C and 85% RH. Overall, these findings were similar to those for Cheddar cheese [340] and indicate that Listeria could also persist for more than 60 days if Chihuahua cheese is prepared from contaminated raw milk.

PICKLED CHEESES The terms pickled and white-brined are often used to describe a group of soft/semisoft, white curd cheeses to which large quantities of salt are added as a preservative. Cheeses belonging to this group are principally manufactured in countries bordering the Mediterranean Sea and include such varieties as feta (Greece), Turkish white-brined cheese (Turkey), Teleme (Bulgaria), Domiati (Egypt), and Kareish (Egypt). Some of these cheeses are frequently prepared from ewe’s, goat’s, or buffalo’s milk. Depending on the cheese variety, salt either can be added directly to the milk or curd, or the finished cheese can be stored in salt brine, salted whey, salted skim milk, or dry salt. The extreme tolerance of L. monocytogenes to high concentrations of salt, along with the organism’s ability to grow at refrigeration temperature, has made these cheeses of particular interest to food microbiologists working with Listeria. Feta In 1989, Papageorgiou and Marth [307] described the fate of L. monocytogenes during manufacture, ripening, and storage of feta cheese. During the course of this work, there was an unconfirmed report of a woman in New York who delivered a stillborn infant in December 1987 after consuming feta cheese contaminated with L. monocytogenes. Hence, this study, which will now be discussed, took on added importance. According to these authors, cow’s milk was inoculated to contain approximately 5 × 103 L. monocytogenes (strain Scott A or CA) CFU/mL. After warming the milk to 35°C, a 1% commercial starter culture of S. thermophilus/L. bulgaricus was added. Forty min after addition of rennet, the coagulum was cut and the resulting curd was transferred to metal hoops. Following 6 h of draining, cheeses were removed from the hoops and placed in a 12% salt brine solution for 24 h. The following day, all cheeses were transferred to 6% salt brine at 22°C for 4 days until the cheese attained a pH of 4.3–4.4. Finally, cheese in the same 6% brine solution was moved to storage at 4°C. Cells of L. monocytogenes were entrapped in the curd matrix during cheesemaking, with populations nearly 10-fold greater in curd than in inoculated milk. Only about 3.2% of the original inoculum was lost in the whey. During whey drainage and the first 1–2 days of ripening at 22°C, numbers of listeriae increased 1.5 log10 CFU/g, with both strains attaining maximum populations of approximately 1 × 106 CFU/g (Figure 12.11). Although growth of both Listeria strains generally ceased at pH values between 4.6 and 5.0, numbers of listeriae remained virtually constant in cheese during 2–5 days of storage at 22°C in 6% brine solution. Both salt brines in which feta cheese was ripened and/or stored were positive for listeriae (these details are discussed with brine solutions at the end of this chapter). Although all feta cheeses older than 5 days maintained a pH of 4.3, both L. monocytogenes strains survived >90 days in finished cheese stored at 4°C (Figure 12.12).

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L. monocytogenes log10 CFU/mL or g

7

7 Average pH

6

6

pH 5

5

Trial 1 Trial 2 Trial 3

4

3 –20

4

3 0

20

40

80

60

100

120

140

Hours

FIGURE 12.11 Fate of L. monocytogenes strain Scott A during manufacture and early brining of feta cheese. (Adapted from Papageorgiou, D.K. and E.H. Marth. 1989. Fate of Listeria monocytogenes during the manufacture, ripening and storage of feta cheese. J. Food Prot. 52: 82–87.)

However, differences between the two Listeria strains were noted, with populations of strains Scott A and CA decreasing 1.28 and 3.07 log10 CFU/g in 90-day-old as compared with 2-day-old feta cheese, respectively. Years later, Papageorgiou et al. [309] used the same protocol to produce feta cheese from pasteurized milk inoculated to contain L. monocytogenes strains Scott A and CA at a level of 1.2 × 106 CFU/mL. After manufacture, the finished cheese containing 3.5 × 107 and 8.0 × 106 CFU/g of strains Scott A and CA, respectively, was periodically analyzed for numbers of listeriae

L. monocytogenes log10 CFU/g

7

6

5

4 Trial 1 Trial 2 Trial 3

3

2 0

20

40

60

80

100

Days

FIGURE 12.12 Survival of L. monocytogenes strain Scott A during storage of feta cheese. (Adapted from Papageorgiou, D.K. and E.H. Marth. 1989. Fate of Listeria monocytogenes during the manufacture, ripening and storage of feta cheese. J. Food Prot. 52: 82–87.)

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during 7.5 months of frozen storage at −38 and −18°C. Regardless of storage temperature, populations of strain CA decreased 9- to 10-fold in cheese samples taken from the surface and interior after 2 to 6 weeks of storage and remained at these levels thereafter. However, strain Scott A was far hardier, decreasing only about 2- and 4- to 6-fold in cheese samples from the surface and interior, respectively, during 7.5 months of frozen storage at either temperature, thus indicating that extended freezing is insufficient to inactivate substantial numbers of Listeria. Turkish White-Brined Cheese Sarumehmetoglu and Kaymaz [351] reported that L. monocytogenes behaved similarly in Turkish white-brined cheese prepared from artificially contaminated raw milk, with numbers of listeriae generally decreasing >100-fold in the finished cheese during 90 days of refrigerated storage. In similar cheesemaking trials by Erkmen [200,201], L. monocytogenes exhibited D-values of 15.7 to 22.4 days in Turkish white-brined cheese stored at 4°C with the shorter D-values associated with cheeses prepared with rather than without a starter culture as well as with cheese brined using a 20 rather than 15% salt solution. Although feta and Turkish white-brined cheese can be prepared from raw milk, ripening such cheese at or above 1.7°C for 60 days will not in any way guarantee that the final product is Listeria-free, with long-term survival of this pathogen highly probable. Hence, it would appear prudent to manufacture these cheeses only from pasteurized milk under good hygienic conditions to decrease the chance of a public health problem involving Listeria. Domiati Cheese Domiati cheese, a popular fresh white-brined cheese most commonly consumed in Egypt and other parts of the Middle East, was prepared by Tawfik [373] using a 1:1 mixture of pasteurized cow:buffalo milk to which 7.5% NaCl, 0.5% Lactobacillus casei, and 106 L. monocytogenes were added. Listeria populations increased approximately 10-fold in the finished cheese as a result of entrapment within the curd matrix and then decreased over time, with the pathogen surviving 4–8 weeks when the cheese was stored in salted whey at 20–25°C. Similar findings were reported by Ahmed et al. [6], with L. monocytogenes strain V7 being inactivated in Domiati cheeses (pH 4.5–5.5) containing 5 and 10% NaCl during 4 weeks of ripening at 30°C. According to Abou-Donia and Al-Medhagi [3], L. monocytogenes also persisted no more than 8 weeks in naturally contaminated retail samples of Domiati cheese. A decrease in cheese pH from 6.1 to 3.8 and an increase in salt content from 6.8 to 11.4% are clearly responsible for limiting growth and survival of L. monocytogenes in this product. Bulgarian White-Pickled Cheese As early as 1964, Ikonomov and Todorov [246] reported manufacturing white-pickled cheese from ewe’s milk containing 102–103 L. monocytogenes CFU/mL. A mixture of 0.1% S. lactis and L. casei served as the starter culture. Although L. monocytogenes persisted 15–30 days in white-brined cheese ripened at 18–22°C, the pathogen survived twice as long when the same cheese was ripened at 12–15°C. In addition to storage temperature, Listeria viability also was partly dependent on the amount of acid produced in the cheese during ripening, with pH values of approximately 4.3 and 4.6 being reported as lethal to listeriae in cheese ripened at 12–15 and 18–22°C, respectively. Sudanese White-Pickled Cheese Working in the United States, Abdalla et al. [1] prepared a traditional Sudanese white-pickled cheese from Lactococcus starter and Lactococcus starter-free pasteurized milk that was inoculated to contain 105 L. monocytogenes Scott A CFU/mL. During cheesemaking, Listeria populations

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increased approximately 10-fold to 106 CFU/g in the cheese, with growth and acid production by the starter culture being prevented by addition of 8% NaCl to the milk during cheesemaking. Consequently, the finished product had a pH of 6.5–7.0, which is optimal for Listeria growth. Relatively stable Listeria populations of approximately 108 CFU/g developed in the cheese after 30–65 days of refrigerated storage in brine containing 8.6% NaCl. Similar Listeria populations were observed when the same starter-free cheese was prepared from pasteurized milk containing 4% NaCl and preserved in a 4% brine solution with addition of 1% potassium sorbate, nisin (25 Mg/mL), or 0.1% hydrogen peroxide to the milk not affecting Listeria survival [2]. Ineffectiveness of these antimicrobial agents was attributed to several factors, including loss during processing, degradation during the early stages of cheese ripening, and suboptimal environmental conditions. In contrast, when a 1% lactic starter inoculum was used, L. monocytogenes populations decreased more than 6 orders of magnitude, with the pathogen no longer being detected in cheese after 50 days of ripening (pH 4.6) at 4°C. As was true for the other pickled cheeses, large numbers of listeriae again leached from the cheese into the brine solution; these findings are discussed in detail at the end of this chapter. Yugoslavian White-Brined Cheese In 1974 Sipka et al. [363] published results from another study in which white-brined cheese was prepared from naturally infected cow’s milk containing 240 L. monocytogenes CFU/mL. Following manufacture, the pathogen grew rapidly in cheese, reaching populations of 7.8 × 105 and 1.0 × 106 CFU/g after 7 and 14 days of brining, respectively. Although maximum Listeria populations were similar to those observed in feta cheese [307], the pathogen appeared to be less hardy in whitebrined than in feta cheese, with populations decreasing to 1.3 × 105 and 1.2 × 102 CFU/g in 24- and 42-day-old fully ripened cheese, respectively. Increased inactivation of listeriae in whitebrined rather than feta cheese apparently is not entirely related to acid development, because the pH of the former cheese, 4.5–5.1, was higher than that of the latter, 4.3. Twenty-one years later, Katic [252] prepared a similar white-brined cheese from artificially contaminated milk containing 0.8% starter culture and found that after 40 days of storage at 4°C, L. monocytogenes populations had increased 100- and 43-fold in cheeses stored in 10 and 16% brine, respectively. In contrast, numbers of listeriae increased only 4- and 12-fold in identical cheeses that were stored in 10 and 16% brine solutions at 8°C, respectively. Thus, given the same brining conditions, higher storage temperatures were, as expected, more detrimental to Listeria survival.

EWE’S

AND

GOAT’S MILK CHEESE

In areas of the world where cows are not plentiful (i.e., northern Scandinavia, Eastern Europe, Mediterranean, Middle East), ewe’s and goat’s milks have been adapted for use either alone or in combination with cow’s milk to manufacture different types of cheese. Representative cheeses in this group include such well-known varieties as French Roquefort and Greek feta cheese, both of which are traditionally prepared from ewe’s milk. Lesser-known cheeses typically prepared from ewe’s and goat’s milk include Egyptian Kachkaval, Italian Fontina, Spanish Manchego, and Italian Pecorino Romano as well as many varieties of white-brined and ethnic goat’s milk cheese, the latter being often produced in mountainous areas of Central Europe and Scandinavia. The occasional presence of L. monocytogenes in milk from healthy ewes and goats has prompted several studies dealing with behavior of this pathogen during manufacture and ripening of some of these less common cheeses. Kachkaval Cheese Work with this group of cheeses dates back to 1964 when Ikonomov and Todorov [246] examined the behavior of L. monocytogenes in Kachkaval cheese (a relatively soft, brine-salted cheese

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manufactured from ewe’s milk in Eastern Europe and the Middle East, pH 5.0–5.8) prepared from raw ewe’s milk inoculated with the pathogen. According to these investigators, L. monocytogenes survived in curd immersed in 5–6% salt brine at 71–76°C during cheesemaking and was still present in Kachkaval cheese (pH 5.0–5.4) after 30–50 days of ripening at 18–22°C. Manchego Cheese The next such study did not appear in the literature until 1987 when Dominguez et al. [180] reported manufacturing four lots of Manchego cheese (a hard aromatic cheese traditionally prepared from ewe’s milk in Spain, pH ~5.8) from a blend of pasteurized ewe’s, goat’s, and cow’s milk (15:35:50) inoculated to contain either 4.0 × 103 or 1.9 × 105 L. monocytogenes CFU/mL. To assess growth of listeriae in cheeses having different rates of acid development, milks were inoculated to contain either 0.1 or 1.0% of a mesophilic lactic acid bacteria starter culture. The coagulum was cut approximately 45 min after addition of rennet; the resulting curd was drained, hooped, and pressed for approximately 10 h. The finished cheese was then brine-salted overnight, ripened 10 days at 15°C/85–90% RH, covered with paraffin, and aged an additional 50 days at 15°C. Numbers of L. monocytogenes increased 10-fold in all cheeses during the first 10 h of manufacture, primarily from entrapment of the organism within curd matrix. The fact that cheeses prepared with either 0.1 or 1.0% starter culture contained similar Listeria populations indicates that behavior of the pathogen was not greatly influenced by the rate of acid production during cheesemaking. After brining the cheese overnight, populations of listeriae decreased approximately 3- to 100-fold, with additional small decreases being observed during ripening of unparaffined cheese at 15°C. Numbers of listeriae remained relatively constant in cheese at pH 5.1–5.8 after paraffining, with populations in 60-day-old cheese approximating the original inoculum in milk from which the cheese was manufactured. However, Rodriguez et al. [332] more recently showed that using a nisin-producing L. lactis subsp. lactis starter culture could markedly shorten survival of L. innocua in Manchego cheese during ripening. Goat’s Milk Cheese Working in Sweden, Tham [291] examined the viability of Listeria in cheese prepared from raw goat’s milk inoculated to contain 105–106 L. monocytogenes CFU/mL. A mixture of mesophilic lactic acid bacteria served as the starter culture. Approximately 45 min after addition of rennet, the coagulum was cut into cubes that were cooked at 37°C, drained, hooped, pressed for 1 h, and brinesalted for 10 h. The finished cheese was then ripened at 12°C for 22 weeks. Actual numbers of L. monocytogenes could be determined in cheeses from only two of six lots using a blood agar/pour plate method. As shown in Figure 12.13, Listeria populations decreased approximately 10-fold in goat’s milk cheese during the first 14 weeks of ripening at 12°C. Extended survival, along with slight growth of listeriae in cheeses ripened longer than 14 weeks, probably is related to an increase in pH from 5.55 to 6.20 during ripening as well as a decrease in numbers of competitive microorganisms that were initially present in the raw milk. Although large numbers of enterococci and other microbial competitors interfered with the quantitative recovery of L. monocytogenes in the remaining four cheeses, the pathogen survived 10–16 weeks in three of these cheeses as determined by cold enrichment. The impact of an active starter culture on the fate of Listeria in Swedish raw milk goat cheese was also later confirmed by Eilertz et al. [183], with L. monocytogenes populations increasing 10- and 10,000-fold in cheeses prepared with and without starter culture, respectively, after 4 weeks of ripening at 4°C. In 2001, Morgan et al. [294] prepared a soft surface-ripened cheese from raw goat’s milk inoculated to contain 102 L. monocytogenes CFU/mL with cultures of Kluyvermyces lactis and Geotrichum candidum used for surface ripening. During cheesemaking, numbers of listeriae increased to 3.3 log CFU/g, with half of this increase resulting from physical entrapment in the

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470

9.0

L. monocytogenes in cheese VI L. monocytogenes in cheese V Total aerobic count in cheese VI Total aerobic count in cheese V

8.0

Added L. monocytogenes per mL milk

7.0 6.0 5.0

0

2

4

6

8

10

12

14

16

18

Weeks

FIGURE 12.13 Survival of L. monocytogenes and total aerobic flora during manufacture and ripening of raw goat’s milk cheese. (Adapted from Tham, W. 1988. Survival of Listeria monocytogenes in cheese made of unpasteurized goat milk. Acta Vet. Scand. 29: 165–172.)

curd matrix, as previously discussed. After 7 days of ripening, cheese samples taken from the surface and interior contained about 2.0 and 3.0 L. monocytogenes log CFU/g, respectively. However, this difference was no longer evident in 28-day-old cheeses with surface and interior samples both yielding about 1.5 log CFU/g. Although the pH of cheese samples taken from the surface and the interior increased from 4.25 to 6.52 and 4.25 to 5.23 after 42 days of ripening, respectively, numbers of listeriae remained at levels near 1.5 log CFU/g. These results are in sharp contrast to those reported for Camembert and most other surface-ripened cheeses, with G. candidum perhaps involved in suppressing the growth of Listeria during cheese ripening as has been previously reported by Dieuleveux and Gueguen [178] A middle-aged English woman developed listerial meningitis in February 1988 after consuming 2–3 oz of commercially produced Anari-type goat’s milk cheese containing 107 L. monocytogenes CFU/g [64]. During a series of follow-up investigations [283], L. monocytogenes populations of <10 CFU/g were discovered in one 2- to 3-day-old Anari cheese and in three 2- to 3-day-old Halloumi cheeses produced by the same manufacturer. After these naturally contaminated cheeses were stored at 4°C for 4–5 weeks, Listeria populations as high as 8 × 104 and 1 × 106 CFU/g developed in Anari (pH 5–6) and Halloumi cheese (pH 6), respectively. Assuming a lag time of zero and an original L. monocytogenes population of 1–9 CFU/g, these authors calculated generation times of 47–56 and 32–37 h for this pathogen in Anari and Halloumi cheese, respectively. Both of these generation times are similar to those previously reported for L. monocytogenes in refrigerated fluid milks (see Chapter 11, Table 11.7). Assuming that these cheeses were on sale for up to 3 months after distribution, potentially hazardous levels of listeriae easily could have developed in such products during retail storage. Although a recent attempt to minimize Listeria growth in soft goat cheese by modified atmosphere packaging under increased levels of CO2 failed [301], highpressure processing of Listeria-contaminated raw goat’s milk cheese at 450 MPa for 10 min or 500 MPa for 5 min reportedly decreased Listeria populations >5.6 log CFU/g without adversely affecting appearance, texture, or flavor [213]. Although a costly microbial reduction strategy, highpressure processing may be one means of enhancing the safety of goat’s milk and other specialty cheeses that command a premium.

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SOFT UNRIPENED CHEESE Soft unripened cheeses include such high-moisture, white-curd varieties as cottage, baker’s, cream, and American-type Neufchâtel cheese. Unlike the groups of cheeses discussed thus far, milk to be manufactured into soft unripened cheese is coagulated through production of acid by the starter culture (or alternatively, by direct acidification of milk to pH 4.6–4.7 with gluconic acid, gluconodelta-lactone, or a mineral acid plus the lactone) rather than by the addition of a coagulant. Hence, these products are sometimes referred to as acid-curd cheese. Because the refrigerated shelf life of most soft unripened cheeses is typically less than 60 days, these varieties must be prepared from pasteurized milk or cream in the case of cream cheese. Although pH values of 4.6–5.0 provide an unfavorable environment for microorganisms that may contaminate soft unripened cheese before, during, or after manufacture, the fact that these cheeses can be consumed immediately after production may pose a public health risk, particularly if psychrotrophic, acid-tolerant organisms such as L. monocytogenes are present. Cottage Cheese Nearly 1 year before the famed cheeseborne listeriosis outbreak in California, Ryser et al. [346] used the short-set procedure to prepare cottage cheese from pasteurized skim milk inoculated to contain 104–105 L. monocytogenes CFU/mL. Following manufacture, half the curd was creamed to contain ≥4% milk fat and half remained uncreamed. Both products were examined for numbers of listeriae during 28 days of storage at 3°C. Numbers of L. monocytogenes remained relatively constant during the initial 5–6 h of cheesemaking, during which the pH of milk decreased from 6.65 to 4.70. These findings agree with those of Schaack and Marth [353], who later demonstrated that growth of L. monocytogenes in skim milk at 30°C is completely suppressed by a 5% inoculum of S. cremoris. After increasing the temperature of the curd/whey mixture to 57.2°C (135°F) over 90 min and cooking the curd at this temperature for an additional 30 min, L. monocytogenes was not detected in samples of curd or whey that were directly plated on McBride Listeria Agar. However, following cold enrichment in Tryptose Broth, L. monocytogenes was detected in four of eight, two of eight, one of eight, and two of eight samples of cooked curd, whey, wash water, and washed curd, respectively, which suggests that some Listeria cells were only sublethally injured during cooking of the curd at pH 4.6–4.7. Such injury may preclude growth on McBride Listeria Agar, which contains both lithium chloride and phenylethanol as selective agents. Examination of the finished product indicated that L. monocytogenes survived in both creamed and uncreamed cottage cheese at levels generally <100 CFU/g during 28 days of refrigerated storage. Although there was no evidence for growth of listeriae in either cheese during storage, probably because of pH values generally <5.5, the pathogen was recovered more frequently and at higher numbers in creamed rather than uncreamed cottage cheese. The higher pH of 3-day-old creamed (pH 5.32–5.45) rather than uncreamed (pH 5.12–5.22) cottage cheese may have been responsible for increasing the repair rate of injured cells, thereby increasing recovery of listeriae. Although behavior of L. monocytogenes in cheese failed to gain widespread attention until 1985, a search of the scientific literature uncovered an earlier study by Stajner et al. [367] that examined the viability of Listeria in unsalted small-curd skim milk cheese (similar to uncreamed cottage cheese) manufactured from naturally infected milk containing approximately 5 × 105 L. monocytogenes CFU/mL. Results from these Yugoslavian investigators support the findings of Ryser et al. [346] in that the pathogen survived at least 7 days in finished cheese (pH 4.55–4.75) stored at 3–5°C. More recently, El-Shenawy and Marth [191] studied the behavior of listeriae in cottage cheese prepared from pasteurized skim milk that was inoculated to contain 106 L. monocytogenes CFU/mL and then coagulated over a period of 3 h using hydrochloric acid, gluconic acid, or bovine rennet

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L. monocytogenes log10 CFU/g or mL

7 6

5 4 3

Rennet Curd Rennet Whey HC1 Curd HC1 Whey GA Curd GA Whey

2

1

0 A

B

C

D

E

F

FIGURE 12.14 Survival of L. monocytogenes in curd and whey obtained during preparation of cottage cheese made with rennet, HCl, or gluconic acid (GA). A: Immediately after cutting; B: after temperature was increased to 48.9°C; C: after temperature was increased to 54.4°C; D: after temperature was increased to 57.2°C; E: after 15 min of cooking; and F: after 30 min of cooking. (Adapted from El-Shenawy, M.A. and E.H. Marth. 1990. Behavior of Listeria monocytogenes in the presence of gluconic acid and during preparation of cottage cheese curd using gluconic acid. J. Dairy Sci. 73: 1429–1438.)

rather than a lactic acid bacteria starter culture, during which time the temperature of the milk was gradually increased from 2 to 32°C. The resulting coagulum was then cut and cooked using the aforementioned procedure of Ryser et al. [346]. As might be expected, acidification of the milk to pH 4.7–4.8 followed by heating was again detrimental to survival of listeriae during manufacture of cottage cheese. Overall, L. monocytogenes populations decreased ~4.5 and > 6.0 orders of magnitude in fully cooked (57.2°C/30 min) curd obtained by adding hydrochloric and gluconic acid, respectively (Figure 12.14). However, using cold enrichment, Listeria was recovered from samples of fully cooked gluconic acid curd. Numbers of listeriae decreased faster in acidified whey than curd, with cold enrichment results indicating that the pathogen was eliminated from gluconic but not hydrochloric acid whey after 30 min of cooking at 57.2°C. Nonetheless, direct acidification of milk (pH 4.7–4.8) for cottage cheesemaking followed by cooking the resultant curd at 57.2°C for 30 min should be more than sufficient to eliminate expected numbers of listeriae (<10 CFU/mL) that might inadvertently enter pasteurized milk as postprocessing contaminants. In contrast to acid curd and whey, populations of listeriae in freshly cut rennet curd and whey were virtually identical to those initially observed in milk (Figure 12.14). Further, slight increases in numbers of listeriae were noted midway through manufacture, with fully cooked rennet curd (pH 6.6) and whey still containing 104 and 103 L. monocytogenes CFU/g or CFU/mL, respectively. Thus, listericidal effects associated with cooking were greatly enhanced under acidic conditions. Subsequent experiments with selective plating media confirmed that substantial numbers of L. monocytogenes cells were sublethally injured during manufacture of cottage cheese, as was suggested by Ryser et al. [346] five years earlier. Sublethal injury was far more evident in whey rather than curd samples, probably because curd afforded some thermal protection to listeriae. Not surprisingly, the degree of sublethal injury was also closely related to coagulant type (i.e., acidity)

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and cooking temperature, with less injury being observed in rennet rather than acid curd and whey, and partially rather than fully cooked samples of curd and whey. Heat alone was primarily responsible for rennet-associated injury, whereas the combined effects of heat and acid led to injury of listeriae in acid curd and whey. With the exception of cottage cheese prepared from milk acidified with gluconic acid, both of these studies demonstrated limited survival of L. monocytogenes in cottage cheese. However, the fact that the pathogen failed to grow in the product and decreased drastically in numbers during manufacture suggests that cottage cheese poses far less of a public health threat than do those varieties that are surface-ripened with molds or bacteria. Lower health risks associated with consumption of cottage cheese are also supported by the extremely low incidence of L. monocytogenes in commercially produced cottage cheese examined in American and European surveys. The likelihood of L. monocytogenes entering cottage cheese during creaming or packaging of the product is far greater than having listeriae present in pasteurized milk at sufficiently high levels to survive in the cooked curd. Although most recently published work has addressed the fate of L. monocytogenes in cottage cheese as a postmanufacturing contaminant, results from these efforts have been somewhat conflicting. Based on the findings of three studies conducted in the United States [313] and England [207,243], L. monocytogenes failed to grow in artificially contaminated, commercially prepared creamed cottage cheese (pH 4.5–5.1), with populations generally decreasing 0.5–1.5 orders of magnitude during 1–5 weeks of storage at refrigeration or abusive temperatures. According to Moir et al. [292], numbers of L. monocytogenes remained relatively stable in commercial, Australian-produced creamed cottage cheese during 1 month of storage at 15°C. This behavior is similar to that observed by Hicks and Lund [243] when creamed cottage cheese was inoculated with an acid-adapted strain of L. monocytogenes previously cultured in Tryptose Phosphate Broth at pH 5.5. In contrast to these findings, at least four additional studies attest to growth of L. monocytogenes [158,171,217] and L. innocua [206] in similar samples of creamed cottage cheese, with populations increasing 0.5–3.0 orders of magnitude during refrigerated storage. Although not readily apparent, such behavioral differences in creamed cottage cheese may be related to differences between Listeria strains as well as differences in acid tolerances [211] and abilities to readily compete with the native microflora. Because L. monocytogenes can persist beyond the normal shelf life of cottage cheese, several options also have been examined for minimizing growth and/or survival of Listeria in this product during refrigerated storage. A few chemical additives, including sorbate [313], 3% sodium lactate [313], 3% calcium lactate [313], and 0.04% lysozyme, were shown to be, at best, only minimally effective, with Listeria populations in inoculated creamed cottage cheese decreasing ≤10-fold during the product’s normal refrigerated shelf life. Bacteriocin-producing starter cultures appear to be a far more promising means of inactivating any listeriae that may inadvertently contaminate the curd after cooking. One approach for introducing these bacteriocins into cottage cheese is the use of a bacteriocin-producing starter culture for cheese manufacture. Applying this strategy, McAuliffe et al. [281] prepared cottage cheese from skim milk using a lacticin 3147-producing strain of L. lactis at an inoculum level of 1%. After manufacture, the curd was inoculated to contain about 104 L. monocytogenes strain Scott A CFU/g at the time of creaming. Following 5 days of storage at 4°C, L. monocytogenes populations decreased below the detectable limit of 10 CFU/g in cottage cheese prepared with the lacticin-producing starter culture compared to populations of 5 × 103 in cheese prepared with a non-lacticin-producing strain of L. lactis. Alternatively, purified or partially purified bacteriocin powders can be directly incorporated into cottage cheese at the time of creaming. Using commercially prepared dry cottage cheese curd to which 7.5 × 103 L. monocytogenes CFU/g were added during creaming, Pucci et al. [319] found that the product (pH 5.1) still contained 1 × 102 L. monocytogenes CFU/g after 7 days of storage at 4°C. However, addition of purified bacteriocin PA-1 powder from Pediococcus acidilactici to identical samples led to complete inactivation of the pathogen within 24 h. Similarly, El-Ziny and

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Debevere [193] found that addition of reuterin (a bacteriocin produced by Lactobacillus reuteri) to the creaming mixture at concentrations of 100, 150, and 250 units/g reduced L. monocytogenes populations 2, 5, and >5 log CFU/g, respectively, after 7 days of storage at 7°C. Using a different brand of dry cottage cheese curd that was inoculated to contain 3 × 105 L. monocytogenes CFU/g during creaming, Benkerroum and Sandine [132] detected very few viable listeriae in the product (pH 4.9–5.0) after 1 or more days of storage at 4°C. This antagonistic effect of cottage cheese (presumably from natural flora or by-products in cottage cheese) toward listeriae was enhanced by adding nisin (2.5 × 103 IU/g), with viable listeriae completely being eliminated from such cheese after 1 day of refrigerated storage. The efficacy of nisin has since been confirmed by Ferreira and Lund [207], who reported that L. monocytogenes populations decreased about 1000-fold in creamed cottage cheese (pH 4.6–4.7) containing 2000 IU nisin/g after 3 days of storage at 20°C. In these studies, nisin, bacteriocin PA-1, and reuterin remained active in creamed cottage cheese during prolonged storage. Thus, it appears that these bacteriocins may also prove useful in limiting Listeria survival in other fermented dairy products, including natural cheeses, cold-pack cheese food, and various cheese spreads [66,74] during the normal shelf life. However, decreased efficacy of nisin as well as PA-1 and reuterin in commercially prepared cheese sauce (pH 6.0), half-andhalf (pH 6.6), and skim milk (pH 6.6) shows that listericidal activity of these bacteriocins is strongly pH dependent. Alternatively, modified atmosphere packaging also has been explored as a means of both extending the refrigerated shelf life of cottage cheese and minimizing Listeria growth. According to Chen and Hotchkiss [158], L. monocytogenes populations in creamed cottage cheese (pH 5.1) packaged under 35% CO2 remained stable during 9 weeks of storage at 4°C, whereas the pathogen increased 1000-fold in aerobically packaged cheese. At 7°C, numbers of listeriae failed to change in CO2-packaged cheese during the first 4 weeks of storage. However, rapid growth occurred within 16 days in aerobically packaged cheese. Fedio et al. [206] also reported that L. innocua failed to grow in creamed cottage cheese (pH 5) during 28 days of storage at 5°C when the product was packaged under 50 or 100% CO2. However, this organism began growing rapidly in identical samples packaged aerobically or under 100% N2 after 7 days of incubation. Although modified atmosphere packaging appears to be useful in minimizing Listeria growth in cottage cheese, inactivation of the pathogen will not occur under such conditions, with the results of Chen and Hotchkiss [158] also indicating that the effectiveness of CO2 is strongly temperature dependent. Cream Cheese In the only other study dealing with the behavior of listeriae in soft unripened cheese, Cottin et al. [168] prepared cream cheese from a chemically acidified mixture of milk and cream that was inoculated to contain 101–107 L. monocytogenes CFU/mL. Using the lowest inoculum, Listeria grew in the finished product (pH ≤6) and attained a stable population of ~103 CFU/g within 2 days of storage at 4°C. Thus, unlike cottage cheese, the pH and moisture content of cream cheese are both sufficiently high to permit limited growth of listeriae in the product during refrigeration.

WHEY CHEESES A few cheeses such as ricotta, Broccio, Ricotone, and Anthotyros are prepared from sweet whey derived from the manufacture of mozzarella, Cheddar, Swiss, Tilsiter, and feta cheese. Manufacture of these whey cheeses is based on the direct acidification of whey, whey and milk, and whey–cream mixtures to pH 5.9–6.0 using food-grade acids (i.e., citric, acetic), lactic starter cultures, or acid whey powder, followed by cooking at 180–190°F to precipitate the whey protein. The fine precipitate that eventually rises to the vat surface is then removed, drained, matted, and marketed either as

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whey cheese or a dairy ingredient. Several additional extremely low-moisture (13 to 18%) whey cheeses, including Gjetost, Mysost, and Gudbrandsdalsost, are unique to Norway and are prepared by thermally concentrating and then boiling a blend of goat’s and cow’s milk whey until the mixture carmelizes and becomes viscous. This plastic mass is then cooled, extruded, and cut into extremely dense blocks for marketing. As was true for mozzarella cheese, L. monocytogenes will be completely inactivated during manufacture of these whey cheeses. However, the potential still exists for contamination during packaging, as is evidenced by at least one Class I recall of ricotta cheese in July 1991. In limited work, Davies et al. [173] manufactured a ricotta-type cheese from raw milk to which 500 ppm potassium sorbate and 50 or 100 ppm nisin were added before the start of cheesemaking. After coagulating the milk by heating at 90°C for 30 min with or without direct acidification to pH 5.9 with acetic acid, the precipitated curd was drained and inoculated with a 5-strain L. monocytogenes cocktail at a level of 2.5 × 102 CFU/g. During 63 days of storage at 6–8°C, Listeria populations in nisin-free control cheeses prepared without and with direct acidification of the milk steadily increased to about 107 and 109 CFU/g, respectively. In contrast, addition of 125 ppm nisin resulted in lag times of 11 and 26 days when the milk was coagulated by heating without and with acidification, respectively, with Listeria reaching maximum populations that were 1 to 2 logs lower than those in the control samples after 63 days of storage. However, regardless of the method of milk coagulation, the Listeria population was held constant for at least 55 days using 250 ppm nisin, indicating that addition of nisin is a viable intervention strategy for preventing growth of listeriae during long-term refrigerated storage of ricotta cheese. Following the report of a small cluster of listeriosis cases traced to Anari whey cheese produced in Cyprus, Papageorgiou et al. [308] examined the fate of L. monocytogenes as a postprocessing contaminant in Greek Myzithra (identical to Anari cheese), Anthotyros, and Manouri cheese, all of which are starter culture-free, soft (50–70% moisture), low-acid (pH 6.0–6.5) whey cheeses. Immediately after commercial manufacture, these cheeses were inoculated to contain ~500 L. monocytogenes strain Scott A or CA CFU/g. Regardless of cheese type or Listeria strain, the pathogen grew rapidly and attained maximum populations of 107–108 CFU/g after 24–30 days, 5–12 days, and 56–72 h of storage at 5, 12, and 22°C, respectively. The study just described prompted investigators in Greece to assess the efficacy of nisin and irradiation for minimizing growth and survival of Listeria in Anthotyros cheese. In the first of these studies, Samelis et al. [350] evaluated nisin as a biopreservative to control L. monocytogenes as a postprocessing contaminant. Anthotyros cheese was prepared from naturally or directly acidified whey to which 100 or 500 IU nisin/mL was added before the start of cheesemaking, with the finished cheese inoculated to contain 104 L. monocytogenes strain Scott A CFU/g. Alternatively, 100 or 500 IU nisin/g was added to the final cheese at the time of Listeria inoculation rather than to the whey used for cheesemaking. Following initial decreases of 1 to 2 logs, Listeria attained levels of 107 to 108 CFU/g in the cheese after 42 days of storage at 4°C when nisin was added directly to the finished cheese or when 250 IU nisin/mL was added to the whey at the start of cheesemaking. However, addition of 500 IU nisin/mL to the whey at the start of cheesemaking effectively prevented Listeria from exceeding the initial inoculum level of 104 CFU/g after 45 days of storage. Thus, nisin was most effective when added to the whey rather than to the finished cheese. In the only other study thus far reported involving Anthotyros cheese, Tsiotsias et al. [384] found that irradiation at 4 kGy decreased populations of L. monocytogenes strain Scott A about 3 logs in freshly made cheese. However, surviving cells were able to recover from the effects of irradiation, with the pathogen increasing 2 to 3 logs in the cheese during 42 days of storage at 4 and 10°C. Thus, irradiation dose not appear to be effective in controlling Listeria in dairy products, with this treatment also negatively impacting the ripening of aged cheeses such as Cheddar and Camembert. Consequently, addition of nisin during cheesemaking and strict hygienic practices during packaging appear to be the most practical means of minimizing the presence and growth of Listeria in Anthotyros cheese.

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3.0

2.5

Log10 Strain/g

2.0

1.5 Scott A 1.0

V7 CA

.5

OH

.0 0

20

40

60

80

100

120

140

160

180

200

Days

FIGURE 12.15 Survival of four strains of L. monocytogenes strains in nonacidified cold-pack cheese food manufactured without preservatives. (Adapted from Ryser, E.T. and E.H. Marth. 1988. Survival of Listeria monocytogenes in cold-pack cheese food during refrigerated storage. J. Food. Prot. 51: 615–621, 625.)

Cold-Pack Cheese Food Unlike the natural cheeses discussed thus far, cold-pack cheese food is typically prepared by comminuting and blending aged Cheddar cheese (or another variety) with nonfat dry milk, dried whey, water, cream, plastic cream (composition similar to butter), salt, acidulants (i.e., lactic or acetic acid), preservatives (i.e., potassium sorbate or sodium propionate), and other optional ingredients into a homogeneous mass without heating. Because all the dairy ingredients and some of the optional ingredients used in manufacturing cold-pack cheese food can potentially harbor Listeria Ryser and Marth [344] investigated the behavior of four L. monocytogenes strains in nine different formulations of cold-pack cheese food inoculated to contain approximately 500 L. monocytogenes CFU/g. During 182 days of storage at 4°C, populations of all four Listeria strains decreased less than 10-fold in nonacidified (pH 5.20) preservative-free cheese food, with the pathogen surviving throughout the product’s entire 6-month shelf life (Figure 12.15). In sharp contrast to these findings, addition of preservatives with or without acidifying agents led to the eventual demise of listeriae in cheese food stored at 4°C (Figure 12.16). In nonacidified cheese food (pH 5.20) preserved with 0.3% sodium propionate, L. monocytogenes survived an average of 142 days as compared to 118, 103, and 98 days in the same product adjusted to pH 5.0–5.1 with lactic, acetic, and lactic plus acetic acid, respectively. Using 0.3% sorbic acid in place of sodium propionate, the pathogen survived an average of 130 days in nonacidified cheese food (pH 5.45) as compared to 112, 93, and 74 days in cheese food acidified to pH 5.0–5.1 with lactic, lactic plus acetic, and acetic acids, respectively. Thus, sorbic acid was consistently more antagonistic to listeriae than sodium propionate. In addition, antilisterial effects of both preservatives were more pronounced in cheese food acidified with acetic rather than lactic acid. Because organic acids are far more bactericidal in the undissociated than dissociated state, increased inactivation of listeriae in the presence of acetic acid probably resulted from the higher proportion of undissociated acetic (~36%) rather than lactic acid (~5.9%) in cheese food acidified to pH 5.0–5.1. These findings indicate that it would be prudent to consider (a) adding preservatives, particularly

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150 140 130

Survival (Days)

120

a

S P L A

Sorbic Acid Na Propionate Lactic Acid Acetic Acid

110 100 90 80 70 60 50 P

S

P+L

S+L

P+A Additive

P+L+A S+L+A

S+A

FIGURE 12.16 Maximum duration of survival of L. monocytogenes in acidified and nonacidified cold-pack cheese food containing preservatives. Each bar represents the average maximum length of survival of all four Listeria strains in one of eight different formulations of cheese food manufactured in duplicate. Any two differing by >20.24 days (length of bar) are significantly different (p < 0.05). (Adapted from Ryser, E.T. and E.H. Marth. 1988. Survival of Listeria monocytogenes in cold-pack cheese food during refrigerated storage. J. Food. Prot. 51: 615–621, 625.)

sorbic acid, to cold-pack cheese food and (b) reducing the pH of the product to 5.0 by adding small amounts of lactic or acetic acid to minimize L. monocytogenes survival in the finished product. Additional information on conditions leading to inhibition and/or inactivation of this pathogen by sorbic, propionic, lactic, and acetic acids can be found in Chapter 6.

PASTEURIZED PROCESSED CHEESE American pasteurized processed cheese was first developed at the turn of the twentieth century and is made by grinding and mixing different natural cheeses with citrate and phosphate-based emulsifiers, with this cheese mixture then heated for 30 sec or more at 150°F to produce a homogeneous mass. This molten cheese is then quickly cooled to form slices or blocks of cheese upon cooling that are ready for sale. Owing to the extreme heat treatment that this cheese receives during manufacture, pasteurized processed cheese continues to have an excellent safety record, with very few foodborne outbreaks of any type associated with this product over the past 50 years. Further, this type of cheese has not been implicated in any Listeria-related recalls or outbreaks of listeriosis, with the pathogen also thus far absent from samples of pasteurized processed cheese analyzed in limited surveys. However, given the relatively common occurrence of L. monocytogenes in food processing environments, Glass et al. [223] chose to assess the ability of L. monocytogenes to proliferate on pasteurized processed cheese as a postprocessing contaminant. In their study, retail slices of pasteurized processed cheese from six different lots (pH 5.61–5.84, 2.35–2.62% salt, 0.918–0.929 aw) were surface-inoculated to contain 103 CFU/g of a 3-strain L. monocytogenes cocktail that included strain Scott A. Overall no growth was evident, with Listeria decreasing 0.6 to 0.7 log CFU/g on these cheese slices after 4 days of storage at an abusive temperature of 30°C. Thus, given these findings, pasteurized processed cheese poses a very minimal health risk to consumers.

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BEHAVIOR OF L. MONOCYTOGENES IN CHEESE AS AFFECTED BY CHEESE COMPOSITION As suggested earlier, the fate of L. monocytogenes and other foodborne pathogens during cheese ripening is determined by the microbiological, biochemical, and physical properties of the particular cheese. Thus, cheese is a very complex system, with the following factors acting simultaneously to determine the behavior of L. monocytogenes during ripening: (a) type, amount, and activity of the starter culture; (b) pH as determined by concentrations of lactic, acetic, formic, and other acids; (c) presence of hydrogen peroxide, diacetyl, and various antimicrobial agents (i.e., nisin and other bacteriocins); (d) levels of nutrients, salt, moisture, and oxygen; and (e) the temperature at which the cheese is ripened. All these factors act together to produce a particular outcome; however, a few conclusions concerning the ability of L. monocytogenes to grow and survive in some of the aforementioned cheeses prepared from contaminated milk can be drawn by examining the behavior of this pathogen in relation to the combined effects of moisture content, water activity (aw), and salt in the moisture phase as well as the pH of the cheese and the temperature at which the cheese is ripened (Table 12.16). Although fully ripened Camembert and feta cheese have widely differing pH values of 7.5 and 4.4, respectively, both cheeses are very similar in terms of moisture content, water activity, percentage of salt in the water phase, and ripening temperature. Thus, rapid growth of L. monocytogenes in Camembert cheese can be largely attributed to the increase in pH of the cheese during ripening, whereas a pH value of 4.4 appears to be responsible for preventing growth of the bacterium during ripening of feta cheese. Inability of L. monocytogenes to multiply in blue cheese during ripening and storage is probably related to the high concentration of salt in the water phase (which results in a low aw), because other workers have confirmed that this organism will not grow in laboratory media [359] and skim milk [303] containing >10 and 12% salt, respectively. As with Camembert cheese, growth of two of four L. monocytogenes strains in brick cheese appears to be directly related to the high pH that the cheese attained during extended ripening. However, inability of the remaining two strains to grow in brick cheese of similar composition is as yet unexplained. When comparing the behavior of L. monocytogenes in Cheddar and Colby cheese, the initial inactivation rate for the pathogen was somewhat slower in the latter cheese. At first glance, it appears that increased viability of Listeria in Colby cheese during the early stages of ripening may be related to the lower percentage of salt in the water phase in this cheese than in Cheddar cheese. However, data in Table 12.16 show that L. monocytogenes was inactivated at similar rates in Colby cheese and cold-pack cheese food, the latter being compositionally similar to Colby cheese except for a higher concentration of salt in the water phase. Hence, factors other than a low concentration of salt in the water phase also must be involved in enhancing the viability of listeriae during ripening of Colby cheese. Lack of growth and decreased survival of L. monocytogenes in Parmesan cheese and in an unidentified hard Italian cheese as compared to other varieties in Table 12.16 correlate well with the lower moisture content/aw of these cheeses during ripening. Barring thermal or acid injury of L. monocytogenes, which is likely to occur during manufacture of cottage cheese and other varieties that undergo substantial heat treatments during manufacture (i.e., mozzarella, Swiss), factors outlined in Table 12.16 are useful in predicting whether or not this pathogen will grow in other cheeses having similar microbiological, biochemical, and physical characteristics. In addition to being present in milk at the time of cheesemaking, Listeria also can easily contaminate the finished cheese during packaging, ripening, and storage. Consequently, Genigeorgis et al. [217] evaluated the fate of Listeria as a postprocessing contaminant by inoculating 49 retail cheeses (24 types/28 brands) with L. monocytogenes and then storing these cheeses at 4–30°C for up to 36 days. As expected, Listeria growth was primarily confined to high-moisture varieties, including Brie, Camembert, ricotta, and the soft Hispanic cheeses, all of which had a pH ≥5.9 and low to moderate levels of salt in the moisture phase (Table 12.17). According to Razavilar [327], L. monocytogenes populations in inoculated Queso fresco cheese curd of pH 6.6 also increased

54.4 38.9 43.0 54.7 37.2 37.2 40.0 32.0 NRc 41.4

Moisture (%) 0.975 0.950 0.990 0.975 0.975 0.975 0.975 0.935 0.950 0.975

Estimated aw

b

Approximately 24 h after the start of cheesemaking. Prepared without preservatives or acidifying agents. c Not reported. d Percentage of salt in solid and water phase.

a

Camembert Blue Brick Feta Cheddar Cheddar Colby Parmesan Hard Italian Cold-pack cheese foodb

Cheese 4.72 11.52 1.89 4.57 4.61 4.61 3.91 4.96 2.12d 4.90

Estimated Salt in Water Phase (%) 4.6 4.6 5.3 4.7 5.1 5.1 5.1 5.1 5.3 5.3

Initiala

pH

7.5 6.3 7.3 4.4 5.1 5.1 5.1 5.1 5.7 5.1

Final 15/6 9–12/4 15/10 22/4 6 13 4 13 4 4

Ripening Temperature (°C) 3.1–3.6 4.0–5.0 3.0–4.7 5.2–6.2 2.5–3.2 2.6–3.4 3.5–4.5 3.3–4.3 4.5–5.1 2.4–2.8

Initiala 6.7–7.5 4.0–5.0 4.6–6.7 5.7–6.2 2.6–3.8 3.0–3.7 3.6–4.6 3.3–4.3 4.5–5.6 2.4–2.8

Maximum

6.7–7.5 1.0–2.3 2.7–6.1 2.8–4.6 0–1.5 0 2.3–4.1 1.0–1.3 2.0 1.1–2.0

Final

Log10 of L. monocytogenes CFU/g

TABLE 12.16 Behavior of L. monocytogenes during Cheese Ripening as Affected by Cheese Composition

65 120 168 90 70–434 70–224 112–140 21–112 35 180

Survival (days)

341 306 345 307 340 340 460 401 161 344

Ref.

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TABLE 12.17 Growth and Inactivation of L. monocytogenes in Surface-Inoculated Retail Cheeses during Storage at 4–30°C Cheese Category and Type Soft mold-ripened Brie Camembert Blue Bacterial surface-ripened Limburger Muenster Soft Italian Provolone String cheese Semisoft and hard-ripened Monterey Jack Colby Cheddar Swiss Hispanic Queso fresco Queso Ranchero Queso Panella Cotija Pickled cheese Feta Ewe’s milk cheese Kesseri Soft unripened Cottage cheese Cream cheese Whey cheeses Ricotta Processed cheese American Monterey Jack Piedmont

pH

% NaCI in Moisture Phase

Growth

6.0–7.7 7.3 5.1

2.5–3.6 2.5 6.1

+ + −

7.2 5.5

4.8 3.8

− −

5.6 5.5

4.6 4.4

− −

5.0–5.2 5.5 4.9–5.6 5.5

1.0–3.0 4.9 2.6–5.4 2.7

− − − −

6.5–6.6 6.2 6.2–6.7 5.5–5.6

4.5–6.6 4.1 2.5–3.9 9.6–12.5

+/− + + −

4.2–4.3

2.2–7.5

−

4.8–5.3

5.5–5.87

−

4.9–5.1 4.8

1.0–1.2 <1.9

+/− −

5.9–6.1

<0.7

+

5.7 5.7 6.4

2.1 4.4 5.1

− − −

Source: Adapted from Genigeorgis, C., M. Carniciu, D. Dutulescu, and T.B. Farver. 1991. Growth and survival of Listeria monocytogenes in market cheeses stored at 4 to 30°C. J. Food Prot. 54: 662–668.

from 104 to 107 CFU/g following 6 and 19 days of storage at 8 and 4°C, respectively. Back et al. [122] also reported temperature-dependent surface growth of L. monocytogenes on several additional retail European soft cheeses including Cambazola, English Brie, Blue Lymeswold, and White Lymeswold, with Listeria populations remaining relatively stable on Blue Stilton, White Stilton, Mycella, and Chaume cheese during short-term storage. In addition, commercial [157] and experimentally produced [209] Arzua cheese (a soft, low-acid Spanish cheese prepared from raw cow’s milk) supported growth of Listeria as evidenced by populations >1000 CFU/g in the finished product. All these findings again point to the high-moisture, low-acid cheeses as being of primary public health importance.

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Going one step further, Bolton and Frank [138] developed a predictive model for growth/no growth of L. monocytogenes in Mexican-style soft cheeses based on salt, pH, and moisture content. In their study, a soft Mexican-style cheese was prepared by direct acidification and subsequently frozen. After thawing, the cheese composition was adjusted to four different moisture contents (42–60%), four different salt contents (2.0–8.0%), and six different pH levels (5.0–6.5), giving 96 treatment combinations. Thereafter, each sample was inoculated with a four-strain cocktail of L. monocytogenes at about 104 CFU/g and analyzed for numbers of Listeria after 21 and 42 days of storage at 10°C. Using a binary and ordinal regression model, none of 60 samples having a 5% probability of supporting Listeria growth (pH 5.0–6.0, brine concentration 8.17–16.00%) supported growth, whereas Listeria grew in 20 of 30 samples having a 50% probability of supporting growth (pH 5.5–6.5, brine concentration 3.23–12.50%) of the pathogen. Furthermore, when applied to the data of Genigeorgis et al. [216] both models provided reasonable predictions for growth/no growth of Listeria in Mexican-style as well as non-Mexican-style cheese, with these models likely to be quite useful in refining current Listeria risk assessments for different cheeses.

FEASIBILITY

OF

PREPARING CHEESE

FROM

RAW MILK

According to current FDA regulations, milk pasteurization or use of a similar heat treatment during cheesemaking is required for the manufacture of 16 cheese varieties, including Brie, cottage, cream, Neufchâtel, Monterey, mozzarella, Scamorza, Muenster, Gammelost, Koch Kaese, and Sapsago [249,388]. Seven varieties of manufacturing cheese (i.e., for use in pasteurized processed cheese, cheese food, cheese spread) require neither pasteurization of the cheesemilk nor a 60-day minimum ripening at ≥1.7°C (≥35°F), whereas the 34 remaining varieties of cheese recognized under current standards of identity must either be manufactured from pasteurized milk or held a minimum of 60 days at ≥1.7°C (≥35°F) to eliminate pathogenic microorganisms. Although statistics on milk pasteurization for cheesemaking are scarce, available evidence indicates that most natural cheese sold in the United States is prepared from pasteurized milk. The mandatory holding period of 60 days at ≥1.7°C (≥35°F) for cheeses manufactured from raw milk was adopted in 1949 [7,249] after researchers demonstrated that viable Brucella abortus, the causative agent of brucellosis, was eliminated from cheese by such an aging process. Although this 60-day holding period was generally deemed adequate to eliminate most foodborne pathogens, later studies demonstrated that Salmonella Typhimurium and other hazardous microorganisms can survive such a cheese-ripening process [227,250]. Furthermore, results in Table 12.16 indicate that L. monocytogenes can survive well beyond 60 days in many natural cheeses held at ≥1.7°C (≥35°F). In keeping with the grave nature of listeriosis as compared to most other foodborne illnesses, the FDA has continued to maintain a policy of “zero tolerance” for L. monocytogenes in all readyto-eat foods. Thus far, no well-documented cases of listeriosis have been associated with consumption of cheeses that were legally prepared from raw milk and held a minimum of 60 days at ≥1.7°C (≥35°F). However, because 4% of the raw milk supply can be expected to contain L. monocytogenes, it would be prudent to manufacture cheeses from pasteurized milk whenever possible. Although Yousef and Marth [401] demonstrated that ripening Parmesan cheese for 10 months, as legally required, is sufficient to produce a high-quality, Listeria-free product, desirable flavor and texture characteristics are not easily attainable in sharp Cheddar and Swiss cheese prepared from pasteurized milk. Hence, alternative means should be developed to enhance the safety of these products. Such methods might include cold sterilization of the milk via microfiltration or addition of various flavor- and texture-enhancing enzymes (or microorganisms) to pasteurized milk, which would allow the cheesemaker to obtain a higher-quality product [251]. However, as important as it is to manufacture cheese from Listeria-free milk, it is equally important to prevent contamination of the product during manufacture, ripening, and storage by using good manufacturing practices. Information concerning problem areas and safeguards during manufacture of dairy products and other foods can be found in Chapter 18.

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TABLE 12.18 Number of Listeriae Recovered from Whey during Manufacture of Various Cheeses Prepared from Pasteurized Milk Inoculated to Contain ~500–5000 L. monocytogenes CFU/mL Cheese Camembert Blue Brick Feta Gouda Colby Cheddar Average

L. monocytogenes CFU/mL of Whey

Percentage of Original Inoculum in Whey

8 43 12 15 5 21 22 18

1.3 3.6 2.5 3.2 1.0 2.4 5.0 21.7

Ref. 261 237 264 238 235 306 259

WHEY Listeria monocytogenes has not yet been isolated from commercially produced cheese whey. However, studies have shown that when various cheeses were experimentally produced from pasteurized milk inoculated with L. monocytogenes, between 1 and 5% of the original inoculum was lost in the whey during cheesemaking (Table 12.18). These findings again demonstrate that the pathogen is concentrated 8- to 10-fold in curd during milk coagulation. Unlike other wheys, populations of L. monocytogenes in acid whey (pH 4.6) obtained from the manufacture of cottage cheese were reduced more than 10,000- fold after cooking the curd–whey mixture at 57.2°C (135°F) for 30 min. However, as previously noted, Listeria was detected in several whey samples after 6 weeks of cold enrichment [346], which, in turn, suggests that some cells were only sublethally injured during cooking of the curd–whey mixture. These observations prompted Ryser and Marth [343] to examine the behavior of L. monocytogenes in wheys from Camembert cheese that were filter-sterilized and adjusted to pH values of 5.0–6.8. All whey samples were then inoculated to contain ~100–500 L. monocytogenes (strains Scott A, V7, CA, or OH analyzed separately) CFU/mL and incubated at 6°C. Although the four L. monocytogenes strains failed to grow in wheys having pH values ≤5.4, the pathogen survived in all samples with populations decreasing ≤10-fold during 35 days of refrigerated storage. In contrast, L. monocytogenes grew in all remaining samples after a 3-day lag period and attained average maximum populations of 7.48, 7.87, and 7.84 log10 CFU/mL in wheys adjusted to pH 5.6, 6.2, and 6.8, respectively, following 35 days of incubation. As previously noted, these Listeria populations in whey were slightly higher than those that would be expected to develop in skim or whole milk during refrigerated storage. Generation times for L. monocytogenes in wheys adjusted to pH 5.6, 6.2, and 6.8 ranged between 25.2–31.6 h, 14.8–21.1 h, and 14.0–19.4 h, respectively, depending on the individual strain. Although doubling times were similar for all strains at the same pH, generation times were significantly longer at pH 5.6 than at pH 6.2 and 6.8. Interestingly, Hughey et al. [244] observed that L. monocytogenes could be inactivated in similar samples of demineralized whey by adding lysozyme. However, this enzyme did not decrease viability of the pathogen in normal whey, which in turn suggests that lysozyme activity is neutralized by whey minerals and/or proteins. Using a different approach to examine the behavior of Listeria in whey, Northolt et al. [299] inoculated heat-treated (68°C/10 s) wheys (pH 6.5) to contain 500–1000 L. monocytogenes CFU/mL and incubated the samples at temperatures between 7 and 30°C. Following a 6- to 24-h lag period,

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the pathogen grew in all samples with doubling times of 12 h, 6 h, 4 h, and 40 min in wheys incubated at 7, 12, 20, and 30°C, respectively. Although incubation of all whey samples was terminated before L. monocytogenes reached the stationary growth phase, the pathogen did attain populations of 104–106 CFU/mL at the conclusion of the experiment. In the only study thus far reported dealing with nonsterile whey, researchers in France [127] produced whey containing 2–7 L. monocytogenes CFU/mL from previously inoculated milk and examined samples for listeriae after 101, 156, and 251 days of storage at 4°C. Moderate growth and extended survival of the pathogen were observed in wheys collected immediately after coagulation of the milk (pH 5.4), with 156- and 251-day-old whey samples at pH 4.8 containing 2.5 × 104 and 7.0 × 102 L. monocytogenes CFU/mL, respectively. As predicted by Ryser and Marth [343], the pathogen also failed to grow in more acidic wheys (pH 5.2–5.3) collected after hooping with listeriae no longer observed in 101- and 156-day-old wheys having pH values of 3.28 and 4.26, respectively. Not surprisingly, increasing the incubation temperature to 6, 14, and 20°C led to faster demise of listeriae in 21 similar wheys (pH 3.75–5.72) initially containing 60–96 L. monocytogenes CFU/mL, with the pathogen being eliminated from all but one sample (pH 5.72) examined after 114 days of incubation at 6°C. These findings demonstrate that L. monocytogenes can grow to high numbers in fluid wheys having pH values >5.4 and remain viable in more acidic wheys during many months of refrigerated storage. Given results of a study described in Chapter 11 in which numbers of L. monocytogenes decreased only 1–5 orders of magnitude during manufacture of nonfat dry milk [181], it appears that this organism also is likely to survive the spray-drying process used in converting fluid whey into whey powder. However, to our knowledge, no Listeria spp. have yet been isolated from dried whey manufactured commercially in Europe or the United States. Although Gabis et al. [210] failed to find any Listeria spp. in 23 environmental samples from whey-processing factories, these authors did isolate L. monocytogenes from a floor drain that was located within a raw-milk-receiving room of a dry-milk-processing factory. Additionally, Listeria spp. other than L. monocytogenes were isolated from several drains and trenches in the powder production area of a second factory that manufactured dry milk products. Considering the widespread use of dried whey (and nonfat dried milk) as an ingredient in numerous products including cheese food, ice cream, sherbet, candy, beverages, and baked goods, strict enforcement of good manufacturing practices for dried whey and nonfat dry milk should be continued to prevent a possible public health problem involving listeriae.

BRINE SOLUTIONS Because L. monocytogenes is quite halotolerant, it is not surprising to learn that brine solutions in which cheeses are salted or ripened also can serve as potential reservoirs for this organism. Current evidence suggests that these brine solutions may become contaminated with L. monocytogenes through direct or indirect contact with the cheese factory environment (i.e., equipment, condensate on walls, floors, and ceilings) as well as actual shedding of L. monocytogenes into the brine solution from Listeria-contaminated cheese [119]. Breer [142] and Terplan [290] reported isolating L. monocytogenes from commercial brine solutions in Europe. In one instance, the pathogen was detected in brine tanks 4 days after soft/semisoft cheeses were removed from the salt brine. Because several recalls in the United States [82] have presumably been traced to contaminated brine tanks [82], interest in the incidence of listeriae in brine solutions has increased with Larson et al. [262] having examined the survival of L. monocytogenes in 38 commercial cheese brines (5.6–24.7% NaCl, pH 4.4–6.8) from Wisconsin and northern IIlinois. In their study, 23 brine samples (16.9–24.7 NaCl, pH 4.9–55.4) were collected from nine different manufactures of mozzarella cheese along with 5, 4, 3, and 3 brine samples from factories producing other Italian, brick, brick/Hispanic, and feta cheese. Each brine sample was inoculated with a six-strain cocktail of L. monocytogenes strains Scott A, OH, CA V7, 87189, and 2811M so as to contain 104 to 105 CFU/mL and then stored at

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4 and 12°C. Overall, 15 of 38 and 3 of 38 samples were still positive for Listeria after more than 200 days of storage at 4 and 12°C, respectively, with length of survival unrelated to the level of background microflora, mineral content, or nitrogen content. However, Listeria survival was substantially shortened in brines containing 10 to 100 ppm sodium hypochlorite, 0.01 to 0.05% hydrogen peroxide, 1% potassium sorbate or 1% sodium benzoate. Because these levels of sodium hypochlorite did not yield any residual sodium hypochlorite in mozzarella cheese after brining, addition of sodium hypochlorite at levels ≤100 ppm should be useful for minimizing Listeria survival in cheese brines. Migration of L. monocytogenes into brine solutions and salted whey during salting of artificially contaminated cheese has been well documented. Ryser and Marth [345] brine-salted brick cheese containing ~103–104 L. monocytogenes CFU/g at 10°C. Cold enrichment of membrane filters through which 50-mL portions of 22% brine solution were filtered indicated that the pathogen leached from the cheese into the salt solution during 24 h of brining. Further, viable listeriae were detected in samples of 22% brine-stored at 10°C at least 5 days after blocks of cheese were removed from the brine. When Abdalla et al. [1] brine-salted Sudanese white pickled cheese in whey containing 8% NaCl, L. monocytogenes once again leached from the cheese into the salted whey, with populations increasing 2.5–3.5 orders of magnitude during 65 days of storage at 4°C. In conjunction with their study on the fate of L. monocytogenes during manufacture, ripening, and storage of feta cheese, Papageorgiou and Marth [307] also examined Listeria viability in the brine solution in which cheese was salted and stored. After 1 day of salting, a 12% brine solution contained an average Listeria population of 2.63 log10 CFU/g, which again indicates that the pathogen leached from cheese into the brine (Figure 12.17). However, no growth of L. monocytogenes was observed in this brine despite ample migration of cheese nutrients into salt brine as well as favorable values for temperature and pH. After transferring feta cheese to a 6% brine solution, the pathogen leached from cheese into salt brine and grew rapidly, with similar populations being observed in both the cheese and brine after 6 days of incubation at 22°C. Although numbers of listeriae decreased in both cheese and salt brine during 90 days of refrigerated storage, the pathogen was inactivated slower in 6% salt brine than in feta cheese.

Strain CA log10 CFU/mL or g

6.0

5.0

Brine Solution

4.0

Feta Cheese 3.0 12%/20°C

6%/4°C

6%/22°C

2.0 0

1

2

3

4

5

6 20 Days

34

48

62

76

90

FIGURE 12.17 Average populations of L. monocytogenes strain CA in 12 and 6% salt brine during ripening and storage of feta cheese. (Adapted from Papageorgiou, D.K. and E.H. Marth. 1989. Fate of Listeria monocytogenes during the manufacture, ripening and storage of feta cheese. J. Food Prot. 52: 82–87.)

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Larsen et al. [261] reported that L. monocytogenes began growing in salt-free whey (pH 5.6) and whey containing 3.6% NaCl and/or KCl after 7–14 days of storage at 4°C, with Listeria growth generally being unrelated to the type of salt used. However, L. monocytogenes populations remained unchanged in identical samples containing 4.8% NaCl and/or KCl. When similar whey samples were incubated at 25°C, L. monocytogenes populations increased 3–4 orders of magnitude, with more rapid growth being observed in wheys containing 3.6 rather than 4.8% NaCl and/or KCl. However, Listeria growth at 25°C could be prevented by adding a L. lactis subsp. lactis or L. lactis subsp. cremoris starter culture before incubation. In 1994, Rajkowski et al. [324] also reported that addition of 4.5% NaCl to ultrahigh temperature (UHT)-pasteurized milk decreased the growth rate of L. monocytogenes in samples incubated at 12–37°C. However, fortification of these same samples with 0.5 or 1.0% polyphosphate did not alter Listeria growth characteristics. Because feta and other white-pickled cheeses such as Teleme, Halloumi, and Domiati are frequently cured in whey or skim milk containing 6 or 12% salt, Papageorgiou and Marth [305] examined behavior of Listeria in salted whey and skim milk. Autoclaved samples of skim milk (pH 6.0–6.2) and deproteinated whey (pH 5.5–5.7) containing 6 and 12% NaCl were inoculated to contain 103 L. monocytogenes (strains Scott A or CA) CFU/mL and incubated at 4 and 22°C. After a lag period of 5–10 days, L. monocytogenes grew rapidly in 6% salted whey and 6% salted skim milk, with the pathogen attaining maximum populations of 107–108 CFU/mL following 50–55 days of refrigerated storage (Table 12.19). In the study discussed earlier, Ryser and Marth [343] reported that these same Listeria strains had shorter lag periods and generation times but achieved similar maximum populations in unsalted, filter-sterilized whey (pH 5.6) after 24 days of incubation at 6°C. Hence, these findings suggest that addition of NaCl or KCl to whey and milk plays a major role in decreasing the growth rate of Listeria. Increasing the incubation temperature to 22°C resulted in lag periods of 6–12 h for both Listeria strains in whey and skim milk containing 6% salt. Generation times were similarly reduced, with both strains exhibiting faster growth rates and higher maximum populations in salted whey rather than salted skim milk (see Table 12.19). These findings agree with those of other researchers, who

TABLE 12.19 Generation Times (GTs) and Maximum Populations (MPs) of L. monocytogenes Strains Scott A and CA in 6% Salted Whey and Skim Milk Incubated at 4 and 22°C Strain Incubation at 4°C Scott A Scott A CA CA Incubation at 22°C Scott A Scott A CA CA

GT (h)

MP (log10 CFU/mL)

Whey Skim milk Whey Skim milk

46.81 45.23 37.49 49.43

7.97 7.58 8.04 7.69

Whey Skim milk Whey Skim milk

3.67 4.31 3.56 4.42

8.02 7.70 8.10 7.89

Product

Source: Adapted from Papageorgiou, D.K. and E.H. Marth. 1989. Behavior of Listeria monocytogenes at 4 and 22°C in whey and skim milk containing 6 and 12% sodium chloride. J. Food Prot. 52: 625–630.

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L. monocytogenes log10 CFU/mL

5 Scott A Whey Scott A Skim milk

4

3

2 CA Whey

1

CA Skim milk 0 –25

0

25

50

75 Days

100

125

150

FIGURE 12.18 Behavior of L. monocytogenes strains Scott A and CA in 12% salted whey and skim milk during extended storage at 22°C. (Adapted from Papageorgiou, D.K. and E.H. Marth. 1989. Behavior of Listeria monocytogenes at 4 and 22°C in whey and skim milk containing 6 and 12% sodium chloride. J. Food Prot. 52: 625–630.)

also observed that L. monocytogenes grew faster and attained higher maximum populations in unsalted whey [343] than in skim milk [334]. Unlike the previous findings obtained with 6% salt, growth of L. monocytogenes was completely inhibited in 12% salted whey and skim milk, with populations of both strains decreasing ≤10-fold during 130 days of storage at 4°C. Although strain Scott A also persisted ≥130 days in 12% salted whey and skim milk incubated at 22°C, strain CA proved to be less salt tolerant, surviving only 80 and 105 days in 12% salted skim milk and whey, respectively (Figure 12.18). Increased destruction of L. monocytogenes in salt solutions held at ambient rather than refrigeration temperatures has been well documented. Results from several of these studies dealing with viability of listeriae in salted Tryptose Broth [360] and cabbage juice [165] are discussed elsewhere. Based on these observations, acidification of brine solutions used in cheesemaking to pH values <5.0 has been recommended to prevent growth of L. monocytogenes, particularly if such solutions contain ≤10% salt. In 1989, Hughey et al. [244] also noted that addition of 0.35% H2O2 to a 23% brine solution caused numbers of L. monocytogenes to decrease by 6 orders of magnitude within 24 h. However, unlike organic acids, the bactericidal activity of H2O2 dissipates fairly rapidly. Hence, addition of H2O2 to salt brine provides only temporary protection against listeriae that might be present within the cheesemaking environment.

REFERENCES 1. Abdalla, O.M., G.L. Christen, and P.M. Davidson. 1993. Chemical composition of and Listeria monocytogenes survival in white pickled cheese. J. Food Prot. 56: 841–846. 2. Abdalla, O.M., P.M. Davidson, and G.L. Christen. 1993. Survival of selected pathogenic bacteria in white pickled cheese made with lactic acid bacteria or antimicrobials. J. Food Prot. 56: 972–976. 3. Abou-Donia, S.A. and A.K. Al-Medhagi. 1992. Detection and survival of Listeria monocytogenes in Egyptian dairy products. J. Dairy Sci. (Suppl. 1) 75: 138. 4. Abou-Eleinin, A.M., E.T. Ryser, and C.W. Donnelly. 2000. Incidence and seasonal variation of Listeria species in bulk tank goat’s milk. J. Food. Prot. 63: 1208–1212.

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5. Ahmed, A.A.-H. 1989. Behaviour of Listeria monocytogenes during preparation and storage of yoghurt. Assuit Vet. Med. J. 22: 76–80. 6. Ahmed, A.A.-H., S.H. Ahmed, M.K. Moustafa, and N.M. Saad. 1989. Growth and survival of Listeria monocytogenes during manufacture and storage of Damietta cheese. Assiut Vet. Med. J. 22: 88–94. 7. Anonymous. 1949. Cheeses, processed cheeses, cheese foods, cheese spreads, and related foods, definitions and standards of identity. Fed. Regist. April 22: 1960–1992. 8. Anonymous. 1985. Code of hygienic practices for soft cheeses to be elaborated. Food Chem. News 27(31): 12. 9. Anonymous. 1985. FDA finds Listeria in 3 brands of General Foods soft cheeses. Food Chem. News 27(25): 33. 10. Anonymous. 1985. FDA is checking facilities of cheese plant after finding Listeria. Food Chem. News 27(24): 25. 11. Anonymous. 1985. FDA is investigating deaths linked to Mexican-style cheese. Food Chem. News 27(15): 42. 12. Anonymous. 1985. Jalisco-Brand Mexican Style Soft Cheese Recalled. FDA Enforcement Report, June 26. 13. Anonymous. 1985. Liederkranz, Camembert, and Brie Cheese Recalled. FDA Enforcement Report, Sept. 4. 14. Anonymous. 1985. Planning program for inspection of imported soft cheese. Food Chem. News 27(30): 35. 15. Anonymous. 1985. Soft cheese manufacturers to be inspected under FDA priority program. Food Chem. News 27(18): 22. 16. Anonymous. 1986. Brie Cheese Recalled. FDA Enforcement Report, April 2. 17. Anonymous. 1986. Brie Cheese Recalled. FDA Enforcement Report, April 9. 18. Anonymous. 1986. Cheese recalls extended by General Foods, U.S. importer. Food Chem. News 27(52): 25–26. 19. Anonymous. 1986. FDA advises import alert on imported soft cheese. Food Chem. News 28(23): 22–23. 20. Anonymous. 1986. FDA finds Listeria in Brie from French certified plant. Food Chem. News 27(50): 35. 21. Anonymous. 1986. FDA finds Listeria in Mexican-style soft cheese. Food Chem. News 28(1): 54. 22. Anonymous. 1986. FDA may block list all soft-ripened cheese from France. Food Chem. News 28(2): 64–65. 23. Anonymous. 1986. FDA proposes soft-ripened cheese testing program to France. Food Chem. News 28(4): 3–4. 24. Anonymous. 1986. FDA to detain all soft-ripened French cheese lacking certification. Food Chem. News 28(10): 9–10. 25. Anonymous. 1986. FDA to sample 20% of soft cheeses from all foreign countries. Food Chem. News 28(6): 27–28. 26. Anonymous. 1986. France agrees to temporary FDA soft-ripened cheese testing program. Food Chem. News 28(7): 24–26. 27. Anonymous. 1986. French Brie Cheese Recalled. FDA Enforcement Report, March 12. 28. Anonymous. 1986. French Brie Cheese Recalled. FDA Enforcement Report, May 14. 29. Anonymous. 1986. French cheeses recalled because of Listeria. Food Chem. News 28(24): 12. 30. Anonymous. 1986. French firm agrees to stop shipments of Brie cheese. Food Chem. News 27(51): 12–14. 31. Anonymous. 1986. French Semi-Soft Cheese Recalled. FDA Enforcement Report, August 20. 32. Anonymous. 1986. French Soft-Ripened Cheese Recalled. FDA Enforcement Report, August 13. 33. Anonymous. 1986. French Soft-Ripened Cheese Recalled. FDA Enforcement Report, Sept. 24. 34. Anonymous. 1986. Listeria causes Class I recalls of ice milk mix, milk. Food Chem. News 28(16): 22. 35. Anonymous. 1986. Listeriosis hazard still being evaluated, FDA tells France. Food Chem. News 18(26): 29–32. 36. Anonymous. 1986. Milk, chocolate milk, half and half, cultured buttermilk, whipping cream, ice milk, ice milk mix and ice milk shake mix recalled. FDA Enforcement Report, June 25. 37. Anonymous. 1986. More recalls made of French cheese, animal feeds. Food Chem. News 28(7): 52–53. 38. Anonymous. 1986. Recall of Brie cheese is extended by FDA. Food Chem. News 28(1): 54. 39. Anonymous. 1986. Soft Mexican-Style Cheeses Recalled. FDA Enforcement Report, April 9. 40. Anonymous. 1986. Soft, Ripened Brie Cheese Recalled. FDA Enforcement Report, April 9.

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Listeria, Listeriosis, and Food Safety Anonymous. 1986. Soft-Ripened French Brie Cheese Recalled. FDA Enforcement Report, April 23. Anonymous. 1987. Annual Report, Swiss Institute for Dairy Research, Liebefeld-Berne, pp. 344–353. Anonymous. 1987. Class I recall made of cheese because of Listeria. Food Chem. News 28(50): 52. Anonymous. 1987. FDA launching two-year pathogen surveillance program. Food Chem. News 29(31): 10–12. Anonymous. 1987. FDA orders sampling of Italian hard cheese for microorganisms. Food Chem. News 29(22): 36. Anonymous. 1987. FDA renews threat of automatic detention for Italian soft cheese. Food Chem. News 29(6): 3–4. Anonymous. 1987. FDA sets separate program for cheese from unpasteurized milk. Food Chem. News 29(8): 17–18. Anonymous. 1987. French Full Fat Soft Cheese Recalled. FDA Enforcement Report, May 13. Anonymous. 1987. French Full Fat Cheese Recalled Because of Listeria. Food Chem. News 29(9): 16. Anonymous. 1987. French Soft Cheese Certification to Be in Place. February 15. Food Chem. News 28(50): 36. Anonymous. 1987. Hard cheese from Italian firm blocklisted. Food Chem. News 29(23): 19. Anonymous. 1987. Mini Bonbel and Gouda Semi-Soft Cheese Recalled. FDA Enforcement Report, June 3. Anonymous. 1987. Old Heidelberg Soft-Ripened Cheese Recalled. FDA Enforcement Report, June 3. Anonymous. 1987. Raw Milk Sharp Cheddar Cheese Recalled. FDA Enforcement Report, August 19. Anonymous. 1987. Salvador-Style White Semi-Soft Cheese Recalled. FDA Enforcement Report, February 11. Anonymous. 1988. Blue Castello Danish Blue Cheese Recalled. FDA Enforcement Report, May 4. Anonymous. 1987. Danish cheese recalled because of Listeria. Food Chem. News 30(4): 42. Anonymous. 1988. FDA finds Listeria in foreign soft cheeses. Food Chem. News 29(49): 36–37. Anonymous. 1988. Ice cream, cheese recalled because of Listeria. Food Chem. News 30(6): 27. Anonymous. 1988. Italian Semi-Soft Cheese Recalled. FDA Enforcement Report, March 30. Anonymous. 1988. L’Amulette Danish Esrom cheese recalled. FDA Enforcement Report, March 23. Anonymous. 1988. L’Amulette Danish Esrom cheese recalled. FDA Enforcement Report, March 30. Anonymous. 1988. L’Amulette Danish Esrom cheese recalled. FDA Enforcement Report, April 6. Anonymous. 1988. Listeriosis: Goats’ Milk Cheese. Communicable Disease Report, February 12. Anonymous. 1988. Mexican-Style, Baby Jack and Monterey Jack Cheese Recalled. FDA Enforcement Report, March 9. Anonymous. 1988. Nisin preparation affirmed as GRAS for cheese spreads. Food Chem. News 30(6): 37–38. Anonymous. 1988. Soft cheese warning because of Listeria monocytogenes. Food Chem. News 29(49): 2. Anonymous. 1989. Controlling Listeria—the Danish solution. Dairy Ind. Int. 54(5): 31–32. Anonymous. 1989. Cyprus Anari Cheese Recalled. FDA Enforcement Report, July 12. Anonymous. 1989. Cyprus Anari Cheese Recalled. FDA Enforcement Report, September 27. Anonymous. 1989. Cyprus Anari Cheese Recalled. FDA Enforcement Report, November 1. Anonymous. 1989. Frozen yogurt recalled. FDA Enforcement Report, November 7. Anonymous. 1989. Home-Made Chorizo Bust in L.A. Year-Long Investigation. Lean trimmings. Western States Meat Association, April 7. Anonymous. 1989. Pasteurized process cheese spread: Amendment of standard of identity. Fed. Regist. 54: 6120–6121. Anonymous. 1989. Salmon recalled because of Listeria; blocklist covers hard cheeses. Food Chem. News 31(37): 27–28. Anonymous. 1989. Umweltministerium warnt vor zwei Weichkäsesorten. Deutsche Molkerei Zeitung 110: 898. Anonymous. 1990. Cyprus Halloumi Cheese Recalled. FDA Enforcement Report, January 17. Anonymous. 1990. Robiola Interbiola Soft Ripened and Semi Soft Cheese Recalled. FDA Enforcement Report, November 7. Anonymous. 1990. USDA, FDA officials report apparent decrease in Listeria isolations. Food Chem. News 32(1): 12–15. Anonymous. 1991. Cheese and Sour Cream Products Recalled. FDA Enforcement Report, March 13. Anonymous. 1991. Jack Semi-Soft Cheese Recalled. FDA Enforcement Report, December 18.

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368. Stauber, N.V., R. Braatz, H. Gotz, G. Sulzer, and M. Busse. 1990. Influence of microorganisms on the growth of Listeria in cheese. Deutsche Milchwirtschaft (Hildesheim) 41: 1126–1130. 369. Stecchini, M.L., V. Aquili, and I. Sarais. 1995. Behavior of Listeria monocytogenes in mozzarella cheese in the presence of Lactococcus lactis. Int. J. Food Microbiol. 25: 301–310. 370. Sulzer, G. and M. Busse. 1993. Behaviour of Listeria spp. during the production of Camembert cheese under various conditions of inoculation and ripening. Milchwissenschaft 48: 196–199. 371. Sulzer, G. and M. Busse. 1991. Growth inhibition of Listeria spp. on Camembert cheese by bacteria producing inhibitory substances. Int. J. Food Microbiol. 14: 287–296. 372. Sun, Y.B., Y.L. Soon, H.L. Dong, H.M. Khung, and M.K. Chang. 2000. Incidence and characterization of Listeria monocytogenes from domestic and imported foods in Korea. J. Food Prot. 63: 186–189. 373. Tawfik, N.F. 1993. Growth and inactivation of Listeria monocytogenes in Domiati cheese. Egyptian J. Dairy Sci. 21: 1–9. 374. Terplan, G. 1988. Factors responsible for the contamination of food with Listeria monocytogenes. WHO Working Group on Foodborne Listeriosis, Geneva, Switzerland, February 15–19. 375. Terplan, G. 1988. Personal communication. 376. Terplan, G., R. Schoen, W. Springmeyer, I. Degle, and H. Becker. 1986. Listeria monocytogenes in Milch und Milchprodukten. Deutsche Molkerei Zeitung 41: 1358–1368. 377. Terplan, G., R. Schoen, W. Springmeyer, I. Degle, and H. Becker. 1986. Listeria monocytogenes in Milch und Milchprodukten. In 27th Arbeitstagung des Arbeitsgebietes “Lebensmittelhygiene” von 9–12, September in Garmisch-Partenkirchen. Giessen/Lahn, German Federal Republic; Deutsche Veterinärmedizinische Gesellschafte V. 378. Tham, W. 1988. Survival of Listeria monocytogenes in cheese made of unpasteurized goat milk. Acta Vet. Scand. 29: 165–172. 379. Tham, W.A. and V.M.-L. Danielsson-Tham. 1988. Listeria monocytogenes isolated from soft cheese. Vet. Rec. 122: 539–540. 380. Tipparaju, S., S. Ravishankar, and P.J. Slade. 2004. Survival of Listeria monocytogenes isolated from soft cheese. Vet. Rec. 122: 539–540. 381. Tiscione, E., R. Donato, A. Lo Nostro, L. Galassi, and B. Ademollo. 1995. Patogeni emergenti nel settore alimentare: qualche osservazione sull’isolamento di microrganismi del genere Listeria in campioni di formaggi freschi e molli. L’Igiene Moderna 103: 19–28. 382. Toorop-Bouma, A.G., H.A.P.M. Jansen, and H. van der Zee. 1995. Listeria monocytogenes in soft cheeses imported in the Netherlands. In Proceedings of the XIIth International Symposium on Problems of Listeriosis, Perth, Australia, October 2–6, p. 491. 383. Toschkoff, Al., A. Lilova-Popova, and D. Veljanov. 1975. Dynamics of multiplication and changes in the virulence of some pathogenic microorganisms in milk and milk products. Acta Microbiol. Virol. Immunol. 1: 40–45. 384. Tsiotsia, A., I. Savvaidis, A. Vassila, M. Kontominas, and P. Kotzekidou. 2002. Control of high Listeria monocytogenes by low-dose irradiation in combination with refrigeration in the soft whey cheese Anthotyros. Food Microbiol. 19: 117–126. 385. Tulloch, E.F., Jr., K.J. Ryan, S.B. Formal, and F.A. Franklin. 1973. Invasive enteropathogenic coli dysentery. Ann. Intern. Med. 79: 13–17. 386. Turtura, G.C. and E.M. Grasselli. 2001. Microbiological charcterization of Caciocavallo Corleonese (Palermo) cheese. Rev. Sci. Aliment. 30: 159–168. 387. United States Department of Agriculture. 1978. Cheese varieties and descriptions. Agricultural Handbook No. 54. Agricultural Research Service, Washington, DC. (Reprinted by National Cheese Institute, Alexandria, VA.) 388. United States Food and Drug Administration. 2003. Title—Food and Drugs, Chapter 133—Cheese and Related Cheese Products. Code Fed. Reg 21(133): 308–359. 389. United States Food and Drug Administration. 2005. Office of Regulatory Affairs. http://www.fda.giv/ ora/orasrch.htm. Accessed 6/5/05. 390. Venables, L.J. 1989. Listeria monocytogenes in dairy products—the Victorian experience. Food Australia 41: 942–943. 391. Villani, F., O. Pepe, G. Mauriello, G. Moschetti, L. Sannino, and S. Coppola. 1996. Behaviour of Listeria monocytogenes during the traditional manufacture of water-buffalo mozzarella cheese. Lett. Appl. Microbiol. 22: 357–360.

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392. Vitas, A.I., V. Aguado, and I. Garcia-Jalon. 2004. Occurrence of Listeria monocytogenes in fresh and processed foods in Navarra (Spain). Int. J. Food Microbiol. 90: 349–356. 393. Vlaemynck, G.M. and R. Moermans. 1996. Comparison of EB and Fraser enrichment broths for the detection of Listeria spp. and Listeria monocytogenes in raw-milk dairy products and environmental samples. J. Food Prot. 59: 1172–1175. 394. Wan, J., K. Harmark, B.E. Davidson, A.J. Hiller, J.B. Gordon, A. Wilcock, M.W. Hickey, and M.J. Coventry. 1997. Inhibition of Listeria monocytogenes by piscicolin 126 in milk and Camembert cheese manufactured with a thermophilic starter. J. Appl. Microbiol. 82: 287–291. 395. Wang, L.-L. and E.A. Johnson. 1992. Inhibition of Listeria monocytogenes by fatty acids and monoglycerides. Appl. Environ. Microbiol. 58: 624–629. 396. Weber, A. von, C. Baumann, J. Potel, and H. Friess. 1988. Nachweis von Listeria monocytogenes und Listeria innocua in Käse. Berl. Münch. tierärztl. Wochenshr. 101: 373–375. 397. Wenzel, J.M. and E.H. Marth. 1990. Behavior of Listeria monocytogenes in the presence of Streptococcus lactis in a medium with internal pH control. J. Food Prot. 53: 918–923. 398. Wenzel, J.M. and E.H. Marth. 1990. Changes in populations of Listeria monocytogenes in a medium with internal pH control containing Streptococcus cremoris. J. Dairy Sci. 73: 3357–3365. 399. Yndestad, M. 1988. Personal communication. 400. Yousef, A.E. and E.H. Marth. 1988. Behavior of Listeria monocytogenes during the manufacture and storage of Colby cheese. J. Food Prot. 51: 12–15. 401. Yousef, A.E. and E.H. Marth. 1990. Fate of Listeria monocytogenes during the manufacture and ripening of Parmesan cheese. J. Dairy Sci. 73: 3351–3356. 402. Yousef, A.E., E.T. Ryser, and E.H. Marth. 1988. Methods for improved recovery of Listeria monocytogenes from cheese. Appl. Environ. Microbiol. 54: 2643–2649. 403. Zuniga-Estrada, A., A. Lopez-Merino, and L. Mota-de-la-Garza. 1995. Behavior of Listeria monocytogenes in milk fermented with a yogurt starter culture. Rev. Lat.-Am. Microbiol. 37: 257–265.

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and Behavior 13 Incidence of Listeria monocytogenes in Meat Products Jeffrey M. Farber, Franco Pagotto, and Chris Scherf CONTENTS Introduction ....................................................................................................................................504 Incidence of Listeria in Meat Products .........................................................................................504 Results from USDA–FSIS Listeria-Monitoring Programs..................................................504 Raw Meat..................................................................................................................505 Cooked and RTE Meat Products..............................................................................505 Recalls and Other Regulatory Actions.....................................................................507 Results from Canadian Listeria monocytogenes Monitoring Programs..............................508 Cooked and RTE Meat Products..............................................................................508 Recalls and Other Regulatory Actions.....................................................................514 Incidence of Listeria spp. in Raw Meat ..............................................................................514 North America ..........................................................................................................515 Non–North American Countries ..............................................................................516 Other Countries ........................................................................................................522 Incidence of Listeria spp. in Sausage and RTE Meat Products..........................................523 North America ..........................................................................................................523 Europe.......................................................................................................................523 Other Countries ........................................................................................................527 Behavior of L. monocytogenes in Meat Products .........................................................................527 Listeriosis in Domestic Livestock........................................................................................528 Localization in Tissues .........................................................................................................528 Raw Beef ..............................................................................................................................530 Growth and Survival.................................................................................................530 Raw Lamb and Pork.............................................................................................................533 Cooked and RTE Meats .......................................................................................................534 Cured Ham................................................................................................................534 Cooked Roast Beef...................................................................................................535 Luncheon Meats .......................................................................................................535 Unfermented Sausage...........................................................................................................538 Fresh Sausage ...........................................................................................................538 Cooked Smoked Sausage .........................................................................................540 Uncooked Smoked Sausage .....................................................................................543 Cooked Meat Specialty Items ..................................................................................543 Fermented Sausage...................................................................................................544

503

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Semidry Fermented Sausage ....................................................................................544 Dry Fermented Sausage ...........................................................................................545 Modified-Atmosphere (MA) Packaging...............................................................................547 Beef...........................................................................................................................548 Sausages....................................................................................................................549 Lamb .........................................................................................................................549 Pork...........................................................................................................................550 Thermal Inactivation in Meats .................................................................................552 Bacteriocins for Controlling Listeriae in Meat....................................................................557 Raw Ground Meat ....................................................................................................557 Fermented Sausages .................................................................................................558 References ......................................................................................................................................560 Internet-Based References .............................................................................................................570

INTRODUCTION Only within the past 15 years have there been cases of human listeriosis traced to meat, with evidence for transmission of listeriosis through consumption of contaminated meat and meat products (see Chapter 3). It is generally accepted among all listeriologists that all Listeria monocytogenes strains should be considered as potentially pathogenic. With this in mind, and given the ubiquity of this pathogen within slaughterhouse and meat-packing environments, it is not surprising that the incidence and behavior of L. monocytogenes in meat products have received increased attention worldwide. This is especially so given that a number of recent foodborne listeriosis outbreaks have been linked to meat products (see Table 10.1, Chapter 10), with products such as pâté, frankfurters, and deli meats being implicated [172].

INCIDENCE OF LISTERIA IN MEAT PRODUCTS RESULTS

FROM

USDA–FSIS LISTERIA-MONITORING PROGRAMS

The USDA–FSIS monitoring and verification program for L. monocytogenes in meat products began in September 1987 with sampling of domestic corned beef, cooked corned beef, and massaged corned beef, as well as imported cooked meats. This program was later expanded to include a wider range of products with meat or poultry salads and spreads added in 1988 [63,111]. During the 1980s, L. monocytogenes began to emerge as a problem in processed meat and poultry products. In the 1990s, state health departments and the Centers for Disease Control and Prevention (CDC) investigated an outbreak of foodborne illness in which hot dogs, and possibly deli (luncheon) meats, were implicated. CDC and FSIS investigators isolated the outbreak strain, a strain of L. monocytogenes, from an opened and a previously unopened package of hot dogs manufactured by a single plant. In total, 101 illnesses were reported, with 15 adult deaths and 6 stillbirths or miscarriages. By 1999, an especially virulent strain of L. monocytogenes emerged. The agency then informed establishments that they should reassess their hazard analysis and critical control point (HACCP) plans. As a consequence, FSIS published a notice advising manufacturers of ready-to-eat (RTE) meat and poultry products of the need to reassess their HACCP plans to ensure that they were adequately addressing L. monocytogenes contamination risks. Data gathered during an outbreak of Listeria-related illnesses during the summer of 2002, combined with other food safety investigations and in-depth verification reviews, led FSIS to conclude that some establishments were not adequately addressing the potential for bacterial contamination in their HACCP plans, sanitation standard operating procedures (sanitation SOP), or other control measures.

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Then, in December 2002, FSIS implemented a directive outlining additional steps to be taken by USDA inspectors to ensure that establishments producing RTE meat and poultry products were preventing L. monocytogenes contamination [156]. Under this directive, plants producing deli meats and hot dogs without validated Listeria programs to eliminate L. monocytogenes on the product, on food contact surfaces, and in the environment were subject to an intensified FSIS testing program. This intensified program included increased product and food contact surface testing, environmental testing in the plant, and more frequent reviews of plant records and data. Subsequently, in February 2003, FSIS released its draft risk assessment on Listeria in RTE meat and poultry products. A public meeting was held on February 26, 2003, to discuss the results. The risk assessment, in conjunction with a previously released FDA/FSIS risk ranking and public comment gathered on the topic (see Chapter 18), provided important data enabling FSIS to design a final L. monocytogenes rule [246]. Raw Meat The USDA–FSIS monitoring and sampling program was not designed to initiate regulatory action against particular firms, but rather to provide the agency with critical background information. During the 26-month period from January 1987 to February 1990, L. monocytogenes was isolated from 122 of 1726 (7.1%) 25-g monitoring samples of domestically produced raw beef [75]. In 1995, the FSIS reported on the levels of bacteria, including L. monocytogenes, in ground beef [25]. The survey, based on 600 1-lb samples of ground beef from 661 plants, reported an 18% incidence of the organism. Cooked and RTE Meat Products Interest in the extent of L. monocytogenes contamination in both domestic and imported cooked and RTE meat products also began in the mid-1980s. The list of these products now includes beef jerky, cooked sausage (both large- and small-diameter), cooked, roast, and corned beef, meat salads or meat spreads, and sliced canned ham and luncheon meat. Each sample collected, consisting of one to six subsamples, represents one lot of product. A portion of each subsample is then pooled to form a composite sample, which is analyzed. If a positive result is obtained for a lot, the composite sample is not reanalyzed to determine the number of positive subsamples in the lot. Results from this program for the years 1993–2000 (Table 13.1) showed that, in general, there was a low incidence of L. monocytogenes in cooked and RTE meat products in the United States, with overall incidences from 0 to 8.1%. The organism was usually absent from beef jerky, as its presence was reported in this product only twice: in 1994 at a 2.2% incidence and in 1998 at a 1.6% incidence. Cooked large-diameter sausage showed a higher prevalence of the organism than cooked small-diameter sausage, with the incidences ranging from 1.8 to 5.3% and 0.4 to 2.1%, respectively. L. monocytogenes was present in only 2.1 to 3.4% of samples of domestic or imported cooked beef, roast beef, and cooked corn beef over this 7-year period. In most instances, these products were removed from their packages after cooking and then repackaged for sale, suggesting that the pathogen most likely entered the product through direct contact with the factory environment, knives, or gowns worn by workers [128]. At least two other firms handled raw and finished product in the same production area of the factory but at different times, which, in turn, indicates the possibility of cross-contamination between raw and finished product. In December 1987, USDA–FSIS officials added sliced canned ham and sliced canned luncheon meat to the monitoring and verification program for the presence of L. monocytogenes. From 1993 to 2000, the pathogen was detected in 4.6 to 8.1% of these samples, which were among the higher incidences among the RTE meat products examined (Table 13.1). In June 1988, monitoring of meat or poultry salads and spreads was begun, with incidences of 1.2 to 4.7% being reported from 1993 to 2000 (Table 13.1).

39 328 472 426 273 149 NAc 0 2.1 5.3 3.0 2.2 8.1 NA

1993 # Lotsb (% pos) 45 438 602 479 580 232 NA 2.2 1.1 4.8 2.1 2.4 5.5 NA

1994 # Lots (% pos) 50 438 611 560 597 99 NA 43 420 561 507 554 91 NA

0 1.0 3.7 3.4 2.2 7.7 NA

1996 # Lots (% pos) 40 371 621 530 206 286 108 0 1.6 2.7 2.1 2.4 4.2 9.3

192 506 746 511 225 263 244

1.5 1.2 3.5 2.2 3.1 4.2 2.9

1998 1997 # Lots (% pos) # Lots (% pos)

278 1,167 2,162 922 435 960 478

0 0.4 1.8 2.7 1.2 4.6 2.1

1999 # Lots (% pos)

0.8 0.5 1.3 2.2 1.0 3.1 1.5

2000 # Lots (% pos)

506

b

0 1.1 4.1 2.7 4.7 5.0 NA

1995 # Lots (% pos)

Ref. 248. Each sample, consisting of one to six subsamples, represents one lot of product. cNA = Not available.

a

Beef jerky Cooked sausage—large diam Cooked sausage—small diam Cooked/roast/corned beef Salads/spreads Sliced ham/sliced luncheon meats Fermented sausages

Meat Product

TABLE 13.1 Incidence of Listeria monocytogenes in USDA–FSIS Monitoring Samples of Cooked and RTE Meat Products, 1993–2000a

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TABLE 13.2 USDA/FSIS Microbiological HACCP Verification Testing Program for RTE Meat and Poultry Products Produced in USDA-Inspected Establishments Product Category Whole sausage-type product, peeled

Whole sausage-type product, unpeeled

Large mass, chopped and formed

Large mass, whole muscle

Small mass, chopped and formed Small mass, whole muscle Salads, pâtés, and spreads Sliced, diced, shredded (with or without sauce) Multi-component products Other

Examples (Not All-Inclusive) Hot dogs, frankfurters, knockwurst, and other products cooked in a casing that is removed by a peeling process after the lethality step and before final packaging Hot dogs, bologna, andouille sausage, pepperoni, salami, and similar products that are shipped in the same casing that exists during the lethality step Turkey roll, loaves, cooked ham, and other products that have been processed before lethality in a manner where exterior bacteria could be transferred into the internal tissues Cooked roast beef, whole chickens, cooked corned beef, cooked turkey breast, bone in ham, prosciutto, dry cured ham, that is, products with only external bacteria before the lethality step. Meatballs, chicken nuggets, patties, breakfast sausage Chicken tenders, whole muscle cutlets, chicken breasts Chicken salad, ham salad, liverwurst, pâté de foie gras Sliced ham, sliced turkey, diced cooked chicken, beef barbeque, sliced pepperoni, chipped beef Dinners, entrees, wraps, pocket sandwiches, egg rolls, pizza Products that cannot be categorized into the other nine categories

In December 2000, the FSIS microbiological monitoring projects based on selected product types were discontinued and a HACCP verification program based on the HACCP process was initiated. Thus, samples were scheduled based on the different RTE HACCP processes used in USDA-inspected establishments and were randomly scheduled in the establishments in which the RTE processes existed (Table 13.2). Therefore, the results for 2001 and 2002 are not directly comparable to those published for previous years (Table 13.3). However, in general, the incidence of L. monocytogenes in these types of products appeared to decrease. A survey of commercially prepared frankfurters from 12 producers over a 2-year period showed that L. monocytogenes contamination can be highly variable from plant to plant, and that seasonality has no effect on the incidence of this pathogen in frankfurters. Recovery rates of L. monocytogenes from two plants were 1.5 and 2.2% for plants producing frankfurters that were made from meat. In total, 104 of 27,300 packages (0.38%) of samples contained L. monocytogenes [231]. Recalls and Other Regulatory Actions After a request from the food industry that the FDA reconsider its policy of “zero tolerance” for presence of L. monocytogenes in foods, the FDA stated that it would need the support of scientific data on infectious levels of the organism to justify any change in its regulation that the pathogen should not be present in RTE foods [15]. Later, the FDA announced that its pathogen-monitoring program would concentrate on selected high-risk foods, including prepared sandwiches [26]. The agency stated that if a prepared sandwich sample tested positive for L. monocytogenes, then a comprehensive follow-up inspection of the firm would be warranted, including sampling of raw materials, plant surfaces, and finished product.

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TABLE 13.3 Prevalence of L. monocytogenes in USDA–FSIS Microbiological HACCP Verification Samples of RTE Meat Products, Cumulative Years (CY) 2001 and 2002 Summary by Product Type CY 2001 % Incidence (Positives/Sample Number)

Product Peeled sausage type product Unpeeled sausage type product Large mass chopped & formed type product Large mass whole muscle type product Small mass chopped and formed type product Small mass whole muscle type product Salads/pâtés/spreads type product Sliced, diced, and shredded type product Multi-component products Other type product Summary by HACCP process type

0.93 0.71 2.34 2.42 0.85 0.67 1.33 2.59 1.48 1.42 1.32

(6/646) (12/1689) (5/214) (12/495) (5/585) (6/896) (3/226) (25/966) (10/675) (2/141) (86/6533)

Summary by Product Type CY 2002 % Incidence (Positives/Sample Number) 1.72 0.63 0.93 0.49 1.05 0.77 0.42 1.96 0.94 0.33 1.03

(13/758) (11/1758) (3/322) (3/609) (6/570) (7/914) (1/237) (25/1274) (8/851) (1/299) (78/7592)

Between January 1994 and October 2006 at least 175 separate Class I or voluntary recalls were issued for cooked and RTE meat contaminated with L. monocytogenes in the United States, including 74 for deli meats, 42 for sausages, 37 for hot dogs, and 22 for other products (Table 13.4). Since 1994 a number of these recalls have involved prepared sandwiches; these recalls have resulted in at least 50,000 sandwiches being removed from the marketplace. In these recalls of prepared sandwiches, the distribution usually involved several states (Table 13.4). Product losses may be higher than indicated because firms holding tested products until results of Listeria analyses become known can “recall” all contaminated lots internally, thereby avoiding a formal Class I recall. Considering the wide variety of tainted sandwiches that have been withdrawn from sale, it appears that L. monocytogenes entered these products during cutting, slicing, or packaging, rather than being initially present in the many different types of sandwich fillings.

RESULTS

FROM

CANADIAN LISTERIA

MONOCYTOGENES

MONITORING PROGRAMS

Cooked and RTE Meat Products Agriculture and Agri-Food Canada initiated and passed on to the Canadian Food Inspection Agency (CFIA) Canada’s L. monocytogenes-monitoring program on processed and RTE meat products from registered processing plants in 1987. At the same time, the Health Products and Food Branch (HPFB), Health Canada, started a similar testing program for L. monocytogenes in RTE meat products from nonregistered establishments [84]. Five subsamples of 150 g (or a convenient package size) each were collected. The quantity analyzed depended on the product; for meats supporting the growth of Listeria spp., 25 g was tested, whereas for meats not supporting growth of Listeria spp., 5 g was tested. During 1989–1990, the incidence of L. monocytogenes in the RTE meat samples tested by the Agriculture Canada monitoring program was high, i.e., an average of about 24% (Table 13.5). During this time, samples taken at establishments with Listeria-positive products showed an incidence of 46% for meat products (data not shown), and an average of 12% for environmental samples. The incidence dropped sharply during 1991–1992 from 0 to 3% for meat products, but contamination in the plant environment remained about the same. After 1992, only environmental samples were taken for testing domestic products, but a more rigorous testing procedure involving three phases was instituted.

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TABLE 13.4 Chronological List of Recalls (Class I or Voluntary) Issued in the United States for Cooked and RTE Meat Products Contaminated with Listeria monocytogenes, 1991–November 2006a Product Sandwichesb Ham saladb Sandwichesb Skinless hot dogsc Ham saladd Frankfurterse Sandwichesf Sandwichesg Beef frankfurters Beef frankfurters Beef cooked salami Linguica sausage Ham, sliced Ham salad Ham, cooked, sliced Smoked sausage Wieners Hot dogs (franks) Ham, sliced Smoked Polish sausage Beef franks Buffalo franks Frankfurters Hot dogs (franks) Bologna ring Sandwiches — ham and cheese, ham salad, etc.b Beef wieners Hot dogs, pork Sandwiches — roast beef, hot dog, burgers, sausage, meatball, ham and cheese, etc.b Cooked beef roast Roast beef, sliced Cooked beef Extra hot beef jerky Ham, chunked and formed Sliced ham Sandwiches — hot dog, ham and cheese, sausage, beef, salami, etc.h Dry sausage (kayseri soujouk) Filzette salami Frankfurters Ham steaks Dry sausage Frankfurters Smoked beef strips Hot dogs/packaged meats Luncheon meat Sliced ham

Date Recall Initiated (mm/dd/yy)

Origin

Quantity in lbs

1991 1991 07/03/91 1991 1991 03/20/92 06/18/92 09/25/92 01/25/94 02/09/94 02/23/94 03/21/94 03/28/94 04/12/94 05/05/94 05/15/94 10/03/94 14/14/94 10/20/94 01/10/95 01/18/95 03/03/95 05/30/95 07/06/95 07/24/95 09/08/95 10/27/95 11/06/95 02/16/96

LA MN LA MI WV CT LA TN CA MI NY RI NE IN MI MS NE NY NM MN PA CO NY BC, Canada PA MI AB, Canada AB, Canada LA

NA 460 NA 3,700 600 3,578 NA NA 1,220 1,600 1,268 100 3,950 1,105 844 250 5,500 432 3,920 36 1,000 260 3,780 11,420 200 NAb 3,510 4,320 NAb

03/13/96 04/03/96 06/14/96 06/20/96 10/24/96 12/09/96 01/22/97

NE IL TX BC, Canada OH NH MS

487 720 2,608 360 400 52 NAh,i

10/03/97 12/18/97 03/19/98 04/17/98 06/04/98 10/22/98 10/29/98 12/22/98 01/15/99 01/22/99

NY MO NY MO CA FL AR MI WI OH

347 507 1,440 635 272 1,734,002 3,600 35,000,000 28,312 348 (continued)

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TABLE 13.4 (CONTINUED) Chronological List of Recalls (Class I or Voluntary) Issued in the United States for Cooked and RTE Meat Products Contaminated with Listeria monocytogenes, 1991–November 2006 Product Various Mortedella Hot dogs, bockwurst Head cheese Frankfurters Pasta with sausage Frankfurters Luncheon meats, various Weisswurst Wieners, frankfurters Franks, skinless Frankfurters Bacon chips Ham, sliced/whole Roasting sausage Hot dogs Chorizos Sausage Beef frankfurters Smoked sausage Hot dogs Hot dogs Polish sausage Beef franks Luncheon meats Pâtés and mousses Roast beef Dry sausage Cooked corned beef and ham Hot dogs Franks Roast beef Sausage, various Sliced cooked meats Sliced cooked meats Sliced cooked meats Pork cretons spread Frankfurters Sausage, various Salami, Westphalian ham Franks Sliced cooked meats Franks Sliced ham Beef bologna Ham Sliced luncheon meat

Date Recall Initiated (mm/dd/yy) 01/22/99 02/05/99 02/05/99 02/17/99 02/18/99 03/02/99 03/18/99 05/14/99 05/28/99 06/01/99 06/04/99 06/18/99 07/27/99 07/29/99 07/30/99 07/30/99 08/26/99 08/27/99 10/13/99 11/12/99 11/12/99 11/18/99 11/19/99 12/09/99 12/14/99 12/17/99 01/14/00 01/28/00 03/01/00 03/15/00 03/24/00 04/06/00 04/13/00 04/26/00 05/03/00 05/05/00 05/10/00 05/11/00 05/12/00 05/19/00 05/24/00 05/24/00 05/29/00 06/07/00 06/07/00 06/14/00 06/14/00

Origin AR ON, Canada WA IL GA IL NJ NC PA ND IL HI IN MI NJ PA PR MA NY LA HI BA MD PA NJ NY AZ Il MI NY PA UT OH MI MI MD ME HI MS BC, Canada MI AL NC MO NY CA HI

Quantity in lbs 35,000,000 456 1,545 2,586 4,460 1,923 18 16,392 60 150 1,285 9,620 126,739 200 200 200 1,640 3,720 2,100,000 1,270 312 1,020 800 4 900 10,064 600 200 80 400 34,500 13,351 850 180 215 450 210 1,125 5,900 400 2,870 45 15,000 60 2,200 1,800 270 (continued)

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511

TABLE 13.4 (CONTINUED) Chronological List of Recalls (Class I or Voluntary) Issued in the United States for Cooked and RTE Meat Products Contaminated with Listeria monocytogenes, 1991–November 2006 Product Beef jerky Hot dogs, RTE deli meats Sliced luncheon meats Wieners Polish sausage Country ham Mexican-style meat products Hungarian salami Sliced, cooked beef RTE beef sausage RTE meat and poultry products RTE bratwurst Cooked smoked ham RTE pork andouille sausage Cooked pork and corned beef Cooked beef products Cooked beef products Luncheon meat Cooked roast beef products Bratwurst Sausage patties Beef and pork sausage products Pork sausage products RTE semi-dry sausage Dried, seasoned beef Cooked, chopped ham Beef and pork sausage RTE souses loaf Frankfurters and hot dogs RTE ham Frankfurters and bologna Sliced ham Cured ham Sausage products Sliced pork hocks RTE braunschweiger Sausage RTE Italian loaf Sliced corned beef Sausage product Cooked, boneless hams Cooked, boneless hams Imported RTE sausage RTE Prosciutto ham Cooked and cured beef loaf RTE ham Pork dumplings

Date Recall Initiated (mm/dd/yy) 07/06/00 08/03/00 08/08/00 10/03/00 10/04/00 12/20/00 01/30/01 02/28/01 03/22/01 03/28/01 04/12/01 07/01/01 07/05/01 09/06/01 09/26/01 10/05/01 10/10/01 10/31/01 12/12/01 12/19/01 12/20/01 01/15/02 01/18/02 02/06/02 02/12/02 02/18/02 03/05/02 04/17/02 04/25/02 05/07/02 05/23/02 06/17/02 06/21/02 06/25/02 07/03/02 07/19/02 07/27/02 08/10/02 08/14/02 08/16/02 08/28/02 08/30/02 09/04/02 09/04/02 09/17/02 10/08/02 10/11/02

Origin CT NY ID TN MN KY IL NJ IL NY OK WA IA WA IN IL IL IA IL MO NC TX FL NY CA CA TX VA OH NV NY MO NY MN NY NY PA NY FL FL AR AR PA CA PA IN HI

Quantity in lbs 125 19,000 380 900,000 240 10,400 3,700 1,570 7,800 14,500,000 100 70 20 5,600 ∼5,000 189,000 700 115 3,300 2,500 150 1,800 22 200 360 190 140,000 300 77,000 10 2,300 250 100 65 22 1,300 19 500 2,200 6,525 1,035 510 185 95 150 (continued)

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TABLE 13.4 (CONTINUED) Chronological List of Recalls (Class I or Voluntary) Issued in the United States for Cooked and RTE Meat Products Contaminated with Listeria monocytogenes, 1991–November 2006 Product RTE pork shoulder RTE pork luncheon meat Cooked pork products RTE souse products Pork sausage Pork sausage Chicken frankfurters Cooked beef products Cooked pork shoulder Cooked pork shoulder Smoked pork chops RTE fully cooked pork and veal bologna Cooked beef sausage RTE chicken salad RTE Thai-style noodle salad containing chicken RTE luncheon meat RTE meat and poultry Beef products RTE sausage and ham products Cooked roast beef Cooked diced chicken breast Chicken salad Frozen beef RTE meat products Beef and pork frankfurters Fully cooked boneless hams RTE fully cooked bologna Fresh deli meat and cheese trays Scrapple Beef jerky Frozen, fully cooked chicken products Frozen, fully cooked chicken products Beef and pork products Wieners Fully cooked ham RTE chicken products Chicken products RTE ham Cooked pork products Chicken salad Chicken products Chicken wrap sandwiches Various sausage products Smoked turkey and pork products Various sausage products Pork blood sausage Various RTE meat products Deli meat wraps “turkey and cooked ham capicolla club wrap”

Date Recall Initiated (mm/dd/yy) 11/20/02 11/26/02 11/27/02 12/03/02 12/04/02 12/12/02 01/03/03 01/22/03 01/28/03 01/30/03 03/06/03 03/22/03 05/05/03 05/05/03 10/04/03 10/10/03 10/12/03 10/24/03 11/05/03 11/18/03 11/25/03 12/11/03 01/11/04 01/28/04 02/17/04 03/16/04 04/05/04 04/12/04 06/03/04 06/29/04 07/01/04 07/21/04 07/27/04 08/04/04 08/10/04 11/03/04 01/31/05 02/09/05 02/28/05 03/15/05 03/25/05 04/05/05 04/05/05 04/11/05 04/11/05 04/19/05 04/21/05 04/26/05

Origin PR IN VA MS FL FL NY SD PR PR PA NY NC IL NJ CA MA WA AR NE GA FL IL PA NY GA PA IL DE PA NC GA NJ UT NY MD NY TN LA NY CA FL MI NY GA CA MO TX

Quantity in lbs 6,800 210 540 500 8,600 200 26,400 2,100 490 590 11 330 180 400 270 550 9,230 200 4,500 110 7,500 2,700 5,190 52,000 540 713 100 135 350 130 404,730 36,980 500 5,360 422 1,275 5,760 47 1,120 250 12,500 3,316 5,117 39,000 10,700 40 1,077 191 (continued)

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TABLE 13.4 (CONTINUED) Chronological List of Recalls (Class I or Voluntary) Issued in the United States for Cooked and RTE Meat Products Contaminated with Listeria monocytogenes, 1991–November 2006 Product Chicken breast wraps RTE ham Various RTE meat products RTE chicken salads Spanish brand sausage (Primera Chorizos) Natural proportion cooked chicken meat Meat and poultry products Barbeque beans with beef and chicken salad Chorizo, blood sausage and blood pudding Chicken frankfurters Cooked country hams Cooked chicken sausage products and beef wieners RTE meat and poultry products RTE beef products RTE chicken product Chicken salad products Turkey, ham, bologna, and chicken lunch makers meals Pork barbeque Dried beef products Ham salad

Date Recall Initiated (mm/dd/yy)

Origin

Quantity in lbs

04/28/05 04/29/05 04/30/05 06/15/05 06/26/05 07/21/05 07/29/05 08/31/05 09/15/05 09/20/05 09/28/05 10/02/05 10/22/05 10/31/05 11/08/05 11/10/05 12/01/05 02/18/06 03/23/06 04/05/06

NY KY Nationwide NY NJ GA Nationwide OK NY NY VA IL MA NY CA PA MO NC TX ME

385 29,000 363,332 5,065 720 170 93,200 23,435 890 23,040 165 1,000 11,200 2,263 275 5,523 2,800,000 30 100 92

a

Reference 247, unless otherwise indicated. Reference 20. cReference 18. dReference 19. eReference 21. fReference 22. gReference 23. hNA = Not available. iReference 27. b

Phase I of the program consisted of taking 10 post-process environmental samples from product contact surfaces. These 10 samples were composited into one analysis. Any positive Phase I analysis triggered entrance into Phase II, consisting of 10 repeat environmental samples, analyzed individually, and a review of Good Manufacturing Practices (GMP) throughout the plant. Phase III is entered if one or more analyses of Phase II samples is found to be positive, in which instance a thorough review of all plant procedures is undertaken before collection of further environmental samples for individual analysis. Between 1992 and 1994, the incidence of L. monocytogenes in the plant environment dropped sharply, indicating the value of this approach [85]. Selected data from three major regions of Canada, the provinces of Ontario, Quebec, and the provinces on the Atlantic coast of Canada, grouped as “Atlantic,” showed a slight overall increase in the incidence of L. monocytogenes from the period 1997–2000 (Figure 13.1). However, it has been shown that the incidence of L. monocytogenes in Canada had decreased to around 10% in 2001–2002 (Table 13.5). The presence of L. monocytogenes in imported RTE meat products during this period remained low, with incidences ranging from 0 to 5% (Table 13.5). The importance to the Canadian consumer of imports from the United States compared to other countries is evident from the relatively large number of American samples tested.

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35

Ontario Quebec 30

Atlantic

% of Unsatisfactory Samples

25

20

15

10

5

0 97/98

98/99

99/00

Work Specification M-205 Sampling Years Assessed

FIGURE 13.1 Change over time of the prevalence of L. monocytogenes in Canadian federally regulated readyto-eat meat establishments, 1997–1998 to 1999–2000, using protocol M-205 for sampling. (From Health Canada Food Safety Assessment Program, Assessment Report of the Canadian Food Inspection Agency Activities Related to Domestic Ready-to-Eat Meat Products, 2002.)

Recalls and Other Regulatory Actions The current Canadian policy on control of L. monocytogenes in foods is risk-based. Thus, for highrisk meats that have been causally linked to listeriosis, such as liver pâté and jellied pork tongue, a Class I recall to the retail level would be put into place. However, all other RTE meats that support growth of L. monocytogenes and that have a refrigerated shelf life of >10 days would only be subject to a Class II recall. The largest number of recalls (20) for RTE meat products in Canada between 1989 and 1997 was for dry, fermented sausages, some of which were smoked (Table 13.6). An almost equal number of recalls were initiated for sandwiches, including substitutes, containing meat. Sliced cold meats of various types were also found contaminated with L. monocytogenes during this period, with contamination likely coming from retail slicers. Wieners and miscellaneous products made up the balance of the recalls. A dramatic decrease in the number of recalls due to meat contaminated with L. monocytogenes since 1997 was revealed through the CFIA food recall database. There were 11 recalls of meat products due to contamination with L. monocytogenes. In particular, there were no such recalls in 1998 or 2000 (CFIA Web site, product recalls, 1997–2002).

INCIDENCE

OF

LISTERIA

SPP. IN

RAW MEAT

Slaughter animals are a recognized reservoir of human pathogens, including L. monocytogenes. Studies indicate an incidence of listeriae ranging from 0 to 9%, both in feedlot cattle [209] and in pork carcasses [2,96,184,197]. However, a 96% incidence of listeriae was reported on pork carcasses from one plant [96], showing the results of poor sanitation. The incidence of listeriae on slaughter plant equipment was also low (0–3%) [96,190], except for the plant mentioned previously, in which

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TABLE 13.5 Incidence of Listeria monocytogenes in Canadian Food Inspection Agency Testing Samples of Domestic and Imported RTE Meat Products, and in Processing Plants, 1989–2002a Domestic Products Number of Samples (% positive) Year 1989 1990 1991 1992 1993 1994 1999–2000 2000–2001 2001–2002

396 66 19 35

(12) (35) (0) (3)

NDc ND

Imported Products Number of Samples (% positive) U.S.A. 50 (4) 1279 (2) 984 (2) 469 (3)

Others 5 (0) 3 (0) 14 (0) 9 (0)

417 (3) 382 (5)

1 (0) 1 (0)

Plant Environment Number of Samples (% positive)

677 (17) 2267 (8) 3314 (13) Phase 1b 3808 (10) 2522 (6) 3629 (2) 77 (9) 416 (11.5) 431 (10)

Phase II 430 (9) 124 (19) 138 (6)

Phase III 164 (13) 20 (0)

a

Ref. 12. Phase I – 10 composited post-process samples of product contact surfaces. Phase II – if Phase I positive, 10 individual environmental samples. Phase III – if Phase II positive, review of plant procedure before further individual sampling. cND = not determined. After 1982, environmental testing only was done for domestic products. b

the incidence was reported at 42% [96]. Levels of the organism were low, ranging from 10 to 100 CFU/g [96]. The only areas where L. monocytogenes was demonstrated at a plant were cold rooms maintained at 5°C, at which temperature this psychrotrophic organism is able to grow.

North America In addition to the government monitoring programs in North America, there have been several reports of nonregulatory surveys of both raw and RTE meat products (Table 13.7). Surveys on raw meat products such as roasts and steaks sampled in the United States indicate that the incidence of Listeria spp. ranges from 0 to 6% (Table 13.7). Comminuted raw meats, however, show a much higher incidence of listeriae, from 24 to 100%, with L. monocytogenes ranging from 0 to 25%. Reported levels of listeriae ranged from 4 to 2.1 × 104 CFU/g. Most beef comes from cattle that are well treated before slaughtering. However, there are times when the stresses of mixing cattle, transportation over long distances, as well as weather and time of year, can reduce the amount of muscle glycogen. The beef that results from this is called dark, firm, dry (DFD) beef, or dark cutting beef. Hooper-Kinder et al. [121] studied susceptibility of this type of beef to growth of L. monocytogenes Scott A. Inoculation of 0, 50, and 100% DFD ground meat samples with L. monocytogenes showed that despite the lower glycogen content, there was no difference between this and normal beef available at the retail level with regard to growth of L. monocytogenes. In raw meat products sampled in Canada, usually ground meats, the incidence of Listeria spp. is high, ranging from 66 to 100%. The incidence of L. monocytogenes ranges from 44 to 100%, indicating that contamination of raw meats with this pathogen is relatively common (Table 13.7).

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TABLE 13.6 Class II Recalls Issued in Canada for Processed and RTE Meat Products Contaminated with Listeria monocytogenes, 1983–November 2002a Number of Recalls Year 1983 1994 1995 1996 1997 1998 2000 2001 2002

Wieners

Dry Fermented Sausages

Sandwiches/Subs

Sliced Roast Beef

2 1 1

45 3

Cooked Ham

Breastb

1 1

2

2 15 1

1

1

2003

4

3 1 1 12 2

2

2

3

2 12 4

2

1

2004 2005

Misc.C

4 2

7

1

5

1

3

26

7

25

6

29

2006 Totals

51

6

a

Ref. 13, 28, 248. Turkey or chicken. cPizza, burrito, cretons, pork ribs, ham salad, sliced turkey, bacon, bologna, salami, meat loaf, pepperoni, smoked beef steak nuggets chicken fried rice, goose rillettes, infant foods. b

These results may be due to contaminated equipment at either the wholesale or retail level. It is noteworthy that there is a much lower incidence of listeriae in the wild animal population than in domesticated animals. Amoril and Bhunia [5] took 20 beef and 20 poultry samples from local grocery outlets to determine the level of L. monocytogenes in raw meats. L. monocytogenes was found in 13 (32.5%) of the samples, which is lower than reported by other researchers [85a,136]. Combined with research data generated by the scientific community, it is surmised that the lower incidence rate is due to efforts of regulatory agencies and industries to decrease the presence of microorganisms in foods. Heredia et al. [117], investigating incidence of Listeria spp. in Mexican ground meat samples, found Listeria spp. and L. monocytogenes in 62 and 16%, respectively, of the 88 samples taken. Non–North American Countries Since 1990, many reports have been published on the prevalence of Listeria spp. and of L. monocytogenes in raw meat and raw meat products prepared in various countries (Table 13.8). In the United Kingdom, MacGowan et al. [162] reported incidences of Listeria spp. ranging from 59 to 88% in beef, lamb, and pork, with L. monocytogenes present in 28–40% of these samples. Wilson [236], based on a larger sampling of beef, lamb, and pork in Northern Ireland, reported an incidence of about 4% for listeriae. Gilbert [93] reported an incidence of L. monocytogenes in raw pork sausage of 49%, whereas MacGowan et al. [162] found a similar incidence of 35%. In a study in Ireland, Sheridan et al. [205] reported high incidences of listeriae, ranging from 45 to 85% in 20 samples

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TABLE 13.7 Incidence of Listeria monocytogenes in Raw Meat and RTE Meat Products in the United States and Canada, 1990–2003

Product Raw meats—U.S.A. Beef roast Pork roast Lamb roast Ground beef Ground pork Pork sausage Lamb patty Beef Pork loin Whole-muscle, store-packaged pork Whole-muscle, enhanced pork Store-ground fresh pork and/or pork sausage Prepackaged ground pork and/or pork sausage Ground pork and/or pork sausage Luncheon meats, 2000–2001, Maryland/California Deli salads, 2000–2001, Maryland/California Ground pork at packing plants/retail grocery Chitterlings (small intestines) at packing plants/retail grocery Swine tissues Raw meats—Canada Ground beef Ground pork Ground veal Ground meat Meat cuts Wild animal meat—moose, deer, bear RTE products—U.S.A. Wieners Wieners Wieners Large sausage, 1998/1999 Small sausage, 1998/1999 Salads/spreads, 1998/1999 Sliced ham/pork, 1998/1999

Number of Samples Analyzed

Percentage of Positive Samples Listeria spp.

LM

Levels of Listeria spp. (CFU/g)

Reference

50 50 10 39 20 17 2 658 135 96

6 6 0 41 30 24 100 ND ND 28.1

NDa 5 ND 8 25 6 0 6 0 14.6

96 96

25 61.5

14.6 22.9

76 76

96

53.1

27.1

76

120

47.5

26.7

76

45,994/4,600

ND

1.2/0.6

104

4,293/4,256

ND

2.4/2.3

104

10b 4–560 4–240 240–21,000 4–56

137 137 137 244 244 244 244 132 197 76

340

50.2

51.9

141

300

9.3

9.3

141

1,849

2.4

2.8

141

22 19 3 11 18 10

100 100 100 100 65 10

77 95 100 63 44 10

87 87 87 160 160 160

93 24 30

10 83 20

8 71 17 1.2/0.4 3.5/1.8 3.1/1.1 4.2/4.6

232 232 24 247 247 247 247 (continued)

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TABLE 13.7 (CONTINUED) Incidence of Listeria monocytogenes in Raw Meat and RTE Meat Products in the United States and Canada, 1990–2003

Product Cooked/roast/corned beef, 1998/1999 Fermented sausage, 1998/1999 Jerky, 1998/1999 Cooked meat products Luncheon meats, 2000–2001, Maryland/California Deli salads, 2000–20001, Maryland/California Frankfurters RTE products—Canada Fermented sausages Cooked meat Wieners Luncheon meats Sausages

Number of Samples Analyzed

Percentage of Positive Samples Listeria spp.

Levels of Listeria spp. (CFU/g)

LM

Reference

2.2/1.4

247 247 247 3 104

369 4,599/4,600

65 ND

2.9/2.1 1.6/0.0 9.2 1.2/0.6

4,293/4,256

ND

2.4/2.3

104

0.4

231

20 0 21 13 0

88 216 216 216 216

27,300 30 16 38 67 9

33 0 26 13 0

Note: LM = L. monocytogenes; ND = not determined; RTE = ready-to-eat.

each of beef, pork, and lamb, with L. monocytogenes present in 15–50% of the samples. Interestingly, the incidence of the organism was lower in comminuted meats (94 samples of frozen beef burgers, 85 samples of ground beef, and 20 samples of sausage), with L. monocytogenes present in 5 to 18% of the samples. An incidence of 5% for L. monocytogenes in ground meat was found in Norway [195], all the organisms belonging to serotype 1. Raw beef products in Germany have a reported incidence of L. monocytogenes of about 50% [132]. As raw minced meat is a popular dish in Germany, the German Ministry of Health decided to restrict sales of the contaminated product [11]. If a sample contained less than 100 CFU/g, there would be further surveillance of the factory or retail outlet, and if the number of listeriae reached 1,000 CFU/g, there would be a public warning. Workers in Italy have reported on the incidence of listeriae in many types of raw meat. According to Maini et al. [164], the incidences of listeriae in 19 samples each of calf, horse, pork, and mutton were 63, 26, 53, and 56%, respectively. Cantoni et al. [49] showed that prevalence of the organism on both sausages and their casing surfaces were roughly equal, 60 and 47% for listeriae and 13 and 12% for L. monocytogenes, respectively. Other samplings of fresh pork and beef for the organism showed incidences ranging from 21 to 47% for listeriae and from 13 to 22% for L. monocytogenes. Serotype 4 was found in 7% of these samples [49]. Comminuted meats and sausages showed higher incidences for Listeria spp. (44–54%), but in the same range for L. monocytogenes (9–19%) [60,61,155]. The main serotype isolated was 1/2c [60]. When present, the organism was at a level of less than 100 CFU/g. Breer and Schöpfer [44] reported that of 209 samples of beef, pork, and pork products collected in Switzerland, listeriae were recovered from 11 to 45% of the samples, with an incidence ranging from 0 to 15% for L. monocytogenes. In comminuted meat products, the corresponding incidences were 40–65% and 8–15%, respectively.

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TABLE 13.8 Incidence of Listeria monocytogenes in Raw Meat in European and Other Countries (1990–1997) Percentage of Positive Samples Country Australia

Product

Number

Beef 50 Lamb 50 Pork 50 Belgium Prepacked blood sausages, 137 25 g Other prepacked meats, 25 g 199 Not prepacked meats, 406 25 g/0.1 g Not prepacked pâtés, 25 g 130 Not prepacked aspic, 25 g 67 Not prepacked blood 18 sausages, 25 g Other not prepacked meats, 132 25 g Raw cured meats Whole muscles (sliced, 516 prepacked), 25 g/0.1 g Beef, 25 g 43 Horse, 25 g 17 Loin, 25 g 121 Raw ham, 25 g 169 Pork bacon, 25 g 153 Minced muscles (sliced or 308 not), dry fermented sausages, etc., 25 g/0.1 g Raw cured meat products, 824 25 g/0.1 g Bosnia/Herzegovina Beef 20 Pork 50 Brazil Dressed lamb carcasses 69 Bulgaria Beef and pork 234 China Pork 25 Beef 10 Lamb 14 Denmark Preserved meat products (not 335 heat-treated), 25 g Heat-treated meat products, 772 25 g Raw meat, 25 g 343 Preserved meat products (not 132/225b heat-treated), 1997/1998 Heat-treated meat products 3,180/3, handled after heat 629b treatment, 1997/1998

Listeria spp. 34 40 30

20 18 26 8 60 70 43

LM

Levels of Listeria spp. (CFU/g) Reference

24 16 10 8.8

131 131 131 222

8 5.2/0.3a

222 222

4.6 1.5 11.1

222 222 222

6.8

222

14.9/6.8a

222

4.7 5.9 19.8 11.8 18.3 11.7/4.22a

222 222 222 222 222 222

13.7/5.8a

222

10 8 3 28 0 0 10.8

158 158 7 186 233 233 233 187

5

187

30.9 14.4/16.5b 7.8/10.1b

187 187 187

(continued)

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TABLE 13.8 (CONTINUED) Incidence of Listeria monocytogenes in Raw Meat in European and Other Countries (1990–1997) Percentage of Positive Samples Country Germany Greece

India

Ireland

Italy

Japan

Korea

Malaysia Spain Norway

Product Ground meat Rinderhack (ground beef) Sliced, vacuum-packed cooked meats Frankfurters Country-style sausages Emulsion-type sausages heated in packs Dry-fermented sausages Cattle Buffalo Sheep Goat Beef Ground beef Pork Lamb Sausage Frozen beef burgers Calf Horse Pork Mutton Sausage Casing surface Pork Beef Ground beef Pork Ground meat Beef Raw meat Sausage Beef Ground beef Pork Ground pork Beef Pork Frozen pork cutlet patties and dumplings Fermented sausage Beef Ground meat Ground meat

Number

Levels of Listeria spp. (CFU/g) Reference

Listeria spp.

LM

21 59 26

27

52 46 8

132 132 198

8 10 12

0 40 0

0 10 0

198 198 198

4 54 54 54 54 20 85 20 20 20 94 19 19 19 18 156 116 67 99 148 153 308 174 82 30 15 5 18 6 43 20 52

0

0 6 6 4 0 5 4 3 10 1 17

198 42 42 42 42 206 206 206 206 206 206 164 164 164 164 49 49 228 228 60 60 61 61 155 155 196 196 196 196 56 56 56

15 12 88 40

85 68 45 60 65 97 63 26 53 56 60 47 33 21 53 35 54 47 44 44 40 80 61 100 11 4 9 2 62

13 12 16 13 9 16 19 12 22 17 13 60 39 67 1 1 1 2 50 16 5

5–500

5–10,000

< 100 < 100

LM < 100 LM < 100

56 30 117 195 (continued)

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TABLE 13.8 (CONTINUED) Incidence of Listeria monocytogenes in Raw Meat in European and Other Countries (1990–1997) Percentage of Positive Samples Country Poland Spain

Switzerland

Taiwan Trinidad

United Arab Emirates

United Kingdom

Product Pork Beef Ground meat Ground pork Ground beef Pork, raw Ground meat Beef Pork Sausage meat Dried meat Ham (uncooked) Beef steak Pork Beef Ground beef Mutton Goat meat Pork Beef Goat Sheep Camel Pork sausage Beef Lamb Pork Sausage Meat Beef Lamb Pork

Number 245 114 168 42 41 5 85 18 31 102 44 19 25 34 76 35 66 70 71 15 17 24 14 59 26 20 32 23 15 1295 37 794

Listeria spp. 19 9 80 64 63 40 33 45 65 16 11

7 11 0 10 1 7 6 21 0 88 80 59 87 93 3 3 4

LM

Levels of Listeria spp. (CFU/g) Reference

11 7 17 29 20 0 15 15 3 8 9 0 24 59 3 6 0 4 0 0

149 149 69 165 165 211 44 44 44 44 44 44 239 239 1 1 1 1 1 103

0 0 0 49 35 40 28 35 53

103 103 103 93 162 162 162 162 115 236 236 236

Note: LM = L. monocytogenes. a

These values are for 2-g and 0.1-g samples. These values are for 1997/1998.

b

In Denmark, Norrung et al. [187] surveyed retail foods for L. monocytogenes during 1997–1998. For heat-treated meat products, 3180 and 3629 samples were tested in 1997 and 1998, respectively. The percentage of unsatisfactory samples (L. monocytogenes between 10 and 100 per g) rose from 7.6 to 9.6% and the percentage of unacceptable samples (L. monocytogenes >100 per g) increased from 0.2 to 0.5% from 1997 to 1998. In the same study 132 and 225 samples of non-heat-treated preserved meats were examined from 1997 and 1998, respectively. The percentage of unsatisfactory samples increased

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from 21 to 21.5% over the 2 years, and the percentage of samples that were unacceptable rose from 0.8 to 1.8% during the same time. In a survey of five French pork slaughtering and cutting plants, Giovanacci et al. [99] recovered 287 isolates of L. monocytogenes from various locations, including live animals, carcasses immediately after slaughter and after chilling, pork cuts (rind and meat), and the environment, including work surfaces, equipment, and various areas of the slaughterhouses. These 287 isolates were grouped into 19 genotypes by three molecular typing methods: random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and PCR-restriction enzyme analysis (PCR-REA). Contamination was present over a period of 1 year in two of the plants, even after cleaning and disinfection procedures. Chasseignaux et al. [55] used molecular typing methods to track L. monocytogenes in two poultry and pork processing plants, and found that 502 isolates of the pathogen could be separated into 50 combined genotypes by ApaI and SmaI digest profiles. Uyttendaele et al. [222] surveyed cooked meat products, raw cured meat products, and other foodstuffs to determine incidence of Listeria contamination at the retail level in Belgium. Incidence of Listeria spp. was significantly higher in raw cured meat products as compared to cooked meat products (13.7 and 4.9%, respectively). Incidence of Listeria spp. in whole cooked meats was higher after slicing than before (6.6 vs. 1.6%), and the incidence rate for cooked minced meats was higher than for whole cooked meats (6.1 vs. 4.0%). Reports from Spain showed that, of 251 samples of ground meat, 63–80% contained listeriae, with L. monocytogenes being recovered from 17 to 29% of the samples [69,165]. A survey by Soriano et al. [211] of 103 samples of raw and RTE foods from restaurants in Spain found an incidence of 2.9% for L. monocytogenes, whereas other species were found more frequently, i.e., L. grayi 13.6%, L innocua 1.9%, L. ivanovii 5.8%, L. seeligeri 3.9%, and L. welshimeri 1.9%. In countries of Eastern Europe, reports have demonstrated similar levels (8–20%) of listeriae to those of Western Europe in 613 samples of fresh beef and pork [149,158,186]. The incidence of L. monocytogenes in these samples ranged from 7 to 10%, with a high prevalence (94%) of serotypes 1 to 3, the remainder being serotype 4 [149]. Other Countries Reports from other countries worldwide indicate that the problem of Listeria-contaminated meat is widespread. In general, the prevalence of Listeria spp., and more specifically L. monocytogenes, is in the same range as that already discussed for North America and Europe. In Trinidad, Adesiyun [1] reported listeriae in 0–10% of samples of fresh beef, mutton, and goat meats, with L. monocytogenes present in 0–4% of the samples. The organisms were prevalent in ground beef samples at similar levels, 11 and 6%, respectively. L. monocytogenes serotypes 1/2c and 4 were found in both local and imported meat. A later study on slaughter pigs demonstrated 7 (5%) of 139 rectal swabs and 3 (1.9%) of 155 carcass samples to be positive for L. monocytogenes [2]. Similar incidences of listeriae have been reported in Asian countries. In the United Arab Emirates, Gohil et al. [103] reported that in fresh beef (15 samples), goat (17 samples), sheep (24 samples), and camel (14 samples), listeriae were present in 0–21% of the samples, and that no L. monocytogenes was found. In Australia, of 50 samples each of fresh beef, lamb, and pork, listeriae were found in 34, 40, and 30%, and L. monocytogenes in 24, 16, and 10%, respectively [131]. In Malaysia, L. monocytogenes was found in 50% of 12 samples of beef [30]. Fifty-four samples each of cattle, buffalo, sheep, and goat were examined by Brahmbhatt and Anjaria [42] in India, with a low incidence of L. monocytogenes (4 to 6%) being found. Ryu et al. [196] found a high incidence of listeriae in Japanese meats, with fresh beef and pork having an incidence rate of 40 and 61% for Listeria spp., and 13 and 39% for L. monocytogenes, respectively. In ground beef and pork samples, the prevalence was higher, with 80 and 100%, and 60 and 67% of the samples being positive, respectively. A study done by Wang et al. [233] in China

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showed a high incidence (from 43 to 70%) of listeriae in pork, beef, and lamb samples. However, the incidence of L. monocytogenes was much lower, from 0 to 28%. Serotypes found were 1/2a, 1/2b, and 1/2c. Meats from Taiwan, however, showed a high incidence of this organism in beef steak and pork, namely, 24 and 59%, respectively [239]. Choi et al. [56] studied the incidence of Listeria spp. in Korean foods. A total of 410 samples were collected, of which 130 were beef, pork, fermented sausage, or frozen foods (frozen pork cutlet patties and dumplings). An overall incidence of Listeria spp. of 20% was found, with 5 of the 26 isolates identified as L. monocytogenes. The individual meat products with incidences of Listeria spp. and L. monocytogenes found and total number of samples, respectively, in parentheses were as follows: beef (11/1/43), pork (4/1/20), frozen foods (9/1/52), and fermented sausage (2/2/15). Only these four food types and chicken contained L. monocytogenes, with this pathogen not found on raw vegetables or in ice cream. Rho et al. [193] looked at the microbial hazards on farms, in slaughterhouses, and in processing lines of swine in Korea. L. monocytogenes was detected in processing rooms in three provinces, with the final pork products being manufactured in the Kyonsang province. Although the aerobic plate count (APC) was low (3.0 log10 CFU/g) at the outset, the final pork products showed relatively high levels of overall contamination at the retail level (data not given).

INCIDENCE

OF

LISTERIA

SPP. IN

SAUSAGE

AND

RTE MEAT PRODUCTS

The prevalence of L. monocytogenes in processed RTE meats is of greater concern than contamination of raw meats. An examination of retail meat slicers revealed a contamination rate of 13% with L. monocytogenes; hence, such machines could be a source of cross-contamination [128]. Two factors, namely, development of proper methods to isolate Listeria spp. from samples containing a diverse microflora and a heightened concern about foodborne listeriosis, have led scientists to examine sausage, pâté, and other RTE meat products for listeriae. Consequently, numerous worldwide surveys have been made since 1990 to determine the incidence of these organisms in such products. North America Since the association of sporadic cases and outbreaks of listeriosis with consumption of uncooked frankfurters [202], there have been several nonregulatory U.S. studies on the incidence of listeriae in packages of retail wieners (Table 13.7). Wang and Muriana [232] reported that, in a survey of 20 brands of retail wieners, 19 brands (93 samples) showed a 10% incidence of listeriae, with an 8% incidence of L. monocytogenes. However, brand no. 20 contained listeriae in 83% of its 24 packages, with a 71% incidence of L. monocytogenes. They reported that listeriae were found in the liquid exudate (purge) at a level of 1–3 CFU/mL, but not in the meat itself, indicating the likelihood of postprocessing contamination. A survey commissioned by the Los Angeles Times found that, in a sample of 30 packages of retail wieners, there was a 20% incidence of listeriae with a 17% incidence of L. monocytogenes [24]. A Canadian study [216] reported that wieners (38 samples) and sliced meats (67 samples) showed an incidence of Listeria spp. of 13–26%, and of L. monocytogenes of 13–21% (Table 13.7). In contrast, Listeria spp. were not isolated from cooked meats (16 samples) and sausages (9 samples). However, fermented sausages (30 samples) studied by Farber et al. [87] showed a 20% incidence of L. monocytogenes. Europe Numerous studies have investigated the incidence of listeriae in RTE meat and sausage products produced in Europe (Table 13.9). Public Health Laboratory Service workers in England and Wales conducted surveys on a large number of samples to determine the incidence of listeriae in

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TABLE 13.9 Incidence of Listeria monocytogenes in Sausages and RTE Meat Products in European and Other Countries (1990–2003)

Country

Product

Australia

Pâté Luncheon meat Pâté Processed meat Luncheon meat VP salami VP corned beef VP ham VP luncheon meat Salami Ham Corned beef Pâté Luncheon meat Cooked meat products Whole muscles, 25 g/0.1 g Cooked ham, 25 g/0.1 g Cooked loin, 25 g Minced muscles, 25 g/0.1 g Minced muscles, 0.1g Prepacked meats, 25 g/0.1 g Prepacked pâtés, 25 g Prepacked aspic, 25 g Not prepacked pâtés, 25 g Not prepacked aspic, 25 g Not prepacked pâtés, 25 g Not prepacked aspic, 25 g Salads Meat (ham) salad, 25-g sample Sliced ham Sliced rolled sausage Sliced smoked pork loin Frankfurters Sausages, pâtés, ham Dry-cured sausage Fermented sausage Smoked sausage Pork sausage Sausage—mixed meat Ground beef rissoles Sausage Würstel

Belgium

Denmark

France Hungary

Italy

New Zealand

Pâté RTE pork

Number of Percentage of Positive Samples Samples Listeria Analyzed spp. LM 7 28 25 25 20 19 72 71 13 132 90 39 7 16 3405 2194 1069 87 1107 1107 701 217 48 130 67 130 67

0 11

Levels of Listeria spp. (CFU/g) Reference

159

20.75

204 204 118 118 217 109 109 109 109 225 225 225 225 225 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222

80 80 78 67 990 136 21 23 55 20 45 82 118

27 33 65 87 90 93 88 39

10 10 23 6 14 10 10 13 24 60 33 28 5

192 192 192 192 151 147 147 147 157 157 157 53 53

48 34

27 50

2 3

5 5 83 41 23 31 39 41 0 5

0 7 8 4 0 0 72 34 15 4 17 10 0 19 4.9 4.0/0.5 1.4/ND 3.5 6.1/0.g (RTE) 0.6 6.7/0.7 (RTE) 2.8 8.3 4.6 1.5 4.6 1.5

LM— 10–224

53 124 (continued)

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TABLE 13.9 (CONTINUED) Incidence of Listeria monocytogenes in Sausages and RTE Meat Products in European and Other Countries (1990–2003)

Country

Product RTE beef RTE lamb Norway VP processed meat South Africa Vienna sausage Ham Cervelat Spain Cured sausage Cooked ham or sausage Luncheon meat Pâté Pork, ready-to-eat Beef, ready-to-eat Switzerland Sliced cured dried beef Salami Mettwurst United Kingdom Pâté RTE meat and poultry Sliced meat—ham Sliced meat—salami Sliced meat—tongue Sliced meat—corned beef Sliced meat—mixed meats Prepared sandwiches Cooked meats Pâté Salad with meat Sandwiches with meat Cook-chill—meat and poultry Salami Pâté Pâté

Yugoslavia

Number of Percentage of Positive Samples Samples Listeria Analyzed spp. LM 18 50 0 3 67 0 35 11 47 4 0 43 14 0 44 7 0 17 18 12 15 7 0 60 42 27 36 14 14 5 0 5 0 26 15 4 59 49 5 14 93 0 696 20 17 3939 15 8 303 8 3 128 17 9 28 7 0 27 15 4 119 5 0 91 17 1551 13 5 239 11 7 15 33 13 237 20 8 736 2 67 16 216 35 1834 10

Pâté

626

4

Cook-chill food—meat

992

Cook-chill food—meat

854

Cured or smoked meat Cooked tripe Cold meats

29 44 2894

0.06

7 9 0.02

Pâté Fermented sausage Hot-smoked sausage VP hot-smoked sausage

1184 21 15 14

0.04 80 28 35

0.02 19 0 21

5

3 9

Note: LM = L. monocytogenes; RTE = ready-to-eat; VP = vacuum-packed.

Levels of Listeria spp. (CFU/g) Reference 124 124 195 229 229 229 34 34 1–100 150 1100 150 211 211 LM—20 218 LM—20 218 218 173 173 226 226 226 226 226 129 <20–1000 16 <20–100 16 <20–100 16 20–100 16 93 93 93 94 LM— <200–106 LM— 94 <200–105 LM— 122 <10–103 LM— 122 <10–103 14 130 78 LM— 104–105 78 46 46 46

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various foods. McLauchlin and Gilbert’s report [173] on a study of pâté (696 samples) and RTE meat and poultry (3939 samples) showed listeriae present in 20 and 15%, and L. monocytogenes in 17 and 8% of the samples, respectively. A report on 605 samples of sliced meats showed a low incidence of 6% for listeriae and 3% for L. monocytogenes, with levels of <200 to <500 CFU/g [177]. Most (91%) of the L. monocytogenes isolates were serotype 1/2, with the remainder being serotype 4. An extensive study done in Yorkshire [16] showed that cooked meats (1551 samples), pâté (239 samples), meat salad (15 samples), and meat sandwiches (237 samples) had incidences for listeriae of 13, 11, 33, and 20%, and for L. monocytogenes of 5, 7, 13, and 8%, respectively. Levels of the organism in this study were 20–1000 CFU/g. Another report on 91 samples of preprepared sandwiches showed a 17% incidence of L. monocytogenes [129]. Of 13 strains typed, 10 were serotype 1/2, and 3 were serotype 4. A study of pâté, with 1834 and 626 samples in 1989 and 1990, showed incidences of L. monocytogenes of 10 and 4%, with levels of the organism up to 106 CFU/g [94]. Serotyping revealed that both serotypes 1/2 and 4 were present. L. monocytogenes was also present in 16% of 67 salami samples [93]. Cooked tripe, which is often eaten without further heating, showed a 9% incidence of L. monocytogenes [130]. In cook-chill catering, often employed in hospitals, food is prepared and cooked in a traditional manner, cooled very rapidly, and maintained chilled (0–3°C) for up to 5 days until reheated for use. In three surveys of cook-chill food containing meat (736, 992, and 854 samples), the incidence of L. monocytogenes was 2, 3, and 9%, respectively [93,122]. Of 28 strains typed, 7 were serotype 1/2, 17 were serotype 3, and 4 were serotype 4 [122]. These results must cause concern, in light of the vulnerability of a hospital population. Gillespie et al. [98] found that of 3494 samples of cold, RTE sliced meats collected from 2579 catering establishments in the United Kingdom, Listeria spp. and L. monocytogenes were present in 13 (0.3%) and 5 samples (0.1%), respectively. In Denmark, 305 samples of wieners and sliced meats showed similar incidences, with 10% of the samples containing L. monocytogenes at a level <100 CFU/g [192]. A study in Norway of vacuumpackaged processed meat showed 11% of 35 samples to be contaminated with L. monocytogenes [195]. In France, a sampling of 18 dry sausages showed that 22% contained L. monocytogenes, whereas in Germany, 9% of 11 mettwurst samples contained the organism [132]. Several studies on sausages in Italy showed similar incidences to those just mentioned. Levre et al. [157], reporting on 120 samples of pork sausage/mixed chicken, turkey, and pork sausage/beef rissoles, found that, overall, as high as 90% of these samples contained listeriae, with 33% positive for L. monocytogenes. Casolari et al. [116], in a study carried out over a 2-year period (1990–1991), demonstrated similar incidences in sausage, with 88 and 28% of 82 samples positive for Listeria and L. monocytogenes, respectively. In 166 samples of meat products (würstel, pâté), overall, 36% contained listeriae, with L. monocytogenes present in 4% of the samples at a level of 10–224 CFU/g. A survey in Switzerland taken during production of cured and air-dried meat products [218] uncovered a substantial number of bündnerfleisch (cured, air-dried beef), salami, and mettwurst that contained listeriae. Overall, of samples taken during production, bündnerfleisch (19 samples), salami (30 samples), and mettwurst (3 samples) had an incidence of 26, 50, and 100% listeriae, and 11, 10, and 0% for L. monocytogenes, respectively (data not shown). The end products showed listeriae at incidences ranging from 15 to 93%, in contrast to incidences ranging from 0 to 5% for L. monocytogenes. The organism was present at levels of <20 CFU/g. Listeriae were isolated only from the surface of bündnerfleisch. L. monocytogenes isolates were of serotype 1/2 (86%), and serotype 4 (14%). Because most L. monocytogenes isolates from human listeriosis patients in Switzerland are of serotype 4b, these data suggest that transmission of the pathogen via meat is relatively uncommon. Recent surveys of RTE meat products in Spain showed that cured or cooked sausages and ham (a total of 32 samples) had a 13% incidence of Listeria spp. [34]. Twelve percent of the cured sausages contained L. monocytogenes, whereas the organism was not isolated from ham or cooked sausage samples [34]. Sliced meat (60 samples) and pâté (36 samples) contained 42 and 14% of Listeria spp., and 22 and 3% of L. monocytogenes, respectively [150]. The organisms were found at a level of 1–100 CFU/g.

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In 1991, a Hungarian researcher, Kovácsné Domjan [147], reportedly recovered listeriae from 37 of 136 (27%), 7 of 21 (33%), and 15 of 23 (65%) samples of dry-cured, fermented, and smoked pork sausage, respectively. L. monocytogenes was present in 10, 10, and 13% of the samples, respectively. Buni [47] in Yugoslavia found that 21 samples of fermented sausage showed a 28 and 19% incidence of listeriae and L. monocytogenes, respectively. In contrast, listeriae were not recovered from hot, smoked sausage. Of 14 surface samples of vacuum-packaged hot-smoked sausage, 35 and 21% contained listeriae and L. monocytogenes, respectively, indicating recontamination before and during vacuum packaging (VP). Overall, the results in Table 13.9 show that a substantial portion of European processed meats are contaminated with listeriae, including L. monocytogenes. The presence of listeriae other than L. monocytogenes in processed as well as raw meats may be indicative of possible contamination with L. monocytogenes. Other Countries A recent report by Vorster et al. [229] in South Africa showed a low prevalence of listeriae in 134 retail samples of processed, RTE meats, with an overall incidence of 8% in Vienna sausage, ham, and cervelat (Table 13.9). L. monocytogenes was not found in any of the samples. A number of reports from Australian workers show, in general, the same incidence of listeriae in processed meats as in North America and Europe. Robertson and co-workers [204, 217] reported that 1 of 20 (5%) and 3 of 28 (11%) samples of sliced meats contained Listeria spp., with L. monocytogenes recovered at rates of 0 and 7%, respectively. No Listeria spp. were recovered from 7 samples of pâté. Hobson et al. [118] reported that in 25 samples each of pâté and processed meat, L. monocytogenes was present in 8 and 4% of the samples, respectively. In a study on 175 samples of vacuum-packaged processed meats, Grau and Vanderlinde [110] reported that 60 of 72 (88%) samples of corned beef, 29 of 71 (41%) samples of ham, 3 of 13 (23%) samples of luncheon meats, and 1 of 19 (5%) samples of salami were contaminated with listeriae, with 72, 34, 15, and 0% of these samples contaminated with L. monocytogenes, respectively. Sixteen of the corned beef samples had listeriae counts >50 CFU/g, and, of these, six counts were >1000 CFU/g. In salami, counts of listeriae were <50 CFU/g, and no L. monocytogenes was found. In an extensive study of 284 samples of RTE meat products, Varabioff [225] found that except for pâté, in which no listeriae were found, the incidence of listeriae ranged from 5 to 41%, with L. monocytogenes ranging from 4 to 19%. Of the isolates recovered, 62% were serotype 1 and 36% were serotype 4. Further investigation of the occurrence of listeriae on cutting utensils and cutting boards showed that most of the contamination occurred at the retail level. A survey of 55 samples of RTE meat products in New Zealand [124] showed incidences of contamination with listeriae ranging from 23 to 67%. Interestingly, mixed-source products had a lower incidence (23%) of listeriae than products made from a single meat (50–67%), whereas the organism was seldom found in single-meat products. The authors also found that significantly more smoked products were contaminated with L. monocytogenes than meats preserved by other methods. Additional information concerning habitats, niches, and relative incidence of listeriae in all facets of the meat industry is needed, as it is now evident that the presence of L. monocytogenes in processed meat products can pose a serious health hazard to certain individuals. Therefore, it is necessary to control all listeriae in meat-processing facilities and to design procedures and treatments that will eliminate L. monocytogenes from RTE processed meat products (see Chapter 17).

BEHAVIOR OF L. MONOCYTOGENES IN MEAT PRODUCTS Although L. monocytogenes was reported during the 1950s in European meat products destined for human consumption, a general failure to positively link cases of human listeriosis to foods other than raw milk provided little incentive to examine the behavior of listeriae in meat. This situation

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was further complicated by a lack of reliable and convenient methods to selectively isolate listeriae from heavily contaminated samples of animal origin, including organ as well as muscle tissue. Thus, the first definitive studies on the behavior of L. monocytogenes in raw meat were not begun until the 1970s. Even then, to accurately quantitate this organism in meat products during extended storage, it was generally deemed necessary to use specially treated “sterile” meat rather than raw meat products containing an expected (i.e., “normal”) microbial background flora. Outbreaks of foodborne listeriosis linked to the consumption of contaminated cheese prompted concerns about the microbiological safety of raw and, particularly, RTE meat products marketed in North America and Europe. The outbreaks also demonstrated an urgent need for methods to rapidly and accurately detect listeriae in a wide range of foods. Subsequent development of the USDA procedure to detect listeriae in meat and poultry products provided researchers with a method to determine the growth and survival of L. monocytogenes in raw and RTE products. Recent meatoriented epidemiological studies along with large listeriosis outbreaks linked to meat products suggest that the behavior and control of L. monocytogenes in meat products are likely to remain an active area of research for some time to come. As in the previous section of this chapter, information concerning the behavior of L. monocytogenes in raw meat will be presented first, followed by a discussion of the fate of this pathogen in processed meat products.

LISTERIOSIS

IN

DOMESTIC LIVESTOCK

Domestic farm animals such as cows, sheep, and pigs can succumb to listerial infections, as well as asymptomatically shed L. monocytogenes in their feces for many months (see Chapter 2). Although virtually all meat from animals exhibiting obvious signs of listeriosis will be condemned and destroyed immediately after slaughter, meat from subclinically infected animals will likely be passed by inspectors as being fit for consumption. Because between 2 and 16% of all healthy cows, sheep, and pigs passively shed L. monocytogenes in their feces, there is ample opportunity for contamination of muscle tissue during slaughter, evisceration, and dressing of the animals. It is likely that meat from domestic livestock will first be exposed to listeriae in the slaughterhouse environment. However, various organs from apparently healthy animals can also occasionally contain L. monocytogenes. In 1972, Höhne [119] reported that approximately 13% of the parotid glands from clinically healthy pigs contained L. monocytogenes. Three years later, Höhne et al. [120] detected L. monocytogenes in intestinal lymph nodes from eight apparently healthy slaughtered animals (five small ruminants, two pigs, and one cow) destined for human consumption. Subsequently, Cottin et al. [62] identified L. monocytogenes in 15 of 514 (3.1%) spleen and lung tissue samples obtained from apparently healthy cattle. According to Amtsberg et al. [6], L. monocytogenes was isolated from the spleen and muscle tissue of an apparently healthy animal. These findings suggest that L. monocytogenes might be transported to tissue via the bloodstream in animals suffering from symptomatic as well as asymptomatic septicemic listerial infections. Hence, to understand the behavior of L. monocytogenes in meat products, it is fitting to begin this section by first examining the localization of L. monocytogenes in organ tissue, and particularly muscle tissue, from infected animals.

LOCALIZATION

IN

TISSUES

In 1988, Johnson et al. [135] reported results from a study of samples of muscle, organ, and lymphoid tissue, as well as feces and blood were obtained from several Holstein cows previously inoculated intravenously with 1010–1011 L. monocytogenes. Samples were examined for the pathogen 2, 6, and 54 days after inoculation, using a combination of direct plating and cold enrichment. As expected, recovery of listeriae varied among animals and was strongly influenced by the time that elapsed between inoculation and slaughter. Overall, 94% of all samples obtained from cows slaughtered 2 days after inoculation contained L. monocytogenes, with 23 of 32 (72%) samples positive by direct plating. More important, the pathogen was routinely detected in muscle

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tissue from the same animals, frequently at levels of 120–280 CFU/g. Despite a marked decrease in recovery of L. monocytogenes from animals examined 6 and 54 days postinoculation, the pathogen was still present at levels of ~140–675 CFU/g in kidney tissue as well as in mesenteric and mammary lymph nodes, with populations about 2 orders of magnitude lower in liver and spleen tissue. Following 2 weeks of cold enrichment, the pathogen also was detected in plate-flank tissue taken from an animal 6 days after slaughter. These findings suggest that consumption of animal organs may constitute a greater health hazard than consumption of muscle tissue, and also readily explains how evisceration of domestic animals can lead to surface contamination of muscle tissue. Assuming that most contamination occurs during handling of carcasses after slaughter, Chung et al. [57] investigated the ability of L. monocytogenes to attach to (or become entrapped) and proliferate on muscle and fat tissue both alone and in the presence of Pseudomonas aeruginosa. Immersion of lean muscle and fat tissue in broth cultures containing 105 or 108 L. monocytogenes CFU/mL for various times followed by thorough rinsing resulted in large numbers of listeriae being attached to (or entrapped in) both types of samples within the first 10 min. Despite large differences in hydrophobicity, pliability, and surface qualities, L. monocytogenes adhered (entrapped) equally well to lean muscle and fat tissue during 50–60 min of incubation at ambient temperature. According to an earlier report by Herald and Zottola [116], attachment of L. monocytogenes to stainless steel and presumably meat surfaces is related to flagellae, fibrils, and exopolymeric substances (i.e., polysaccharides), all of which are readily produced by Listeria during extended incubation at room temperature. However, lean muscle tissue supported faster growth of the pathogen than fat tissue when samples were stored 1 day at ambient temperature, followed by 7 days at refrigeration temperature. Following attachment of L. monocytogenes and P. aeruginosa to lean meat at a concentration of ~106 CFU/4 cm2, Listeria populations increased approximately 100-fold during 24 h of incubation at room temperature and remained at this level during 7 days of refrigerated storage, regardless of the presence or absence of P. aeruginosa. In contrast, populations of P. aeruginosa on lean meat increased >100-fold during initial storage at room temperature, but then decreased, with levels frequently 10 times lower than the Listeria population following 7 days of refrigerated storage. Dickson [71] simulated contamination of raw beef during processing, handling, and storage by placing surfaces of heavily inoculated lean and fat beef tissue (~2 × 106 L. monocytogenes CFU/cm2) in direct contact with uninoculated tissue. Overall, transfer of listeriae was largely dependent on the type of tissue, with minimum and maximum transfer observed from fat-to-fat and lean-to-fat tissue, respectively. However, bacterial transfer was also influenced by adsorption time of the original inoculum and contact time with uninoculated tissue. Adsorption times of <60 min generally led to the highest Listeria transfer rates, particularly between inoculated and uninoculated lean beef tissue. These findings indicate that most listeriae are likely to be in suspension in water films on tissue surfaces shortly after inoculation. Following an adsorption time of 30 min, approximately 30 and 50% of the original Listeria inoculum migrated from lean and fat tissue, respectively, to lean tissue after 5 min of direct contact at ambient temperature. When contamination between fat and lean tissue was simulated using shorter contact times of 15–60 sec, a greater percentage of listeriae migrated from inoculated fat to uninoculated lean tissue, reflecting transfer of cells in unadsorbed water from hydrophobic fat to hydrophilic lean tissue. More important, the fact that bacterial transfer also occurred at 5°C, with 0.6–9.5% of the original Listeria population migrating from inoculated to uninoculated lean and/or fat tissue after an 18-h adsorption period, provides a reasonable explanation for spread of this pathogen to Listeria-free meat during storage in walk-in coolers. These findings attest to the hardy nature of L. monocytogenes on the surface of raw meats and to the need for effective means of reducing surface contamination on carcasses. Regarding the latter, Chung et al. [58] reported that wash solutions containing nisin effectively delayed growth of L. monocytogenes on surfaces of raw meats, particularly when such products were incubated at refrigeration, rather than ambient temperatures. Although nisin-producing bacterial starter cultures have been used in the dairy industry for many years, with the exception of certain types of cheese spread, present laws in North America still prevent direct addition of nisin to most foods, including raw meat.

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Warriner et al. [234] examined attachment of various bacteria to beef from steam-pasteurized carcasses. There was no significant difference in attachment of bacteria to steam-pasteurized carcasses versus loin cuts that were not given any treatment. Furthermore, there did not appear to be any significant difference in attachment strength between the different bacteria tested (Pseudomonas fragi NCTC 10689, L. monocytogenes BL5/2, Salmonella typhimurium LT2, and Escherichia coli JM109). Populations of enteric pathogens (i.e., Salmonella spp., enteropathogenic E. coli, and Yersinia spp.) on raw meats can be sharply reduced by exposing the water phase of meat surfaces to 0.2 M lactic acid (pH 2.5) at 21°C [135]. Although L. monocytogenes is generally recognized as being more acid tolerant, it can be inactivated at pH values less than 4.0. Hence, provided that L. monocytogenes is exposed to lactic acid for sufficient time, acid washes may be somewhat helpful in decreasing Listeria populations on the surface of animal carcasses. As noted by Johnson et al. [135], L. monocytogenes was routinely detected in muscle tissue from cows that were killed 2 days after being inoculated intravenously with the pathogen. Although some contamination of muscle tissue might have occurred during sampling, results from this study suggest that L. monocytogenes can enter muscle tissue via the bloodstream. To further investigate this hypothesis, Johnson et al. [133] examined muscle, liver, and spleen tissue from two lambs and one calf that had been inoculated intravenously with L. monocytogenes. Microscopic examination of tissues stained with immunoperoxidase or Azure A revealed L. monocytogenes cells at levels of 103–104 CFU/g in muscle tissue and 103–106 CFU/g in both liver and spleen tissue. Although L. monocytogenes appeared to be associated with phagocytes in liver and spleen tissue, the pathogen was observed in loose connective tissue between muscle fibers and also within the muscle fibers themselves. Using the USDA enrichment method, Johnson et al. [137] subsequently detected L. monocytogenes serotype 1/2a at 10 CFU/g in aseptically removed interior samples from 2 of 50 (4%) and 3 of 50 (6%) retail whole-muscle beef and pork roasts, respectively. One beef sample also was positive for L. innocua and L. welshimeri. Although the presence of these two nonpathogenic (i.e., noninvasive) Listeria spp. within a whole-muscle roast suggests possible contamination during sampling, other results from Johnson et al. [135–137] strongly support at least limited transmission of L. monocytogenes from the bloodstream into muscle tissue. Niebuhr and Dickson [185] studied the impact of pH enhancement, i.e., raising the pH to ~9.6, on survival of L. monocytogenes on boneless beef trimmings. Populations were reduced by 3 log10 cycles after exposure to ammonia gas, which was used to raise the pH [176].

RAW BEEF Growth and Survival Interest in behavior of Listeria in raw meat products dates back to at least 1966 when Sielaff [208] inoculated beef, pork, and rabbit with L. monocytogenes immediately after slaughter and examined these samples for listeriae during extended storage at 3–4°C. Under these conditions, the pathogen survived at least 15 days in all three products. Although this study was among the first to examine the ability of L. monocytogenes to survive in raw beef, information concerning actual growth of this pathogen in raw beef was not available until 1978. In that year, Gouet et al. [105] reported results from a study that examined multiplication of L. monocytogenes in “sterile” minced beef alone and in combination with a defined microflora. L. monocytogenes failed to grow alone in minced beef (pH 5.8) stored at 8°C, and populations decreased <10-fold during 17 days of incubation. In contrast, numbers of listeriae decreased approximately 100-fold in samples of minced beef (pH 5.8) that were simultaneously inoculated with Lactobacillus plantarum and held at 8°C for 17 days. These researchers also found that higher concentrations of L. monocytogenes (106 CFU/g) enhanced growth of L. plantarum. When samples

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of minced beef were simultaneously inoculated to contain equal numbers of L. monocytogenes, P. fluorescens, and E. coli, Listeria populations decreased approximately 10-fold after 24 h of incubation at 8°C, with numbers rapidly increasing after day 7. Rapid growth of listeriae during the latter half of incubation was likely prompted by proteolysis of meat proteins by P. fluorescens, which, in turn, led to a gradual increase in pH of the meat from 5.8 to 6.8. With a complex microflora consisting of ~103 CFU/g each of L. plantarum, P. fluorescens, E. coli, Micrococcus sp., Clostridium perfringens, and Streptococcus faecalis, behavior of L. monocytogenes was similar to that previously observed for the pathogen in the presence of P. fluorescens and E. coli. Listeriae populations reached approximately 6 × 105 CFU/g in minced beef following 17 days at 8°C. A rapid increase in numbers of P. fluorescens before growth of L. plantarum again appeared instrumental in raising the pH from 5.8 to 7.2, which, in turn, stimulated growth of listeriae. In a similar investigation completed 11 years later, Kaya and Schmidt [143] also found that growth of L. monocytogenes in artificially contaminated sterile minced meat during extended incubation at 8–20°C was suppressed by the addition of 106 Lactobacillus CFU/g, but was unhindered in the presence of 106 Pseudomonas sp. CFU/g. However, in contrast to the previous study by Gouet et al. [105], this strain of L. monocytogenes readily grew in the absence of other microorganisms, with populations increasing approximately 2 and 4 orders of magnitude in sterile minced meat after 10 and 1–5 days of storage at 4 and 8–20°C, respectively. In this particular instance, proteolysis of meat proteins by pseudomonads apparently was not a prerequisite for growth of L. monocytogenes in sterile minced meat with an initial pH value of 5.8–6.0. During 1988 and 1989, three additional studies were done to determine the behavior of listeriae in ground beef. However, unlike previous investigations, the meat was not pretreated to eliminate the normal background flora. When ground beef at pH 5.6–5.9 was inoculated to contain 105 and 106 CFU/g of L. monocytogenes Scott A or V7, packaged in oxygen-permeable or oxygen-impermeable film, and held at 4°C, Johnson et al. [113] found that the numbers of organisms as well as the pH remained relatively constant during 14 days of incubation, regardless of the film’s degree of permeability. In contrast to the previous study by Gouet et al. [105], in which the pH of minced beef containing a complex microflora increased from 5.8 to 7.2 during extended incubation at 8°C, the pH of all packaged ground beef samples in this study remained in the range of pH 5.6–5.9 throughout storage. Thus, the lower pH of the meat in this study, along with a lower incubation temperature (4 vs. 8°C) are likely to have been at least partly responsible for inhibiting growth of listeriae in packaged samples of “retail-like” ground beef. In keeping with these findings, Shelef [205] also reported that L. monocytogenes failed to grow in artificially contaminated ground beef or ground liver during 1 and 40 days of incubation at 25 and 4°C, respectively. However, in contrast to results of the previous study by Johnson et al. [134], the pathogen failed to grow in ground beef despite a final pH value of 7.8. Although the reasons for this behavior remain obscure, inability of L. monocytogenes to multiply in ground beef under certain conditions is likely related to the type and load of inherent microflora in the product. This hypothesis regarding the microflora in foods is supported by a West German study in which Kaya and Schmidt [143] showed that an increase in the natural bacterial flora of ground beef from 105 to 107 CFU/g led to increased inhibition of L. monocytogenes in artificially contaminated meat. When ground beef was inoculated to contain ~105 L. monocytogenes CFU/g, the pathogen grew after 1–5 days at 7–20°C in samples harboring ~105 non-Listeria contaminants per g but failed to multiply in corresponding samples containing higher levels of naturally occurring organisms. Although a similar growth pattern was observed for samples naturally contaminated with 102 Listeria per g and 105 non-Listeria per g, growth was markedly hindered in naturally rather than artificially contaminated samples, with Listeria populations remaining constant in the former during 14 days at 8°C. However, behavioral differences were no longer observed at lower temperatures, with the organism failing to grow during 14 days of incubation at 4°C in both artificially and naturally contaminated ground beef containing similar background populations.

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Work by Barbosa et al. [33] with vacuum-packaged ground beef indicates that the organism grows better in beef with a pH >6.0. The study also showed that in addition to pH, strain type can affect the fate of the organism in ground beef. The authors used four different strains of L. monocytogenes for inoculation and observed slightly different responses with each. For example, in normal pH (5.47) ground beef L. monocytogenes serotypes 3a and 3b increased 2.3 and 1.8 logs, respectively, after 35 days of storage at 4°C, whereas after 56 days the levels of strain 1/2a remained constant. Interestingly, the Scott A strain (serotype 4b) decreased by 1 log in numbers. In general, the organism multiplied slowly on all vacuum-packaged samples, but growth was better on ground beef of pH 6.14 than of pH 5.47. For example, after 28 days of storage at 4°C, serotypes 3b, 3a, and 1/2a had increased by 2.87, 2.64, and 2.24 logs, respectively, in high-pH ground beef, whereas the Scott A strain did not change significantly in numbers [33]. The authors felt that the antimicrobial effect of low pH may also be an indirect one, altering the natural microbial flora while promoting growth of bacteriocin-producing lactic acid bacteria. L. monocytogenes appears to behave in a similar manner on surfaces of fresh intact beef muscle. In two studies conducted shortly before L. monocytogenes emerged as a serious foodborne pathogen, Lee et al. [153,154] dealt indirectly with the incidence and subsequent behavior of Listeria spp. along with many other psychrotrophic as well as mesophilic organisms present on the surface of hot-boned and conventionally boned beef. In their study, hot-boned beef was obtained from five steers 2 h after slaughter, and then was vacuum-packaged and cooled from 32 to 21°C. Conventionally processed beef was obtained from carcasses that were hung in a cold room at 2°C for 2 days after slaughter. Both types of beef were then examined for mesophilic and psychrotrophic organisms at day 0 when the surface temperature had decreased to 21°C (<1 h for conventionally processed beef) and again following 14 days of storage at 2°C. Nearly 1250 bacterial isolates were subsequently identified by computer analysis of 116 miniaturized tests/isolate. Although Listeria spp. were never isolated from slow or moderately chilled, hot-boned beef at day 0, 9.1 to 13.5% of all microorganisms present on the surface of such beef after 14 days were identified as Listeria spp., some isolates of which were likely L. monocytogenes. Overall, only one isolate from conventionally processed beef was identified as belonging to the genus Listeria. From these data one can infer that Listeria spp. can grow on the surface of vacuum-packaged hot-boned beef, but not on the surface of unpackaged conventionally processed beef during 2 weeks of storage at 2°C. Because the high water-binding properties of hot-boned beef make this product particularly well suited for sausage making, widespread use of hot-boned beef by the processed-meat industry may be partially related to the relatively high incidence of listeriae in RTE meat products. In a more definitive Australian study reported in 1988, Grau and Vanderlinde [108] examined the ability of L. monocytogenes to grow on the surface of artificially contaminated (102–103 CFU/cm2) vacuum-packaged, nonsterile beef striploin during extended incubation at 0 and 5.3°C. Although L. monocytogenes populations increased in all samples (and in the purge that developed in packages) during storage, the extent of Listeria growth was markedly influenced by incubation temperature, pH of the sample (5.6 vs. 6.0), and type of tissue (lean vs. fat). Overall, higher Listeria populations consistently developed on fat tissue, with growth also being more rapid at the higher of the two incubation temperatures and pH values. Numbers of listeriae on fat tissue of pH 5.6 increased from 5 × 103 to 3 × 107 CFU/cm2 during 16 days of incubation, whereas the pathogen was just beginning to grow on corresponding samples after 7 to 14 days of storage at 0°C. These researchers also noted that Listeria populations increased <10- and ~1000-fold on vacuum-packaged meats of pH 5.6 and 6.0, respectively, after 10 to 11 weeks of storage at 0°C. Thus, it appears that two conditions, (a) a storage temperature of 0°C and (b) a product pH value of 5.6, must be met simultaneously to prevent significant growth of L. monocytogenes in vacuum-packaged raw meats. Grau and Vanderlinde [110] extended their studies by using two models, the modified Arrhenius and square-root models, to examine aerobic growth of L. monocytogenes on lean and fatty raw beef tissue. For both lean and fatty tissue, the modified Arrhenius model gave better fits and estimates of growth rates. The effect of temperatures between 0 and 30°C on the growth rate of the organism

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could be described by a modified Arrhenius equation: Ln (gen/h) = −205.73 +1.2939 × 105/K − 2.0298 × 107/K2, where K = Kelvin. The combined effect of temperature and pH on the growth rate of the organism on lean beef was best described by the following equation: Ln (gen/h) = −232.64 + 1.4041 × 105/K − 2.1908 × 107/K2 + 1.1586 × 102/pH − 4.0952 × 102/pH2. For lean meat at pH values of about 5.5–5.6 and 6.0–7.0, the latter equation applied between approximately 2.5 and 35°C, and 0 and 35°C, respectively. There was considerable scatter in the measured lag periods for both types of meat, and therefore, a poor fit was observed for both models. In two trials, both models predicted growth on lean tissue where no growth was observed experimentally. In the first, L. monocytogenes failed to grow on lean tissue with a mean pH of 5.61 during 13 weeks of storage at 0°C, whereas in the other, no growth was observed after 48 h of incubation at 43.2°C, pH 5.46. However, growth of the organism was observed on lean tissue having a mean pH of 5.61 at 2.5°C, on lean meat of pH values 6.0, and all fatty tissue (pH 5.5–5.7), regardless of storage temperature. One should be aware, however, that in this study the investigators used only one strain of L. monocytogenes (which was not a meat strain), and that meats in which the contaminating flora exceeded 10% of the Listeria count were discarded, thus partially eliminating the effects of background microflora on growth of the organism. Growth rates predicted by the best equations for lean tissue were compared with the literature data for aerobic growth of L. monocytogenes on several foods. Growth rates predicted for lean meat were higher than those found for corn, clarified cabbage juice, and milk, whereas foods supporting similar growth rates to the lean beef included UHT milk, raw and cooked chicken, and cooked ground meat [37,110]. Other researchers have found that some of the models derived from growth of L. monocytogenes in broth cannot reliably predict growth of the organism in raw pork [97]. For example, L. monocytogenes grew on pork fat tissue without lag at −0.3°C and higher temperatures at rates that were greater than those at lower temperatures in unacidified tryptic soy broth (TSB). The better growth of the organism on fat tissue as compared to TSB suggests that fat tissue may contain micronutrients that are not present in TSB [97]. In addition, the organism grew on pork muscle tissue only at temperatures of ≥15.4°C at a slower rate than in acidified (pH 5.5) TSB, implying that pork muscle tissue may contain growth inhibitors that are either absent or present in very low levels both in TSB and fat tissue. All published models done to date in broth predict that the organism will grow at pH 5.5 at 5°C, in contrast to the failure of L. monocytogenes to grow on normal pH raw pork muscle even at temperatures as high as 15°C. There has only been one published study showing that the organism can grow on raw meat (beef muscle) of normal pH at low temperatures [110].

RAW LAMB

AND

PORK

Most investigations have focused on behavior of Listeria in raw beef; however, several reports exist on the fate of this pathogen in raw lamb and pork. As early as 1973, Khan et al. [144] published results from a study in which aseptically obtained “sterile” raw lamb meat was inoculated to contain ~105 CFU/g of L. monocytogenes, packaged in gas-permeable or gas-impermeable film, and examined for survival of listeriae during extended storage at 0 and 8°C. Although Listeria populations remained relatively constant in lamb meat packaged in gas-permeable film during 20 days at 0°C, numbers of listeriae decreased approximately 10-fold in corresponding samples that were packaged in a gas-impermeable film. These results are similar to those obtained by Johnson et al. [134], who concluded that L. monocytogenes also was unable to grow in refrigerated ground beef that was packaged in gas-permeable or gas-impermeable film. Following 12 days of storage at 8 rather than 0°C, populations of listeriae in lamb increased >1000-fold; however, unlike meat packaged in gas-permeable film, the pathogen exhibited a 2-day lag period and grew markedly slower in gas-impermeable packages. Various physical differences of the meat, combined with an increased concentration of CO2 (and presumably a lower pH) in gas-impermeable packages, may have been responsible for partially inactivating the pathogen at 0°C and delaying the onset of growth at 8°C.

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Two years later, Khan et al. [145] examined the behavior of L. monocytogenes in preparations of sarcoplasmic pork, beef, and lamb protein inoculated to contain ~104 CFU/mL of L. monocytogenes and held at 4°C. According to these investigators, the pathogen grew readily in preparations of pork and beef protein, reaching levels of approximately 105 and 107 CFU/mL, respectively, after 12 days of refrigerated storage. Although Listeria populations remained relatively constant in corresponding preparations of lamb protein held at 4°C, the pathogen grew readily in lamb meat stored at 8°C. Hence, it appears that raw pork, beef, and lamb can support rapid growth of listeriae, particularly when these products have undergone temperature abuse, as frequently occurs at the retail level. These early observations were confirmed by Lovett et al. [159], who found that L. monocytogenes reached levels of at least 108 CFU/g in inoculated samples of retail lamb, pork, and beef after 14 days at 7°C. L. monocytogenes exhibited both a 2-day lag period and a slower rate of growth in beef and pork than in lamb. Such variations in growth rate might be related to differences in concentrations of various amino acids (particularly lysine, serine, and valine, reportedly essential for growth of L. monocytogenes), as first suggested by Khan et al. [144] in 1973. A survey of microbiological contamination of retail pork products by Duffy et al. [76] revealed that L. monocytogenes was detected in 26.7 and 19.8% of plant and retail samples, respectively, with contamination was found more often in ground products. In comparing methodologies for detecting Listeria spp. and L. monocytogenes in pork, Kanuganti et al. [141] compared two methods, i.e., an enrichment and subculturing method (method I, UVMII) versus an enrichment followed by plating on PALCAM agar (method II, PALCAM). Results showed that PALCAM was slightly more sensitive for detecting L. monocytogenes in hog intestine (chitterlings) samples (8.3% in method I vs. 9.3% for method II). In a sampling of 1849 swine tissues, 16 and 44 samples were found to be contaminated with L. monocytogenes using methods I and II, respectively. The researchers also used these methods for detecting L. monocytogenes in ground pork and from small intestines (raw chitterlings, 300 samples) from three plants and one retail grocery store. Out of 340 samples of ground pork, 152 (45%) and 171 (50.2%) yielded L. monocytogenes, using methods I and II, respectively. Interestingly, although one plant had a high rate of contamination (73 and 80% contamination according to the respective methods), the highest rate was at the retail level, i.e., 85 and 92%, for methods I and II. Raw chitterlings were not sampled from retail establishments.

COOKED

AND

RTE MEATS

Cured Ham Investigators in Europe and the United States determined behavior of L. monocytogenes in ham during processing and extended storage. Working in The Netherlands, Stegeman et al. [212] examined the thermal resistance of listeriae in experimentally produced hams to which 2 or 3% NaCl and 120 or 180 ppm sodium nitrite were added during manufacture. After inoculating the product with ~104 CFU/g of L. monocytogenes, all hams were canned, heated to an internal temperature of 68.9–71.0 (or 64.0°C) within 5 h to simulate normal and underprocessing conditions, respectively, cooled, and sampled after 5 days of storage at 4°C. Three different enrichment procedures failed to recover viable listeriae from any samples. Results from Stegeman et al. [212] appear to indicate that standard thermal treatments are more than sufficient for producing Listeria-free ham. However, once removed from protective packaging, all cooked/RTE meats can become contaminated with listeriae during slicing and further handling. To simulate postprocessing contamination, Glass and Doyle [100] inoculated the surface of commercially produced ham slices as well as five other meat products (further discussion follows) to contain approximately 0.2 or 500 CFU/g of L. monocytogenes. All samples were then vacuumpackaged and periodically examined for numbers of listeriae during prolonged incubation at 4.4°C. Regardless of the original inoculum, L. monocytogenes attained populations of 105–106 CFU/g on

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organoleptically acceptable ham (pH 6.3–6.5) after 4 weeks of refrigerated storage, indicating that manufacturers cannot rely on the combination of VP and refrigeration for control of listeriae on ham. Samelis and Metaxopoulos [198] examined the incidence and main sources of Listeria spp. and L. monocytogenes in processed meats and a meat processing plant. The results showed that many of the tumblers were heavily contaminated, because the need to operate continuously prevented proper cleaning and disinfection. Listeriae were found to survive in tumbled meats that were cooked in boilers, where the core temperature was below 70°C. Cooked Roast Beef Glass and Doyle [100] evaluated the potential for growth of L. monocytogenes on the surface of vacuum-packaged samples of cooked roast beef having initial pH values of ∼5.9. Unlike sliced ham, cooked roast beef supported far less growth of listeriae, with populations increasing 2 orders of magnitude on organoleptically acceptable product after 4 weeks of refrigerated storage. Slower growth of the pathogen on precooked roast beef correlated well with a decrease in product pH to values of ≤5.15 in 4-week-old samples. Luncheon Meats Because previous studies found that luncheon meat, cooked ham, and cooked turkey breast meat were the most frequently contaminated cooked meat products in The Netherlands, these products were used in a study to determine survival and growth of L. monocytogenes [38]. Products were inoculated with low levels (10 CFU/g) of the organism and stored under vacuum or in an atmosphere of 30% CO2/70% N2 at 7°C for 4–6 weeks. Growth on vacuum-packaged product was similar to that on modifiedatmosphere-packaged stored meats, with counts increasing up to 108 CFU/g after 35 days. High numbers of lactic acid bacteria were present, but did not affect growth of the pathogen. However, Listeria decreased in number on saveloy (fermented sausage, pH 5.5–5.7) and raw Coburger ham (pH 4.3–4.5), most likely from the acidity of these products. Grau and Vandelinde [109] examined growth of L. monocytogenes on both naturally contaminated and artificially inoculated processed corned beef and ham. For corned beef stored at 0°C, L. monocytogenes grew at about half the rate of the other microflora, whereas at 9°C, both groups of organisms grew at similar rates. Similarly, on ham stored at 5°C, the organism grew at one-third the rate of the other flora. The composition of the meats, i.e., pH, salt, and residual nitrite, played a role in determining the growth potential of the organism. For example, at 0°C, the organism did not grow on ham containing 170 ppm residual nitrite, but did grow on ham with 11 ppm nitrite. Similarly, although the organism grew at similar rates on ham stored at 15°C, as the storage temperature decreased, the organism grew slower on ham containing the higher level of residual nitrite. The organism grew fastest on corned beef of pH 6.2, aw 0.97, and <5 ppm nitrite, and slowest on ham of pH 6.6, aw 0.97, and 170 ppm nitrite. From inoculated studies, equations were developed to describe the growth of both L. monocytogenes and other flora, i.e., lactic acid bacteria and Brochothrix thermosphacta on both ham and corned beef. For naturally contaminated corned beef, good agreement was obtained between predicted and actual growth of the organism. As predicted, L. monocytogenes was not able to grow on naturally contaminated ham stored at 0.1°C, although slight growth (i.e., from approximately 0.3 CFU/g to 6.2 CFU/g) of the organism was observed on product stored at 4.8°C. Juneja et al. [138] examined the potential for outgrowth of various foodborne pathogens from cooked ground beef during cooling from 54.4 to 7.2°C within 6, 9, 12, 15, 18, or 21 h. Ground beef was first inoculated with L. monocytogenes at levels of around 103 CFU/g; the meat was then heated in a linear fashion to 60°C within 1 h, and then cooled as described earlier. The organism was not detected in any of the beef samples examined. Presumably, the slight heating and cooling regime was sufficient to reduce levels of the organism below the detectable level, levels at which the organism remained during the duration of the cooling period [138].

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The fate of L. monocytogenes on unirradiated and irradiated cook-chill roast beef and gravy was examined at 5 and 10°C [107]. The organism grew well on both products, increasing 5 logs in number on unirradiated beef and gravy over 15 days at 5°C, and by 6 logs in irradiated products stored at 10°C for 23 days. Although the observed lag phase of L. monocytogenes was longer on irradiated as compared to unirradiated product, the specific growth rates were similar at each storage temperature, suggesting that the background microflora on the beef and gravy did not interfere with growth of L. monocytogenes. The authors concluded that there would be no increased risk of listeriosis if cook-chill roast beef and gravy were to be irradiated with 2 kGy [107]. The organism grows well on cooked beef (Table 13.10) [123]. Interestingly, the organism had similar growth rates aerobically and anaerobically, although the authors claimed that samples stored under aerobic conditions probably became anaerobic during the course of the experiment [123]. L. monocytogenes was able to survive pasteurization (e.g., 91 and 96°C for 3 or 5 min) of precooked beef roasts and then grow when products were stored at 4 and 10°C for up to 56 and 12 days, respectively [113]. Products stored at 10°C supported much better growth of the surviving listeriae than those stored at 4°C, i.e., at 10°C L. monocytogenes reached levels in 12 days that took up to 8 weeks to attain at the lower storage temperature. It is well known that the organism can repair itself much better at higher temperatures. Van Laack et al. [223] examined the effect of three packaging treatments, i.e., vacuum-packaged directly after hot boning (hot-packaged),

TABLE 13.10 Generation (GT) and Lag Times (LT) for L. monocytogenes in Meats Food

Temperature (°C)

Roast beef

−1.5a 3a 3b 0 5 5 10 15 5 10 7 12.5a 10a 5 5 5 5

Corned beef Cooked meat Ham

Cooked beef Pâté Pâté Pork Pork Pork Pork

belly, belly, belly, belly,

water pH 7.1–7.3 1% LADc pH 7.0 2% LADc pH 7.0 5% LADc pH 7.0

GT (h) 100.0 26.7 80.9 110.0 44–61 33.2 13.4 6.1 18.6–22.6 8.5–9.0 19.7 1.6 9.12 15.2 17.1 23.4 27.6

LT (h) 173.7 59.0 477.1

80.6–83.4 22.6–30.4 48 24 27.6 <1 <1 1 4

Reference 125 125 109 210 109 109 109 123 123 68 89 224 224 224 224

a

Vacuum packs. CO2 packs. cLactic acid decontaminated. b

Source: Adapted from Carlin, F., C. Nguyen-the, and A.A. Da Silva. 1995. Factors affecting the growth of Listeria monocytogenes on minimally processed fresh endive. J. Appl. Bacteriol. 78: 636–646; Hudson, J.A., S.J. Mott, and N. Penney. 1994. Growth of Listeria monocytogenes, Aeromonas hydrophila, and Yersinia enterocolitica on vacuum and saturated carbon dioxide controlled atmosphere-packaged sliced roast beef. J. Food Prot. 57: 204–208; Snyder, O.P. 1996. Use of time and temperature specifications for holding and storing food in retail food operations. Dairy, Food, Environ. Sanit. 16: 374–388.

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vacuum-packaged after chilling for 1 day (cold-packaged), or unpackaged, on survival of L. monocytogenes on pork loins stored at 1°C for up to 9 days (Table 13.11). Numbers of the organism increased by about 1 log, demonstrating the ability of the organism to grow on raw refrigerated pork in the presence of numerous competitors. Although a statistical analysis of the data was not done, the type of packaging did not appear to greatly influence growth. The issue of whether L. monocytogenes can grow on raw or cooked meats is complex. It is evident that factors such as pH, aw , background microflora, sodium nitrite and NaCl, length and temperature of storage, strain characteristics, and product history all play a role in determining the fate of Listeria on a meat surface. Another seemingly important factor that is often overlooked is the initial inoculum on the product. Although early work from modeling experiments done in broth suggested that the initial numbers of organisms present on a food surface had little or no influence on subsequent outgrowth and growth rate, some research with naturally contaminated product does not substantiate this theory. For example, Farber and Daley [84] found that when L. monocytogenes was present in very low numbers on meats such as sliced ham, turkey breasts, wieners, and pâté stored at 4°C, the pathogen did not increase in number. Similar results have been observed with foods other than meats and poultry [Farber, unpublished results].

TABLE 13.11 Influence of Packaging Treatment (Hot Packaging [HP], Cold Packaging [CP], and Cold Unpackaged [CU]) on Numbers of L. monocytogenes on Pork Loins during 9 Days of Storage at 1 ± 1°C Average log10 CFU/cm2 Sampling Day

Packaging Treatment

Trial 1

Trial 2

0

HP

2.42 (8/8)

CP CU

2.68a (5/8)b 2.48 (5/8) 2.65 (6/8)

2.86 (8/8) 2.86 (8/8)

1

HP CP CU

2.98 (6/8) 2.48 (5/8) 2.91 (7/8)

2.86 (8/8) 2.86 (8/8) 2.73 (8/8)

2

HP CP CU

3.36 (8/8) 2.68 (5/8) 2.64 (6/8)

3.11 (8/8) 2.77 (7/8) 2.91 (7/8)

5

HP CP CU

3.77 (7/8) 2.92 (7/8) 2.73 (8/8)

3.11 (8/8) 3.36 (8/8) 3.19 (7/8)

9

HP CP CU

3.73 (8/8) 3.19 (7/8) 3.48 (5/8)

3.23 (8/8) 3.61 (7/8) 3.23 (5/8)

a

Average of MPN values from positive samples. The number between parentheses indicates the proportion of samples from which L. monocytogenes was recovered.

b

Source: Adapted from Van Laack, R.L.J.M., J.L. Johnson, C.J.N.M. Van der Palen, F.J.M. Smulders, and J.M.A. Snijders. 1993. Survival of pathogenic bacteria on pork loins as influenced by hot processing and packaging. J. Food Prot. 56: 847–851.

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Midelet and Carpentier [178] examined the transfer of microorganisms from various materials to beef and discovered that L. monocytogenes biofilms had a higher attachment strength to polymers than other bacteria, and it is these biofilms that are the highest source of contamination. The higher the biofilm population, the higher the CFU detached. L. monocytogenes appears to have the ability to persist on meat processing equipment, as evidenced by Lunden et al. [161]. A dicing machine that diced cooked meat products was the source of L. monocytogenes contamination, as revealed through molecular typing with pulsed-field gel electrophoresis (PFGE). The contamination was traced from one plant to another, and the dicing machine that was transferred between plants was implicated as the source.

UNFERMENTED SAUSAGE Even though potentially contaminated raw meats find their way into enormous quantities of sausage products, with over 200 varieties manufactured in the United States alone, no information pertaining to behavior of L. monocytogenes during manufacture and storage of these popular meat products appeared in the scientific literature before 1988. Although the California listeriosis outbreak of 1985 eventually led to the aforementioned surveys in which Listeria was detected in raw and processed meats, including sausage, the early consensus was that consumption of such products did not pose a serious threat of contracting listeriosis, as shown by a lack of any confirmed cases of meatborne listeriosis. However, this situation changed in December 1988, following the report of a breast cancer patient who developed listerial meningitis and eventually died after consuming turkey frankfurters that were contaminated with L. monocytogenes. Unfermented sausages are best classified according to the following five categories, which are based on the method of manufacture: (1) fresh sausage (e.g., fresh pork sausage, bratwurst), (2) cooked sausage (e.g., liver sausage, Braunschweiger), (3) cooked smoked sausage (e.g., frankfurters, bologna), (4) uncooked smoked sausage (e.g., Mettwurst, smoked country-style pork sausage, Kielbasa), and (5) cooked meat specialty items (e.g., head cheese). Research efforts have dealt primarily with the behavior of Listeria in sausages belonging to the first three categories. Fresh Sausage Although fresh pork sausage is by far the most widely manufactured fresh sausage, this category also includes other well-known varieties such as fresh Italian, breakfast, and beef sausage as well as fresh bratwurst, Thuringer, and bockwurst. The latter two are most popular in Germany. All varieties of fresh sausage are normally prepared from coarse or finely comminuted pork, beef, or veal to which water is added along with an array of spices that varies with the type of sausage to be produced. In the United States, certain varieties of fresh sausage also may contain binders and/or extenders (e.g., cereal, vegetable starch, nonfat dry milk, and dried whey) at levels not exceeding 3.5% by weight. After being stuffed into natural or artificial casings, the product is twisted and cut to form individual sausage links, which are cooled rapidly to preserve freshness and flavor. Unlike cooked and fermented sausages, fresh sausages have a short shelf life and must be constantly refrigerated to prevent growth of spoilage organisms, including lactic acid bacteria and micrococci. When commercially prepared fresh bratwursts were surface-inoculated to contain approximately 0.1 or 600 L. monocytogenes CFU/g, vacuum-packaged, and stored at 4.4°0 C, Glass and Doyle [100] found that the pathogen attained populations of 106 CFU/g on organoleptically acceptable 4-week-old bratwursts, regardless of the initial inoculum. As with ham, profuse growth of listeriae on fresh bratwurst was attributed to a pH value >6.0, which was maintained by the product throughout the first 4 weeks of refrigerated storage. In another study involving fresh sausage, Hughey et al. [127] investigated the ability of lysozyme to prevent growth of L. monocytogenes in bratwurst prepared from coarsely ground pork. After addition of commercial bratwurst spice, distilled water was added with or without 100 ppm

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lysozyme and 5 mM EDTA, a generally recognized as safe chelating agent that enhances antibacterial activity of lysozyme. This meat mixture was then inoculated to contain ~4 × 103 CFU/g of L. monocytogenes and stuffed into natural hog casings that were subsequently linked, separated, vacuum-packaged, and stored at 5°C for 45 days. As expected, based on the previous study, Listeria populations increased rapidly in fresh bratwurst (pH ≅ 6.0) without added lysozyme or EDTA, reaching levels of >106 CFU/g following 10 days of refrigerated storage. L. monocytogenes also behaved similarly in bratwurst containing lysozyme alone; however, the presence of EDTA alone resulted in a 15-day lag period, thus preventing the pathogen from reaching populations of 106 CFU/g until nearly 30 days of storage. In contrast, lysozyme and EDTA acted synergistically to retard growth of L. monocytogenes in fresh bratwurst. Under these conditions, the pathogen exhibited a lag period of nearly 21 days, i.e., approximately 7 days beyond the normal shelf life of the product, and reached populations of <105 CFU/g following 44 days of refrigerated storage. Although only listeriostatic, the combined use of lysozyme and EDTA appears to be an effective means of controlling Listeria growth during the normal shelf life of fresh bratwurst. Furthermore, once growth is prevented, low levels of L. monocytogenes that occasionally appear in fresh bratwurst (<103 CFU/g) should be readily eliminated by proper cooking. Glass et al. [102] looked at the use of sodium lactate and sodium diacetate as antilisterial agents on wieners and cooked bratwurst. The results showed that dipping wieners in lactate-diacetate solutions was not effective in controlling L. monocytogenes, but addition of these inhibitors to weiner and bratwurst formulations limited growth for up to 12 weeks on cured, smoked bratwurst containing 3.4% lactate and 0.1% diacetate. Sodium lactate levels of 3% and combinations of sodium lactate and sodium diacetate prevented listerial growth on wieners stored for 60 days at 4.5° C (Figure 13.2). Seman et al. [203] modeled the growth of L. monocytogenes in different RTE meats by manipulating levels of sodium chloride, sodium diacetate, potassium lactate, and product moisture content. The cured meat products used were light bologna, wieners, smoked or cooked ham, and cotto salami. Sodium lactate and sodium diacetate treatments (1.5 or 2.5% sodium lactate with 0.15% sodium diacetate) limited growth of the pathogen at 4°C throughout the 18-week test period 11

1.3% Lactate 2.0% Lactate 2.5% Lactate 3.0% Lactate 3.5% Lactate 1% Lactate, 0.1% Diacetate 1% Lactate, 0.25% Diacetate 2% Lactate, 0.1% Diacetate

Log10 CFU/Package

10 9 8 7 6 5 4 0

7

14

30

45

60

Time (d)

FIGURE 13.2 Inhibition of L. monocytogenes by sodium lactate and sodium diacetate on cured, smoked weiners stored at 4.5°C. Population numbers reported are averages for triplicate packages. (Adapted from Glass, K.A., D.A. Granberg, A.L. Smith, A.M. McNamara, M. Hardin, J. Mattias, K. Ladwig, and E.A. Johnson. 2002. Inhibition of Listeria monocytogenes by sodium diacetate and sodium lactate on wieners and cooked bratwurst. J. Food Prot. 65: 116–123.)

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Lm Count (log10 CFU/g)

540

Time (weeks)

FIGURE 13.3 Plot of L. monocytogenes growth on slices of light bologna inoculated with a five-strain L. monocytogenes cocktail over 18 weeks of storage at 4°C. Controls contain no potassium lactate or sodium diacetate. T2 contains 1.5% potassium lactate solids and 0.15% sodium diacetate. T3 contains 2.5% potassium lactate solids and 0.15% sodium diacetate. (Adapted from Seman, D.L., A.C. Borger, J.D. Meyer, P.A. Hall, and A.L. Milkowski. 2002. Modelling the growth of Listeria monocytogenes in cured ready-to-eat processed meat products by manipulation of sodium chloride, sodium diacetate, potassium lactate and product moisture content. J. Food Prot. 65: 651–658.)

(Figure 13.3). The model provided a good approximation of the actual situation, but the authors acknowledge that these results are very specific to these products, and there would likely be different results in other meat systems. Cooked Smoked Sausage This group of sausages, which includes the ever-popular frankfurter (hot dog) as well as bologna and various luncheon meats, is prepared from mixtures of comminuted beef and/or pork to which salt, sugar, sodium nitrite, and spices are normally added. When making frankfurters, this meat and ingredient mixture, commonly referred to as the sausage emulsion, is stuffed into natural or artificial casings, which are then twisted to form sausage links. This string of frankfurter links is cooked to an internal temperature of 71.1°C (160° F) to (1) coagulate protein, (2) fix the color, and (3) pasteurize the product. Although not absolutely required, frankfurters and other similar sausages are frequently hung in smoking rooms either before or after cooking. Alternatively, commercially available liquid smoke products can be added to the sausage emulsion or applied directly to the surface of frankfurters before or during heating. In either event, besides imparting a pleasant smoked flavor to the finished product, some smoke components (i.e., formaldehyde, acetic acid, creosote, and phenols with high boiling points) are actually bacteriostatic and/or bactericidal toward many microbial contaminants. After cooking, frankfurters are carefully cooled, packaged, and refrigerated during shipment to wholesale and retail markets. Skinless frankfurters, which are very popular, are produced in a similar manner except that the casing is mechanically peeled from the sausage after cooking or smoking.

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Epidemiological data from the Centers for Disease Control and Prevention (CDC) showing an apparent association between listeriosis and undercooked frankfurters prompted several studies examining the thermal resistance of L. monocytogenes in this sausage. Zaika et al. [245] prepared frankfurters from a sausage emulsion inoculated to contain ~108 CFU/g of L. monocytogenes. After stuffing, all frankfurters were thermally processed (without smoke) according to a standard commercial heating schedule. These USDA officials found that L. monocytogenes populations decreased approximately 1000-fold in frankfurters that were heated to an internal temperature of 71.1°C (160°F). Based on these data, cooking frankfurters to an internal temperature of 71.1°C would probably eliminate maximum levels of L. monocytogenes (<103 CFU/g) that could conceivably occur in raw frankfurter emulsions. Data gathered by the American Meat Institute in 1988 pointed to frankfurters as being likely carriers of L. monocytogenes and also suggested that poor environmental conditions before packaging could play a major role in contaminating the finished product [8]. Moreover, Glass and Doyle [100] reported that this pathogen can proliferate on vacuum-packaged, artificially contaminated (~0.01 L. monocytogenes CFU/g) retail frankfurters during incubation at 4.4°C, with populations 2 to 5 orders of magnitude higher on organoleptically acceptable samples after 4 weeks of refrigerated storage. Similarly, L. monocytogenes increased in number from 5 × 102 to 2.1 × 105 MPN/g on vacuum-packaged frankfurters stored at 4°C for 20 days. Interestingly, noninoculated control products contained 1.2 × 102 MPN/g after the 20-day storage period, after initially being negative for the organism [46]. In a more detailed study examining growth of L. monocytogenes on vacuum-packaged all-beef, poultry, or beef/pork wieners at 5°C for up to 28 days, McKellar et al. [171] found that of a total of 61 wieners analyzed, 40 (65.6%) supported growth of L. monocytogenes. For those samples supporting growth, an average increase of 1.26 logs was observed within a 14-day period. Initial lactic acid bacteria concentrations, as well as phenol and nitrite levels varied considerably during storage, unlike NaCl levels. In addition, average pH levels decreased significantly during storage by 0.19 pH units. Several statistical models were derived in an attempt to adequately describe growth and death of the organism in all wiener samples. Although no one model was sufficient, the best model obtained implicated initial and final lactic acid bacteria counts and initial pH as factors that influence growth of L. monocytogenes. Prevention of Listeria contamination and inhibition of growth is further complicated by present consumer demands for reduced amounts of salt and preservatives, along with longer shelf life, smaller packages, and greater convenience. All these will require the processed meat industry to develop even stricter requirements for processing, cooking, handling, packaging, and refrigeration of products. Several studies were initiated by the food industry to examine the feasibility of using heat to eliminate L. monocytogenes from the surface of finished frankfurters. In one such study [10], frankfurters were dipped in a broth culture of L. monocytogenes (106–108 CFU/mL) to simulate postprocessing contamination. Listeria populations on the surface of the frankfurters decreased only 100-fold after 8 min of heating at 86.1–87.8°C (187–190°F). Furthermore, this heat treatment rendered the sausages organoleptically unacceptable for most consumers. Hence, “postprocess pasteurization” may not be a viable means of eliminating L. monocytogenes from the surface of frankfurters that have been contaminated after manufacture. Additional efforts to control Listeria contamination on the surface of frankfurters have focused on the bactericidal properties of commercially available liquid smoke products. In one study [177], beef frankfurters were immersed in a culture containing 1 × 103 CFU/mL of L. monocytogenes, removed, thoroughly air-dried, and then dipped in full-strength commercially available liquid smoke solution (CharSol C-10). Although Listeria populations remained unchanged in control frankfurters that were dipped in phosphate buffer, vacuum-packaged, and analyzed after 72 h at 4°C, numbers of listeriae decreased 60 to ≥99.9% 15 min after the frankfurters were treated with liquid smoke. Furthermore, the pathogen was never detected in smoke-treated sausage following 72 h of refrigerated storage. Even though dipping frankfurters in full-strength liquid smoke eliminated L. monocytogenes, this treatment produced an extremely intense smoky-flavored product that would likely be organoleptically unacceptable to most consumers.

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Preliminary results from additional experiments dealing with the antilisterial effects of less concentrated liquid smoke solutions were reported by Wendorff [235]. Initially, beef frankfurters were dipped into a concentrated broth culture of L. monocytogenes, thoroughly dried, dipped into aqueous liquid smoke solutions containing 10–40% CharSol C-10 or Poly-10 (concentrations normally used in frankfurter production) and then analyzed for listeriae after 72 h of refrigerated storage, using the Gene-Trak DNA Hybridization assay. Although L. monocytogenes was eliminated from the surface of frankfurters dipped in 40% solutions of CharSol C-10 or Poly-10, the pathogen was still detected on frankfurters treated with 10 and 25% solutions of CharSol C-10. Demonstrating that these liquid smoke compounds lost their activity against Listeria on the surface of frankfurters when added directly to sausage emulsion before stuffing, Wendorff [235] examined the ability of liquid smoke compounds to inactivate listeriae on surface-inoculated skinless frankfurters that were sprayed with five levels of CharSol Poly-10 and CharSol Supreme (twice the strength of CharSol C-10) just before VP (Table 13.12). When used at organoleptically acceptable concentrations, CharSol Supreme was more effective than CharSol Poly-10, with Listeria populations on the surface of frankfurters decreasing >40% following 72 h of refrigerated storage. When this study was repeated with a more realistic L. monocytogenes inoculum level (~103 CFU/g), approximately 89% of the Listeria population was inactivated on frankfurters treated with CharSol or CharSol Poly-10 at levels of 2.0 and 4.0 oz/100 lb of frankfurters, thus indicating that none of these treatments can guarantee a Listeria-free product. Hence, to avoid contamination of finished product, efforts must be made to develop microbial monitoring, sampling, and HACCP programs that can effectively address problems pertaining to a lack of separation between raw and finished processing areas, as well as procedures used to clean and sanitize the factory environment and equipment such as grinders, mixers, and particularly sausage peelers. In the only other reported study involving cooked smoked sausage, Glass and Doyle [100] examined behavior of L. monocytogenes on vacuum-packaged, artificially contaminated slices of commercially produced bologna. As was true of ham and bratwurst, the pathogen also grew well on bologna (pH 6.1–6.4), with populations generally 3 to 4 orders of magnitude higher than initially on organoleptically acceptable samples held 4 weeks at 4.4°C. These findings stressed the importance

TABLE 13.12 Fate of L. monocytogenes on the Surface of Beef Frankfurters Sprayed with CharSol Poly-10 or CharSol Supreme Liquid Smoke and Stored at 4°C for 72 h L. monocytogenes Treatment Control CharSol Poly-10

CharSol supreme

Level (oz/100 lb Frankfurters)

Initial Inoculum (CFU/g)

Inactivation after 72 h (%)

5.28 5.30 5.28 5.16 4.84 4.67 5.02 5.04 4.73 4.41 4.30

0 Growth 0 23.6 63.1 75.2 44.7 42.1 71.5 86.3 89.5

0 1.7a 3.4a 5.1a 8.5 12.0 1.8a 3.6a 5.4 9.0 12.6

a

Organoleptically acceptable concentration of liquid smoke.

Source: Adapted from Wendorff, W.L. 1989. Effect of smoke flavorings on Listeria monocytogenes in skinless franks. Seminar presentation, Department of Food Science, University of Wisconsin–Madison, January 13.

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of following good manufacturing practices, which will, in turn, greatly reduce the possibility of listeriae contaminating RTE meats during slicing and packaging. Uncooked Smoked Sausage According to incidence data (Table 13.9), one variety of West European uncooked smoked sausage, namely Mettwurst, appears to be particularly prone to contamination with Listeria spp., including L. monocytogenes. These observations prompted a 1989 study by Trüssel and Jemmi [219], in which an experimentally produced Mettwurst emulsion (pH 5.3) containing ~2.6% NaCl and 100 ppm sodium nitrate was artificially contaminated with L. monocytogenes at levels of 103 or 107 CFU/g and examined for numbers of listeriae during manufacture, after 7 days of ripening at 14°C, and after 3 weeks of subsequent storage at 4°C. Overall, Listeria populations remained relatively constant during manufacture and ripening, which, in turn, reflects the apparent inability of this organism to multiply in RTE meat products having pH values <5.5. As expected from the results of European surveys, this pathogen also survived well in Mettwurst during the product’s entire refrigerated shelf life, with 4-week-old samples still containing approximately 102 and 106 CFU/g of L. monocytogenes. Given these findings, it would be prudent for Mettwurst producers to review their manufacturing practices and develop procedures for decreasing Listeria contamination during all stages of production. Cooked Meat Specialty Items A significant amount of beef jerky is consumed annually in the United States. It is popular because it is easy to prepare, lightweight, and stable without refrigeration. However, some health concerns have arisen with this product, and outbreaks of salmonellosis have been reported [54]. A recent study examined the fate of several foodborne pathogens, including L. monocytogenes, during preparation and storage of beef jerky [114]. Half of the inoculated beef loin strips tested were marinated at 4°C overnight and then dried at 60°C for 10 h. The remaining half of the samples were first heated in marinade to 71.7°C, and then dried. L. monocytogenes populations decreased by 1.8 and 6.0 logs after 3 and 10 h of drying, respectively. Cooking to 71.7°C before drying led to a 4.5-log decrease in numbers of the organism, with a further 2-log reduction in numbers occurring during the 10-day drying period. After 8 weeks of storage at 25°C, none of the pathogens (E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes) were recovered from the beef jerky [114]. Much attention has been drawn to pâté as it has been incriminated in at least two foodborne outbreaks. There is still controversy in the literature regarding the growth potential of L. monocytogenes on pâté. De Boer and van Netten [68] found inoculated retail pâté to be a good menstruum for growth of the organism, which reached levels as high as >8.3 log CFU/cm2 after 7 days at 12.5°C, when the background microflora was low (<2.3 CFU/cm2). There was an inverse correlation between growth of the organism and presence of lactic acid bacteria, i.e., when high numbers of lactic acid bacteria were present, the pH was low (average of 4.9), and L. monocytogenes increased slightly or decreased in numbers during 1 week of storage at 7° or 12.5°C. Morris and Ribeiro [180] also found that L. monocytogenes could grow on some naturally contaminated pâtés with refrigerated storage for 21 days, yielding levels as high as 2 × 108 CFU/g, whereas on other samples, growth was not evident. Other investigators found that L. monocytogenes could not multiply on retail inoculated pâté stored at 4°C for up to 3 weeks [84]. To more fully assess the health hazard posed by L. monocytogenes in liver pâté products, multifactorial design experiments were conducted to examine the influence of temperature (4 and 10°C), NaCl (1 and 3%), sodium nitrite (0 and 200 ppm), sodium erythrobate (0 and 550 ppm), and spice (0 and 0.4%) on growth of the organism on experimental pâté [89]. A total of 16 different liver pâté formulations made experimentally were stored at 4 and 10°C for various periods of time. Analysis of variance was used to determine the effect of the various factors on maximum growth rate. L. monocytogenes grew well on the experimental pâtés, with temperature being the only factor exerting an effect on the growth rate.

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The generation and lag times for L. monocytogenes on pâté are shown in Table 13.10. Potential growth of L. monocytogenes is related to pâté composition and pH, the numbers of lactic acid bacteria present, and the storage temperature and time. Fermented Sausage Sausages classified as fermented undergo a controlled lactic-acid-type fermentation, usually through the action of a commercially produced starter culture added to the meat. Although all fermented sausages can be further classified according to moisture content as either semidry or dry, manufacturing procedures for both types are generally similar until the point of drying. Fermented sausages are normally prepared from comminuted beef and/or pork to which sugar and various spices are added along with sodium or potassium nitrate and/or nitrite. This meat preparation, known as a mix rather than an emulsion, is inoculated with a commercial mixture of lactic acid bacteria, which frequently includes species of Pediococcus (particularly P. cerevisiae and P. acidilactici), Lactobacillus, and Leuconostoc. After stuffing the inoculated sausage mix into natural or artificial casings, the strings of sausage links are hung in ripening or “green rooms” at 27–40°C with 80 to 90% relative humidity (RH). Within 2–3 days, sugar added to the mix is fermented to lactic acid by the starter culture, which, in turn, decreases the pH to ~5.1 and produces the characteristic tangy flavor found in fermented sausages. As in cheese making, controlled lowering of sausage pH to levels near the isoelectric point of meat protein is important for proper removal of water during later stages of sausage manufacture. Following fermentation, sausages destined to become semidry varieties containing ~50% moisture (e.g., Cervelat-type sausages and Lebanon bologna) are normally placed in smokehouses where they are smoked and cooked to internal temperatures of 60–68°C. In contrast, dry sausages which will ultimately contain ~35% moisture (e.g., pepperoni, Genoa, and Milano salamis) are moved to drying rooms (10–17°C/65–80% RH), where they remain for various times, depending on the type and size of sausage. Some varieties also may be exposed to cool smoke before drying; however, unlike semidry varieties, dry sausages are never cooked. Although fermented sausages keep well because of their relatively high salt content, low pH, low aw, and moisture content, both varieties, particularly semidry, should be stored at refrigeration temperatures. Semidry Fermented Sausage The relatively severe heat treatment that semidry sausages receive during manufacture generally has been regarded as sufficient to eliminate most commonly encountered non-spore-forming foodborne pathogens. Hence, despite concerns regarding presence of listeriae in meat products, behavior of L. monocytogenes during manufacture and storage of semidry fermented sausage has received relatively little attention. Although L. monocytogenes is unlikely to survive during the manufacture of semidry sausage, ample opportunity exists for this pathogen to contaminate the finished product during slicing and packaging. To simulate postprocessing contamination, Glass and Doyle [100] inoculated slices of commercially produced, fermented semidry sausage to contain approximately 0.01 or 100 L. monocytogenes CFU/g, followed by vacuum packaging. Quantitative examination for listeriae during prolonged incubation at 4.4°C was undertaken. Unlike ham, bologna, and frankfurters, the pathogen failed to grow on fermented semidry sausage of pH 4.8–5.2, with populations generally decreasing ≤10-fold on organoleptically acceptable samples after 6 to 12 weeks of refrigerated storage. Recognizing the likelihood of listeriae being introduced into semidry sausage during slicing or packaging and surviving throughout the normal shelf life of the product, Cirigliano et al. [59] investigated the possibility of eliminating L. monocytogenes from inoculated slices of German- and Polish-type beef sausage (104–105 CFU/g L. monocytogenes strain Scott A or V7) by exposing

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vacuum-packaged product to temperatures of 32.2–51.7°C (90–125°F) for up to 72 h. According to the authors, L. monocytogenes populations failed to change in product held at 32.2°C (90°F); however, numbers of both Listeria strains decreased approximately 100-fold after product was held at 37.8°C (100°F) for 72 h, with a slightly faster rate of inactivation observed in Polish- than German-type sausage. Although increasing the temperature to 43.3°C (110°F) led to elimination of strain V7 from Polish- and German-type sausage after 8 and 48 h, respectively, strain Scott A was not eliminated from either product until completion of a 72-h heat treatment. When exposed to 48.9 and 51.7°C (120 and 125°F), strain V7 was inactivated in Polish- and German-type sausages within 4 and 24 h, respectively. Although somewhat more heat resistant, strain Scott A was eliminated from both products after 24 h at 51.7°C (125°F). In some instances, objectionable fat losses were observed for both product types; however, the authors concluded that mild heat treatments could be used to salvage Listeria-contaminated German- or Polish-type sausage without seriously affecting product quality. In fermented “tea” sausages inoculated with 8 × 106 MPN/g of L. monocytogenes, the organism decreased about 1.5 logs in number during the initial 4-day ripening period when the pH dropped from 5.47 to 4.80, decreased another 1.5 logs in number during the 9-day drying period, and then remained constant in number during the 20-day storage period at 18 to 22°C [47]. Similar results were obtained for sausages inoculated with lower levels of the organism. The initial aw value, as well as those after drying and after storage were 0.974, 0.933, and 0.861, respectively. Dry Fermented Sausage Unlike semidry varieties, dry fermented sausages are never exposed to temperatures that would be expected to inactivate even small numbers of listeriae. Consequently, dry sausages have attracted more attention, as shown by several recent studies that examined the fate of L. monocytogenes during the fermentation, drying, and storage of hard salami, pepperoni, and different varieties of sausage. In the first such study, Johnson et al. [135] prepared hard salami from naturally contaminated (i.e., meat from cows inoculated intravenously with L. monocytogenes) as well as artificially inoculated ground beef, both of which contained ~104 CFU/g of L. monocytogenes. Glucose, spices, sodium nitrite, and salt were added to the ground beef along with a glucose-fermenting strain of Pediococcus acidilactici. After stuffing the mix into casings, all sausages were fermented at 40°C for 24 h, dried at 13°C for 9 days, vacuum-packaged in gas-impermeable film, and stored at 4°C for 12 weeks. Listeriae populations decreased approximately 10- and 100-fold during fermentation (40°C/24 h) of hard salami prepared from naturally contaminated and artificially inoculated ground beef, respectively. Inactivation of listeriae during the fermentation appeared to be primarily attributable to production of lactic acid (or other metabolites) by the starter culture, with pH values decreasing from approximately 5.7 to 4.4 by the end of fermentation. Although numbers of listeriae remained relatively constant in naturally contaminated hard salami following 9 days of drying (13°C/65% RH), listeriae populations decreased nearly 100-fold during drying of product prepared from artificially inoculated ground beef. L. monocytogenes was detected in both products during 8 weeks of refrigerated storage. Higher levels of listeriae were recovered from naturally contaminated rather than artificially inoculated hard salami, suggesting that the behavior of this pathogen is best studied using sausage prepared from naturally contaminated rather than artificially inoculated ground beef. Compositionally, both products were very dry, having aw values of 0.79–0.81 as compared to ~0.91 for commercially produced hard salami. Although L. monocytogenes might be expected to survive more readily in higher-moisture commercial products, growth of the pathogen in retail hard salami appears unlikely given the presence of 5–7% NaCl and 100–150 ppm sodium nitrite, combined with a pH of 4.3–4.5 and a relatively low storage temperature. Trüssel and Jemmi [219] reported a different finding, in that L. monocytogenes populations in salami prepared from a mix inoculated to contain approximately 103 or 107 CFU/g of L. monocytogenes

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decreased ≤10-fold in product of pH ≤5.6 during 7 days of ripening at 12–22°C/82–95% RH. After 8 weeks of drying at 10–17°C/78–82% RH, numbers of listeriae in salami of pH 5.4–5.7 decreased to <10 CFU/g, regardless of the initial inoculum. However, when an enrichment procedure was used, the pathogen still could be detected in these sausages after an additional 6 to 11 weeks of drying at 10–17°C/35–50% RH to aw values of 0.68–0.69. Thus, as was true of certain fermented dairy products (see Chapter 12), small numbers of L. monocytogenes cells can also persist in fermented dry sausages for at least 14–19 weeks. Farber et al. [88] examined the fate of L. monocytogenes during production of uncooked German, American, and Italian-style fermented sausages. Similar results were obtained with both the German and American sausages, in which levels of the organism decreased about 2 to 3 logs after fermentation and smoking and a further 1 to 2 logs after drying. This contrasted with how L. monocytogenes behaved in Italian-style fermented sausages, the only types not made with starter culture. In these latter sausages, L. monocytogenes increased slightly during the fermentation period, remained constant in number during drying, and then decreased slightly during the 4-week holding period at 4°C. This study points to the importance of using starter cultures during production of dry fermented sausages, and also emphasizes the necessity of having a well-designed and operating HACCP plan (plus GMPs) for each style of sausage made. Work also has been done to examine the fate of L. monocytogenes in fermented sausage made from poultry meat inoculated with approximately 104 CFU/g followed by fermentation at 20°C. The sausages were formulated to contain two levels (2.0 and 2.5%) of sodium nitrite and gluconodelta-lactone (GdL). Starter cultures were added to some formulations. L. monocytogenes survived the 28-day ripening period, with numbers decreasing by about 1 log in those formulations containing 2.5% sodium nitrite plus either GdL or starter culture, and staying constant in the other batches (one exception was the batch containing 2% sodium nitrite, where a 2-log decrease in numbers was observed). It would appear that the hurdles, consisting of preservatives, starter culture, aw (0.90–0.92), and pH (final 4.8–5.9) were not sufficient to inactivate the listeriae cells present [168]. Recognizing that L. monocytogenes may survive the typical process used to manufacture hard salami, Glass and Doyle [101] attempted to identify various heat treatments that could be used to inactivate L. monocytogenes during manufacture of dry fermented sausage. Work with “beaker sausage” prepared from ground beef/pork containing 3.5% salt, 103 ppm sodium nitrite, and approximately 5 × 103 CFU/g of L. monocytogenes indicated that numbers of listeriae decreased >10-fold following an active fermentation (32.2°C/16 h) with P. acidilactici during which the pH decreased from approximately 6.3 to 4.8. Prolific growth of L. monocytogenes in a similar lot of beaker sausage prepared without P. acidilactici confirms the importance of an active starter culture in preventing growth of listeriae in fermented sausage. Furthermore, these findings suggest that 3.5% salt and 103 ppm sodium nitrite are of virtually no value in preventing growth of listeriae in sausage mix. Subsequent holding of fermented beaker sausage at 46.1°C for 8 h or heating to an internal temperature of 51.7 or 57.2°C failed to eliminate listeriae, with the pathogen still present in all samples examined according to the USDA enrichment method. Although holding samples at 51.7°C for 8 h or 57.2°C for 4 h reduced Listeria populations >100-fold, enrichment data indicated that the pathogen was still present in either of two samples that received both heat treatments. Only after heating beaker sausage to an internal temperature of 62.8°C was the pathogen no longer detected either by direct plating or enrichment. Subsequently, Glass and Doyle [101] investigated the fate of. L. monocytogenes in pepperoni during normal processing and storage and during heating to an internal temperature of 51.7°C for 4 h immediately after fermentation or drying. After inoculating commercially prepared pepperoni mix to contain 104 CFU/g of L. monocytogenes, populations of listeriae decreased approximately 100-fold following fermentation (35.6°C/12 h) by P. acidilactici, which caused the pH to decrease from 6.0 to 4.7. These findings are similar to those of Johnson et al. [135], who found that Listeria populations decreased 10- to 100-fold during fermentation of hard salami. Following 5 days of drying at 12.8°C, numbers of listeriae decreased to <10 CFU/g in normally processed pepperoni; however, with the USDA enrichment procedure, L. monocytogenes could still be detected in 82-day-old refrigerated

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samples of vacuum-packaged pepperoni. Heating the same pepperoni to an internal temperature of 51.7°C between fermentation and drying had relatively little effect on L. monocytogenes, with viable populations decreasing only about 10-fold. Although holding pepperoni for 4 h at 51.7°C reduced Listeria populations to undetectable levels (using direct plating and enrichment), the pathogen was sporadically recovered with the USDA enrichment procedure from 5- to 22-day-old sausage. Subsequent holding of the same pepperoni (pH 4.6) at an internal temperature of 51.7°C for 4 h immediately after 26 days of drying at 12.8°C led to complete inactivation of the pathogen as determined by direct plating and enrichment procedures. Additional experiments conducted on pepperoni containing 5.3 × 103 CFU/g of L. monocytogenes after 19 days of drying verified that a minimum heat treatment of 4 h at 51.7°C was required to obtain a Listeria-free product. Thus, although normal processes used to manufacture pepperoni are not sufficient to eliminate L. monocytogenes from heavily contaminated product, holding pepperoni and possibly other dry sausages at an internal temperature of 51.7°C for at least 4 h may prove to be a viable means of salvaging contaminated product. The antibotulinal properties of nitrate, and particularly nitrite, have been recognized for years, yet current scientific literature contains little information concerning the effect of these preservatives on Listeria behavior in dry fermented sausage. Junttila et al. [140] examined the ability of L. monocytogenes to survive in dry Finnish sausage containing various levels of potassium nitrate, sodium nitrite, and salt. All sausage was prepared from a mixture of ground beef and pork to which sugar, spices, and 3 or 3.5% salt were added along with 50 to 1000 ppm potassium nitrate and/or sodium nitrite. After inoculation to contain approximately 105 CFU/g of L. monocytogenes and a starter culture consisting of Staphylococcus carnosus and Lactobacillus plantarum, the sausage mix was stuffed into casings. All sausage links were fermented 2 days at 23°C, smoked 5 days at 20–22°C and dried 1 week each at 18 and 10°C. Listeria monocytogenes populations in sausage containing commonly used levels of salt (3.0%) and sodium nitrite (120 ppm) decreased 1.14 orders of magnitude over 21 days. Similar findings also were reported with 3.5% salt. Increasing levels of sodium nitrite (200 ppm) and potassium nitrate (330 ppm) to those commonly used 30 years ago led to a somewhat faster inactivation of listeriae in dry fermented sausage, with inactivation again most pronounced during the later stage of drying. Over the same 21-day period, Listeria populations decreased approximately 3.3 orders of magnitude in sausage containing 3.5% salt and 1000 ppm potassium nitrite; however, this concentration of potassium nitrate is no longer permitted in dry fermented sausage. Growth of L. monocytogenes in this product was apparently suppressed by the combination of salt, sodium nitrite, and a pH of 4.7; however, given the pathogen’s known tolerance to salt, acid, and low temperatures, addition of commonly used levels of sodium nitrite to fermented sausage was only marginally effective in inactivating listeriae. Samelis et al. [200] did a microbiological ecology study regarding the stability and safety of traditional Greek salami. All of the batches of fermented sausages had naturally occurring Listeria spp., which disappeared over time. The results showed that although 60% of the incoming materials had listeriae, L. monocytogenes was completely eliminated from the sausages 7 days after fermentation. The study used local traditional starter cultures rather than the premanufactured cultures as they are typically produced in northern Europe, which may be appropriate for that climate, but the climate in Greece is very different, and consequently the microbiological components of the starter culture would be different. Thus, although this study and others have provided valuable information concerning the behavior of L. monocytogenes in sausage products, an understanding of interactions between various factors such as starter cultures, food additives, and various heat treatments is still needed to develop suitable methods to eliminate L. monocytogenes from fermented sausage and other processed meat products.

MODIFIED-ATMOSPHERE (MA) PACKAGING Modified-atmosphere packaging (MAP) can extend the shelf life of many perishable foods including meats and poultry. The CO2-enriched atmosphere that is created within a meat pack can inhibit the

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normal spoilage flora and select for organisms such as the lactic acid bacteria [82]. Concerns have been raised about the ability of L. monocytogenes to outgrow the normal spoilage flora on MApackaged foods. In addition, the relatively long shelf life of MA-packaged foods can allow psychrotrophic foodborne pathogens such as L. monocytogenes to grow to high levels. As seen earlier, the organism can grow on vacuum-packaged meats including beef, lamb, and pork. The effects of intermediate to high levels of CO2 on the survival and growth of the organism on meats and poultry are not clear [91]. Beef Growth of L. monocytogenes was observed on samples of vacuum-packaged high-pH (>6.0) beef stored at 0, 2, 5, and 10°C, but not on those vacuum packs stored at −2°C. In general, rather long lag periods were observed, and the organism grew at a slower rate than spoilage flora [81]. The organism only grew in CO2 packs stored at 10°C, and not at any of the lower storage temperatures tested. However, when normal ultimate pH beef (pH 5.3–5.5) was used as the food menstruum, L. monocytogenes was not able to grow on beef stored in CO2 packs at 5 or 10°C [27]. It is likely that the lower pH of normal as compared to DFD meat, combined with the high-CO2 environment, was sufficient to cause inhibition and a slight decline in numbers of the organism. As in the findings of Grau and Vanderlinde [109], the organism was able to grow well on vacuum-packaged meat stored at 5 and 10°C. It was interesting that in the study by Avery et al. [31], L. monocytogenes grew at a faster rate than the spoilage flora on vacuum-packaged beef, which is in contrast to the results obtained by Gill and Reichel [95]. Perhaps L. monocytogenes can compete better with the spoilage microflora at a lower pH. A follow-up study by Avery et al. [32] was designed to study the effects of previous high CO2 exposure of L. monocytogenes to its subsequent growth during abusive retail display. Beef steaks of normal pH were first inoculated with the organism, individually packaged in CO2 packs, and then stored at −1.5°C for <3 h, 5 or 8 weeks. At each of the latter three time intervals, samples were removed, overwrapped, and placed on retail display at 12°C for up to 140 h. Even after only a brief (<3 h) exposure to CO2 , L. monocytogenes grew slightly or not at all during retail display, with demonstrated lag phases of >75 h. There were no comparative controls, however, to show the growth of the organism when inoculated into normal-pH beef and then stored at 12°C. Experiments were also done whereby steaks were removed from storage, and then rinsed to remove some cells of L. monocytogenes, which were reinoculated into freshly cut beef steaks to simulate cross-contamination. In this instance, inocula from steaks stored in CO2 packs for 5 or 8 weeks did not grow on the cross-contaminated steaks. It was concluded that (1) prior exposure of beef steaks contaminated with L. monocytogenes to high-CO2 environments will not increase the risk of growth of the organism when steaks are placed on retail display and possibly temperature-abused, and (2) there is not a large risk of growth when cross-contamination occurs from high-CO2 stored beef to fresh raw beef before retail display. Experiments have also been done to examine survival and growth of the organism on sliced roast beef stored under vacuum or CO2 at −1.5 and 3°C. The organism was not able to grow under CO2 at −1.5°C, but did grow under all other test conditions. At 3°C, the organism grew three times faster on vacuum-packaged as compared to CO2-stored roast beef (see Table 13.10). When growth occurred, maximum numbers of the organism were attained only at the end of shelf life of the product. Tsigarda et al. [220] examined behavior of L. monocytogenes as well as autochthonous flora on sterile and naturally contaminated beef fillets stored under aerobic, vacuum, and modifiedatmosphere packaging (MAP; 40% CO2, 30% O2, and 30% N2) conditions at 5°C, with or without the presence of oregano essential oil (Figure 13.4). In this particular study, the type of packaging film was the critical factor for L. monocytogenes growth and survival, and for determining the dominant microflora. L. monocytogenes increased in number when the pseudomonads dominated, which was under aerobic storage and under MAP/VP conditions using a high-permeability film. B. thermosphacta comprised the majority of the microflora when MAP/VP low-permeability

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4

3.5

3

Log CFU/g−1

2.5 0% Oregano Oil 0.8% Oregano Oil

2

1.5

1

0.5

0 0

2

3

4

6

7

9

11

14

16

Time (d)

FIGURE 13.4 Growth and survival of L. monocytogenes on naturally contaminated beef fillets with 0 and 0.8% oregano essential oil, packaged in 40% CO2, 30% O2, and 30% N2 within low-permeability film at 5°C. Each number is the mean of two samples taken from different experiments. Each sample was analyzed in duplicate (coefficient of variation <5%). (Adapted from Tsigarda, E., P. Skandamis, and G.-J.E. Nychas. 2000. Behaviour of Listeria monocytogenes and autochthonous flora on meat stored under aerobic, vacuum and modified atmosphere packaging conditions with or without the presence of oregano essential oil at 5°C. J. Appl. Microbiol. 89: 901–909.)

films were used and there was no growth of L. monocytogenes. Oregano essential oil (0.8% v/w) reduced the initial bacterial population by 2–3 logs, with limited aerobic growth and survival or death of L. monocytogenes observed under both MAP and VP, in both types of film at 5°C. These researchers discovered that use of low-permeability film controlled growth of L. monocytogenes throughout storage, regardless of the gaseous atmospheres. Sausages Experiments were done to determine survival of L. monocytogenes on sliced frankfurters incubated under 20–80% CO2 at 4, 7, and 10°C for up to 6 weeks of storage [148]. Within the commercial minimum shelf life of 3 weeks at 4°C, L. monocytogenes populations increased in vacuum packs, 20% CO2, and 30% CO2 by 2.5, 1, and 0.5 logs, respectively, but growth was inhibited in the presence of both 50 and 80% CO2. Upon continued storage for another 3 weeks, the organism grew in the presence of 50%, but not 80% CO2. However, increasing the storage temperature to 7°C allowed for growth of the organism within the 3-week storage period even in the presence of 80% CO2. Therefore, under commercial conditions (4 to 10°C, 3-week shelf life), only 80% CO2 was able to inhibit growth of the organism. However, this level of CO2 caused undesirable organoleptic changes in the product, and it was suggested that as a compromise, perhaps CO2 levels between 50 and 80% should be used [148]. Lamb Sheridan et al. [207] examined growth of L. monocytogenes on both raw and minced lamb pieces stored under various gas atmospheres (vacuum; 80% O2:20% CO2; 50% O2:50% CO2; and 100%

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TABLE 13.13 Mean Total Aerobic (TA) and L. monocytogenes (LM) Counts (Log10 CFU/g) at 42 Days on Lamb Pieces and Mince Packaged in Different Gas Atmospheres at 5°C Pieces

Meat Type Gas atmosphere Air Vacuum pack 80% O2/20% CO2 50% CO2/50% N2 100% CO2

Mince

TA

LM

TA

LM

8.86 7.53 8.91 8.15 7.55

6.07 3.81 4.91 4.22 1.35

9.2 8.65 8.97 7.75 8.07

5.37 1.74 2.68 3.68 0.58

Although not clearly stated, the initial total count appears to be 2.8 × 101 CFU/g.

a

Source: Adapted from Sheridan, J.J., A. Doherty, P. Allen, D.A. McDowell, I.S. Blair, and D. Harrington. 1995. Investigations on the growth of Listeria monocytogenes on lamb packaged under modified atmospheres. Food Microbiol. 12: 259–66.

CO2) at 0 and 5°C. In all instances, the organism did not grow on lamb stored at 0°C. Moreover, at 5°C under vacuum, L. monocytogenes grew on lamb pieces, but not on minced lamb (Table 13.13). After 42 days of storage at 5°C, besides the air-stored samples, the organism grew best on lamb and mince pieces stored under 80% O2:20% CO2 and 50% O2:50% CO2, respectively. Again, L. monocytogenes did not grow on lamb stored under 100% CO2. Pork In studies done to examine the microbial ecology of fresh MAP pork stored at various temperatures, listeriae were found to be one of the predominant organisms on product stored at −1°C, but not on samples stored at 4.4 or 10°C [174]. In fact, most of the flora, producing bacteriocins with a wide spectrum of activity, were isolated from samples stored at the two higher temperatures. Growth of the organism on fresh pork longissimus dorsi was examined. No growth of the organism was observed at 1°C, regardless of storage atmosphere. At 7°C storage, L. monocytogenes grew on the air-stored samples, but not on those stored under 100% N2, 80% O2:20% CO2, or 60% O2:40% CO2 [166]. No additional hazards could be observed by using modified atmosphere (MA) for packaging pork of normal pH. Although no results were actually presented, Davies [67] found that under an atmosphere of 80% O2:20% CO2, growth of L. monocytogenes on cooked ham was not greater than that in the aerobically stored controls. Manu-Tawiah et al. [167] examined the influence of CO2 levels of 20 and 40% on growth of L. monocytogenes and Yersinia enterocolitica on fresh pork chops stored at 4°C for 35 days. In general, levels of L. monocytogenes in air- or vacuum-packaged pork were not significantly different from numbers in chops packaged in the gas atmospheres, with levels increasing about 1.5 to 2.0 logs to close to 106 CFU/cm2 (initial count of approximately 3.7 CFU/cm2) after 35 days. The growth rate of the organism, and of the aerobic psychrotrophic spoilage flora, was faster in air as compared to the gas atmospheres, and in all instances was slower than that of the aerobic psychrotrophic spoilage flora. In contrast to the results of Wimpfheimer et al. [237], no differences were observed in numbers of the organism on chops packed in 60% N2, 0% O2:40% CO2, or 50% N2:10% O2:40% CO2. Y. enterocolitica grew much better than L. monocytogenes in all gas atmospheres tested, increasing to close to 108 CFU/cm2 after 35 days at 4°C. The fact that Yersinia grew much better in MA-packaged than air-stored chops is disconcerting from a public health standpoint.

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The application of additional hurdles in conjunction with MAP is a strategy that will be more commonly used in the future. In an interesting study, the effect of nisin and MAP on growth and survival of L. monocytogenes on cooked pork tenderloin stored at 4 and 20°C was examined [80]. It was found that the organism was able to grow on pork tenderloin stored under 100% CO2 at both storage temperatures. However, when nisin (104 IU/mL) was added, the combination proved to be bactericidal (Table 13.14). A lower concentration of nisin (103 IU/mL) also proved to be effective for pork stored at 4°, but not at 20°C. At the same time, growth of Pseudomonas fragi, a spoilage organism, was observed. In general, numbers of pseudomonads were reduced in MA-stored samples, but growth of the organism was not affected by nisin [79]. The authors used the general concept of a “safety index,” which compares relative numbers of spoilage organisms to pathogens. In MApackaged plus nisin-treated samples, numbers of P. fragi increased with time relative to growth of L. monocytogenes. However, this was not observed in those samples not containing nisin. MAP has also been used in conjunction with irradiation in an attempt to control the growth of foodborne pathogens on raw pork [106]. In nonirradiated pork stored under an atmosphere of 75% N2:25% CO2, the organism increased about 2 logs in number in 9 days at 10°C, whereas the irradiated (1.75 kGy) control was at undetectable levels. Using a higher initial inoculum (106 vs. 103 CFU/g), similar results were obtained; that is, after 9 days at 10°C, total Listeria counts were around 4.5 × 104 and 1 × 108 CFU/g in the irradiated and nonirradiated treated samples, respectively. The benefits of using irradiation after meat packaging were evident, as during storage the lactic acid microflora outgrew L. monocytogenes as well as some other pathogens tested [106]. It was theorized that because Lactobacillus saké usually predominates in irradiated MA-packaged pork, sakacin A, a bacteriocin produced by this organism, may have been responsible for inhibiting the growth of L. monocytogenes. However, in both irradiated and nonirradiated samples, L. monocytogenes increased in number by about the same amount. Devlieghere et al. [70] found that growth of L. monocytogenes was not influenced when the level of lactic acid bacteria was <107 CFU/mL, but once

TABLE 13.14 Cumulative Changea in log CFU/g for L. monocytogenes on Cooked Tenderloin Stored in MAP at 4 and 20°C MAP TimeC (days)

6 12 18 24 30

100% Air with Nisinb

100% CO2 with Nisin

100% Air with Nisinb

100% CO2 with Nisin

0

104

0

104

Timed (days)

0

104

0

104

2.78 4.28 5.03 4.97 4.81

−2.11 −2.1 –2.12 –2.15 –2.18

0.65 2.16 4.97 4.74 4.67

–2.18 –2.18 –2.19 –2.18 –2.18

1 2 4 7 10

4.2 5.24 5.36 5.64 6.07

–2.1 –2.11 –2.13 –2.14 –2.15

1.9 4.32 5.57 5.67 5.53

–2.1 –2.11 –2.1 –2.13 –2.14

(Log CFU/g at day x − Log CFU/g at day zero) for two samples per treatment per day. The concentration of L. monocytogenes on the cooked tenderloin surface at day zero was 3.18 log10CFU/g. bThe unit for nisin activity was IU/mL. cStorage temperature of 4°C. dStorage temperature of 20°C. a

Source: Adapted from Fang, T.J., and L.-W. Lin. 1994. Growth of Listeria monocytogenes and Pseudomonas fragi on cooked pork in a modified atmosphere packaging/nisin combination system. J. Food Prot. 57: 479–485.

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numbers reached greater than 107 CFU/mL, the concentration of lactic acid increased with a concurrent significant decrease in pH (data not shown). In this study, the researchers also compared mathematical models for prediction of growth and actual results. One can conclude that in most instances, L. monocytogenes will be able to grow at levels of CO2 up to 50%. Growth of the organism at levels greater than this will depend mainly on the interplay between the gas atmosphere, pH, temperature, and microbial competition. Thermal Inactivation in Meats Interest in the heat resistance of listeriae in raw meat products dates back to at least 1980 when Karaioannoglou and Xenos [142] reported on survival of Listeria in grilled meatballs. In their experiments, minced beef inoculated to contain 102, 103, 104, or 105 L. monocytogenes CFU/g, was combined with eggs, bread, onion, garlic, salt, and spices, and then fashioned into meatballs weighing 35–40 g each. Meatballs were then placed on a coal-fired grill at 110 to 120°C and cooked for 15 min until they attained an internal temperature of 78–85°C. After grilling, L. monocytogenes was isolated from all meatballs that originally contained 104–105 listeriae per g. However, the pathogen was discovered in only one of four meatballs inoculated to contain 103 listeriae per g and was absent from meatballs that originally contained 102 listeriae per g. Because data from the European surveys indicate that retail raw beef occasionally may contain up to 103 Listeria CFU/g (some of which are likely to be L. monocytogenes), thorough cooking of raw meat is presently advised to eliminate L. monocytogenes as well as salmonellae, pathogenic strains of E. coli, and other organisms that have been associated with foodborne illness. Although these findings attest to the hardy nature of listeriae in fresh ground beef, L. monocytogenes also was equally tenacious in artificially contaminated frozen ground beef, with populations remaining unchanged at 105 CFU/g during 6 months of storage at −18°C. Concern about the possible resistance of L. monocytogenes to pasteurization of milk, along with detection of this pathogen in cooked meats, prompted interest in the possibility of its surviving thermal processing steps commonly used to convert raw meat into RTE products. Boyle et al. [41] investigated the thermal destruction of L. monocytogenes strain Scott A in ground beef (approximately 20% fat) by submerging sealed tubes containing ground beef with 108 listeriae per g in a water bath at 75°C until the internal temperature of samples was 50, 60, 65, or 70°C. Samples then were examined for listeriae by direct plating and selective and cold enrichment. According to these researchers, Listeria levels did not decrease in samples heated to an internal temperature of 50°C over 6.2 min. Numbers of listeriae decreased 4.4 to 6.1 orders of magnitude in samples of ground beef during 8.4 and 10.6 min of heating to 60 and 65°C, respectively; similar results also were reported in 1988 by Farber et al. [86]. Ground pork heated to 62°C over 25 min was sufficient to inactivate 5.8–7.35 logs/g of L. monocytogenes, depending on the pork formulation. In general, it was found that most of the additives such as kappa-carrageenan, sodium lactate, and algin or calcium binders used in the ground pork formulations did not influence thermal inactivation of the organism [240]. Similarly, Yen et al. [242] found that sodium phosphates, sodium erythorbate, and added water had little or no effect on survival of various strains of L. monocytogenes during heating of ground pork. Interestingly, the same authors found that although, as noticed previously [83,241], addition of cure resulted in 2.0–2.2 logs less inactivation of the organism in ground pork cooked to 62°C, this protective effect was only seen at temperatures below 67°C. There have been several studies done to determine the D- and z-values of L. monocytogenes in various ground and whole-meat products (Table 13.15). D60°C values for most products are in the range of 1.8 to 8.3 min. Carlier et al. [51] examined destruction of L. monocytogenes during the cooking of whole hams to an internal temperature of 58.8°C. During storage of the hams for 2 months at 9°C, survivors were found among the hams inoculated with around 4 × 105 CFU/g, but not in those inoculated with <10 CFU/g. It was suggested that a minimum core temperature of 65°C be attained in these products, with an F70°C-value of at least 40 min. Survival of L. monocytogenes

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TABLE 13.15 Heat Resistance of L. monocytogenes in Meats Product Ground beef Fermented sausage mix Ground beef roast Ground beef Beaker sausage Meat slurry Meat slurry Beef Beef steak Liver sausage slurry Lean ground beef Fatty ground beef Meats (predicted value) Ham Ground pork Ground pork Minced beef, in vacutainer Minced beef, in vacuum pack Vacuum-packed minced beef, late logarithmic LM pH 5.6/6.2 Vacuum-packed minced beef, late stationary LM pH 5.6/6.2 Hot dog batter, composite culture, stationary phase Hot dog batter, composite culture, starvation Hot dog batter, outbreak strain, stationary phase Hot dog batter, outbreak strain, starvation Beef, pH 5.4, log, no heat shock Beef, pH 5.4, log, heat-shocked Beef, pH 5.4, stationary, no heat shock Beef, pH 5.4, stationary, heat-shocked Pork, heating 1.3°C/min Pork, heating 2.2°C/min Pork, heating 8.0°C/min Sausage, 23% beef, 77% pork Sausage, pork only Minced beef in vacutainers Minced beef in vacutainers Minced beef in vacuum pack Minced beef with LM NCTC11994, no prior heat treatment Minced beef with LM NCTC11994, prior heat treatment

Temperature (°C)

D-value (°C/min)

z-Value (°C)

60 60 60 60 60 60 70 60 60 70 60 70 60 62.8 62.8 60 70 60 60 62 60 60 60 60

3.12 16.7 4.47 1.62 9.13 2.54 0.23 7.3 3.8 0.14 8.32, 6.27 0.20, 0.14 2.42 0.6 1.2 3.82 0.13 1.82 3.48a 6.5–7.7 1.14–1.7 0.31 0.15 2.87/4.39

5.3 4.6

60

8.23/8.79

62 62 62 62 60 60 60 60 60 60 60 60 60 60 60 60 55

3.2 ± 0.5 3.3 ± 0.3 1.8 ± 0.5 1.8 ± 0.6 3.07 3.83 8.65 7.88 9.2 6.2 5.5 9.13 7.3 0.33 0.31 0.15 6.66

55

5.57

Reference 86 201

41 6.8 7.2

191 163

5.98, 5.98

92

6.2 9.3 11.4 6.8

39 81 163

5.05 6.74

50 146

5.05–5.45

188 40 112

5.9 6.0 5.6 5.2

10.0 6.8

169 169 169 169 74 74 74 74 74 74 74 74 74 72 40 40 230 230

(continued)

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TABLE 13.15 (CONTINUED) Heat Resistance of L. monocytogenes in Meats Product Minced beef with LM M63, no prior heat treatment Minced beef with LM M63, prior heat treatment Minced beef with LM NCTC 12480, no prior heat treatment Minced beef with LM NCTC 12480, prior heat treatment Minced beef with LM M102, no prior heat treatment Minced beef with LM M102, prior heat treatment Beef with 0% sodium lactate and 0.0% sodium diacetate Beef with 2.4% sodium lactate and 0.1% sodium diacetate Beef with 4.8% sodium lactate and 0.1% sodium diacetate Franks (L. innocua) Beef patties (L. innocua) Beef–turkey patties

Temperature (°C)

D-value (°C/min)

z-Value (°C)

55 55 55

4.54 3.98 5.13

230 230 230

55

4.58

230

55 55 60

5.53 4.57 4.67

230 230 139

60

10.37

139

60

12.31

60 60 60

5.52 3.25 6.66

ND

Reference

139 183 183 183

a

Cells heat-shocked at 42°C for 1 h.

in vacuum-packaged, nitrite-free beef roasts prepared with brines containing selected antimicrobial agents was examined [221]. The brines used included sodium chloride, sodium tripolyphosphate, Brifisol 414™, acetic acid, sodium lactate, Lauralac™, and potassium sorbate. Meats were cooked in a bag once or twice to 62.8°C under conditions simulating product contamination from pumping brines or from postcooking slicing or cutting. Some of the salient findings from this study were that (1) survival of L. monocytogenes on the surface of the beef roasts was surprisingly high, considering that cells had possibly been exposed to temperatures in the 80°C range for more than 30 min; (2) regardless of the number of cookings, the survival rates for listeriae were similar in internally cooked inoculated roasts for all brine treatments except NaCl-sodium tripolyphosphate, giving rise to the possibility that sublethal heating may have induced a heat shock response in these organisms; (3) the highest incidence of listeriae-positive samples was seen in those roasts processed to most closely resemble product currently being marketed, i.e., standard NaCl-phosphate brines; and (4) greatest destruction of the organism was observed with brines containing a phosphate blend and sodium lactate or glycerol monolaurin in combination with one cooking, and more so with two cookings [221]. Steam surface pasteurization of meats is gaining acceptance as a means of reducing levels of pathogenic microorganisms on meats. Some advantages of using steam pasteurization include the following: (1) the entire surface of a carcass can be uniformly heated, (2) irregularly shaped surfaces can be uniformly covered, (3) wastewater accumulation is not an issue, and (4) the process is not subject to operator misuse. A study was designed in which frankfurters inoculated with L. innocua were steam-pasteurized in a small pasteurizer designed in-house. The heating chamber was evacuated for 15 sec, and then the product was exposed to steam at a set pressure until the desired treatment time was reached. Treatment times of 32 and 40 sec at 136 and 115°C, respectively, led to a 4-log reduction in counts of L. innocua on the meat surface, with only a slight effect on color or weight [66]. Another study compared steam pasteurization (S; 15 sec) to traditional methods of reducing pathogens on meat surfaces [189]. The latter methods, which were tested both individually

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TABLE 13.16 Effectiveness of Combination and Individual Decontamination Treatments in Reducing L. monocytogenes on Surfaces of Freshly Slaughtered Beef Treatmenta TW TWS WS VW VWS TWLS VWLS

Initialb 5.52 5.57 5.46 5.56 5.49 5.51 5.54

± ± ± ± ± ± ±

0.22 0.15 0.15 0.12 0.04 0.19 0.13

Mean Red.c

Treatmenta

± ± ± ± ± ± ±

T W V S VWLS*5 VWLS*10

4.96 4.56 4.40 3.49 3.84 5.07 5.01

0.34d 0.34de 0.34def 0.34f 0.34ef 0.34d 0.34d

Initialb

Mean Red.c

± ± ± ± ± ±

2.54 ± 0.33f 1.28 ± 0.33g 3.33 ± 0.33ef 3.44 ± 0.33ef 4.51 ± 0.33d 4.23 ± 0.33de

5.26 5.27 5.39 5.38 5.18 5.21

0.33 0.35 0.20 0.16 0.35 0.33

Order of treatment within abbreviation indicates order of application. T = trim; W = 35°C water wash; S = 15-sec steam pasteurization; V = hot water/steam vacuum spot cleaning; L = 2% lactic acid spray; S*5 and S*10 = 5- and 10sec exposure time, respectively, for steam pasteurization. bMean initial pathogen population (log CFU/cm2) from four replications ± standard error of mean. cMean reduction in pathogen population (log CFU/cm2) from four replications ± standard error of mean. d,e,f,gMeans having the same superscript within columns are not significantly different (P < 0.05).

a

Source: Adapted from Phebus, R.K., A.L. Nutsh, D.E. Schafer, R.C. Wilson, M.J. Rieman, J.D. Leising, C.L. Kastner, J.R. Wolf, and R.K. Prasai. 1997. Comparison of steam pasteurization and other methods for reduction of pathogens on surfaces of freshly slaughtered beef. J. Food Prot. 60: 476–484.

and in combination, included knife trimming (T), water washing (W; 35°C), hot water/steam vacuum spot cleaning (V), and spraying with 2% vol/vol lactic acid of pH 2.25 at 54°C (L). All combination treatments were effective in reducing the numbers of L. monocytogenes on the meat surface, with TWLS, VWLS, and TW treatments (the order of treatment within the abbreviation indicates the order of application) being the most and the VW and VWS the least effective (Table 13.16). The authors concluded that higher levels of pathogen reduction can be attained through use of combinations of decontamination treatments, with knife trimming and/or steam vacuum spot cleaning (to remove visible contamination), followed by steam pasteurization, being a very effective intervention strategy that can be used by the meat industry [189]. Dorsa et al. [73] also examined effects of steam vacuuming (SV) and a hot water spray wash of 74 ± 2°C (W) on L. innocua present on the surface of beef carcass tissue. Initial reductions of 2.0, 2.2–2.5, and 2.6–2.7 CFU/cm2 were observed after the SV, W, and SV + W treatments, respectively. However, after storage of the meat for 21 days at 5°C, numbers of L. innocua on all treated meat samples were the same as those of the untreated control. It should be mentioned, however, that the initial loads of listeriae on meat were high (106 CFU/cm2) and that with lower initial levels, outgrowth of the organism may not have occurred or may have been minimal. Bréand et al. [43] studied the influence of time and mild temperature on L. monocytogenes survival curves in trypticase soy broth. They applied 11 different temperatures to stationary-phase L. monocytogenes CIP 7831 (ATCC 35152) ranging from 53 to 60°C for 0 to 250 min for temperatures between 53 and 55°C, and 0 to 120 min for temperatures between 56 and 60°C. Results showed that the linear relationship between the logarithm of the death rate and the temperature inducing death was also valid for mild temperatures. The study also demonstrated that mathematical modeling permitted building a new strategy to determine the best mild heat treatment in which the pasteurization tables based on the Bigelow law [39a] are no longer appropriate.

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Doherty et al. [72] performed studies on heat inactivation of L. monocytogenes in minced beef and minced beef homogenate in vacuum bags or vacuum containers. Results showed no significant differences in the D-values at 50, 55, or 60°C. However, these D-values were lower than previously reported for L. monocytogenes in ground meat by Mackey et al. [163]. Studies by several researchers [72,182,194,230] have shown that postpackage pasteurization of meats proved effective in reducing or eliminating L. monocytogenes from inoculated samples. Muriana et al. [182] looked at the effect of heat on inactivation of L. monocytogenes on roast beef and smoked ham deli products. Inactivation rates differed, depending on the temperature and substrate (Figure 13.5). Miller et al. [179] used a cold shock treatment of 0 to 15°C for 1 to 3 h to increase the thermal sensitivity and thermal inactivation of L. monocytogenes. Taormina and Beuchat [214] found that the alkaline-stress response in L. monocytogenes may induce a response which leads to greater resistance to otherwise lethal thermal processing conditions, and that exposure to chlorine increased thermal sensitivity. In a later study, these researchers discovered that alkalistressed cells that are not alkali-adapted have lower D59°C-values than alkaline-cleaner-exposed cells [215]. Growth on frankfurters at 4 and 12°C was more delayed with alkali-adapted cells than with cells exposed to an alkaline cleaner [215]. Doyle et al. [74], in a review of the heat resistance of L. monocytogenes, mention that heat resistance varies depending on the strain, age of culture, growth conditions, recovery media, and characteristics of the particular food, such as salt content, aw , pH, and presence of inhibitors. They also describe L. monocytogenes having a higher heat resistance than most other foodborne pathogens that are non-spore-forming and that criteria aimed at the elimination of E. coli or Salmonella spp. may not be sufficient for elimination of L. monocytogenes. In summary then, several points regarding the heat resistance of L. monocytogenes in meat can be stated: (1) the organism appears to be about four times more heat resistant than salmonellae in meats; (2) attention should be paid to those meats that are heated slowly, a process that may induce the production of heat shock or stress proteins in these and other organisms; (3) D-values obtained for Roast Beef

-1

-1

-2

-2

-3 -4 -5

-3 -4 -5 -6

-6 -7

Smoked Ham

0

Log reduction

Log reduction

0

0

10 20 30 40 50 60 70 80 90 100 110 120

-7

0

10

20 30

40 50

Time (s) D145F= 95.2 D150F= 54.1 D155F= 12.5 D160F= 4.0

seconds seconds seconds seconds

60 70 80

90 100 110 120

Time (s)

62.8

65.6

68.3

71

D145F= 67.6 D150F= 29.6 D155F= 15.4 D160F= 5.5

seconds seconds seconds seconds

----------

----------

Zroastbeef = 10.3°F (5.7°C)

Zham = 14.2°F (7.9°C)

FIGURE 13.5 Regression trend lines and associated D- and z-values for L. monocytogenes heated in purge from ready-to-eat (RTE) roast beef and smoked ham deli products. Heating regimens were performed at 145°F (62.8°C), 150°F (65.6°C), 155°F (68.3°C), and 160°F (71°C). (Adapted from Muriana, P.M., W. Quimby, C.A. Davidson, and J. Grooms. 2002. Postpackage pasteurization of ready-to-eat deli meats by submersion heating for reduction of Listeria monocytogenes. J. Food Prot. 65: 963–969.)

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meats can vary quite widely, depending on the prior history of the food and organism, recovery methodology, and strain type; (4) in general, the D-values for L. monocytogenes in meat are higher than those for dairy products; (5) cured meats require greater heating than noncured products to ensure the same level of protection; (6) combinations of decontamination treatments work best for eliminating the organism from carcass surfaces; and (7) heat-in-bag thermal processes which avoid the possibilities of in-plant postprocessing contamination should be encouraged.

BACTERIOCINS

FOR

CONTROLLING LISTERIAE

IN

MEAT

There have been several review articles describing the potential for using bacteriocins and/or lactic acid bacteria for controlling Listeria spp. in foods [181], and more specifically in meats [177,199,213]. The general use of bacteriocins to control listeriae has been dealt with in other chapters of this book. Therefore, a summary of the information related specifically to meat products follows. Raw Ground Meat Buchanan and Klawitter [45] were among the first investigators to look at the application of bacteriocin-producing cultures to control L. monocytogenes in foods, investigating the potential for using Carnobacterium piscicola strain LK5 to inhibit growth of the organism in various foods. Foods were inoculated with 103 CFU/g L. monocytogenes, either with or without 104 CFU/g of the LK5 strain. In sterile raw ground beef, strain LK5 inactivated the organism at 5°C and prevented its growth at 19°C. C. piscicola did not have any effect on the organism in nonsterile ground beef (or chicken roll); however, L. monocytogenes did not grow on the control products. In general, the bacteriocin-producing strain was most effective when the background microflora on the foods was low. Similar studies with sterile and nonsterile ground beef were done with another lactic acid bacterium, Lactobacillus casei as well as its associated bacteriocin, lactocin 705 [227]. Meat was inoculated with either the pure bacteriocin or the L. casei strain and then stored at 20°C for 24 h. In general, the reduction in numbers of L. monocytogenes was greatest with the highest levels of lactocin 705 tested (800 AU/mL) [16], with the fewest numbers of L. monocytogenes present in the meat slurry, and with autoclaved ground beef. Starter cultures also have been assessed for their ability to inhibit L. monocytogenes in minimally heat-treated vacuum-packed beef cubes either with or without gravy and/or glucose. For these experiments, Lactobacillus bavaricus strain MN was inoculated into beef at 105 or 103 CFU/g, along with 102 CFU/g L. monocytogenes, then vacuum-sealed, and stored at 4 or 10°C [238]. Several results were obtained: (1) strain MN grew and produced bacteriocin at refrigeration temperatures, although it was much more effective at inhibiting L. monocytogenes at the lower storage temperature; (2) the higher inoculum level used was significantly more effective in reducing levels of L. monocytogenes; (3) bacteriocin production was independent of the presence of glucose in the meat; (4) addition of sugar enhanced the antilisterial activity even though the pH was not greatly reduced in those meats with gravy and glucose; (5) the antilisterial activity was greater in those meats containing gravy with glucose, suggesting that the gravy may have enhanced diffusion of the bacteriocin; and (6) bavaricin production occurred during the early stages of growth of strain MN. The authors concluded that the food industry may find the use of bacteriocinogenic lactic acid bacteria attractive. Bacteriocin-producing lactic acid bacteria have also been used to control growth of L. monocytogenes on frankfurters. In these experiments, either high (107 CFU/g) or low (103–104) levels of a bacteriocin-producing strain of Pediococcus acidilactici, as well as its plasmid-cured, bacteriocin-negative derivative (bac−), were inoculated separately onto wieners along with a fivestrain cocktail of L. monocytogenes. Frankfurters were stored both aerobically and anaerobically at 4 and 15°C. In general, presence of P. acidilactici on the frankfurters inhibited listeriae to various degrees, depending on the pediococci levels, storage temperature, and package atmosphere [36]. Yousef et al. [243] examined the ability of L. monocytogenes to grow in wiener exudates. The organism

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grew in two of the five exudates tested, with the best growth being observed in those exudates containing the lowest amount of phenols, the lowest indigenous levels of lactic acid bacteria, and the highest pH. However, exudates stored at 4°C did not support growth of the organism. Those exudates that did not support growth of the organism proved to be listericidal. Previous work done by Glass and Doyle [101] showed that L. monocytogenes could grow on wieners stored at 4.4°C. Differences in results between the two studies could have resulted from loss of wiener exudate during manipulation, uneven distribution of exudate on the wiener surface, or variations in the intensity of smoking within and among different brands [243]. Both P. acidilactici H and pure pediocin AcH inactivated listeriae that were inoculated into one brand of wiener exudate which had been stored at 25°C for 8 days. In the control samples, L. monocytogenes increased in number from approximately 104 to 107 CFU/g within 4 days. Following up on this work, the latter authors used the same lactic acid bacteria in an attempt to control the growth of L. monocytogenes in temperature-abused vacuum-packaged wieners stored at 4 and 25°C. At 25°C, the presence of P. acidilactici strain LB42 inhibited growth of L. monocytogenes, but was not listericidal. In contrast, P. acidilactici strain JBL1095 was listericidal, decreasing counts of L. monocytogenes by 2.7 logs. This inactivation was not caused solely by pH, as pH values in wieners inoculated with both strains of pediococci were similar. The only difference between the two strains was that one (JBL1095) produced pediocin AcH and the other did not, strongly suggesting that this bacteriocin enhanced the antilisterial activity of lactic acid bacteria in vacuum-packaged wieners. Amezquita and Brashears [4] examined the use of lactic acid bacteria for competitive inhibition of L. monocytogenes in RTE meat products. Three cultures of lactic acid bacteria (LAB) were used as they were shown to exert the greatest effect on L. monocytogenes growth, exhibiting not only antilisterial activity, but an inhibitory capacity as well. With cooked ham, LAB cultures proved to be bacteriostatic, but with frankfurters, LAB were antilisterial. In frankfurters, LAB had an antilisterial effect whether they were used singly or in combined cultures. Several researchers [102,170,203] have examined the potential antilisterial effects of sodium lactate and sodium diacetate in meat, wieners, and RTE meats. Overall, there was a direct correlation between concentration of inhibitory substances and inhibition of L. monocytogenes. In addition, there was a synergistic effect when these additives were used in combination (Figure 13.3). Fermented Sausages A number of studies have examined the effects of starter cultures and their bacteriocins on survival and growth of L. monocytogenes on sausages. Foegeding et al. [90] used pediocin-producing strains of P. acidilactici (along with an isogenic mutant as a control) to control growth of L. monocytogenes on dry-fermented American-style sausages. Recovery of the organisms during the fermentation was made easier by using antibiotic-resistant strains of both the pediococci and listeriae. The study was unique in showing that in combination with other fermentation end products, inhibition of L. monocytogenes was enhanced by bacteriocin production in situ during fermentation and drying. Berry et al. [35] had also previously shown the benefits of using P. acidilactici as a starter culture to control growth of listeriae during sausage fermentation. According to these authors, a commercial summer sausage mix was inoculated to contain ~106 CFU/g of L. monocytogenes and fermented with either bacteriocin-producing or non-bacteriocin-producing strains of P. acidilactici. Following 12 to 14 h of fermentation at 37.8°C, populations of listeriae decreased approximately 100- and 10-fold in summer sausage fermented with bacteriocin-producing and non-bacteriocin-producing strains of P. acidilactici, respectively. The pathogen also was inactivated in sausages with slower acid production (pH > 5.5), which suggests that bacteriocin production occurred independent of carbohydrate fermentation. However, L. monocytogenes was still detected in 9 of 90 (10.0%) sausage samples that had been heated to an internal temperature of 64°C and then refrigerated for up to 2 weeks. Thus, although use of this bacteriocin-producing starter culture led to a dramatic decrease in numbers of viable listeriae in summer sausage, it did not completely eliminate the

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pathogen in finished product prepared from sausage mix containing 106 CFU/g of L. monocytogenes. However, because the presence of >103 L. monocytogenes/g in commercially prepared sausage mix is highly unlikely, it appears that current heat treatments are adequate to produce a Listeria-free semidry sausage. Another starter culture, L. saké, was found to control growth of L. monocytogenes on German-style fresh sausages. Results varied depending upon the initial pH of the “fresh Mettwurst,” i.e., at initial pH values of 6.3 and 5.7, and bacteriostatic and bacteriocidal (1-log reduction) effects on L. monocytogenes, respectively, were observed. In another study using the same starter culture [126], a bacteriocin-producing (sakacin K) strain of L. saké, isolated from a naturally fermented sausage, was able to reduce numbers of L. monocytogenes in dry-fermented sausages by 1.25 logs, as compared to the isogenic bac− control strain. Lactobacillus plantarum has also been used as a starter culture to study the fate of L. monocytogenes in naturally and artificially contaminated salami [48]. In this study, the starter cultures prevented growth of listeriae, but were not listericidal. In inoculated samples, little difference was observed between samples inoculated with bac+ or bac− strains of L. plantarum. However, in naturally contaminated samples, the only samples that were free of listeriae were those inoculated with the bacteriocin-producing strain of L. plantarum. Lahti et al. [152] examined survival and detection of E. coli and L. monocytogenes during manufacture of dry sausage, using two different starter cultures. Starter culture A contained Staphylococcus xylosus DD-3, bacteriocin-producing P. acidilactici PA-2 and Lactobacillus bavaricus MI-401, and starter culture B contained S. carnosus MIII and L. curvatus Lb3. Sausage batter (35% pork, 35% beef, 30% fat) was inoculated with high (5.05–5.41) or medium (2.92–3.35) log10 CFU/g levels of L. monocytogenes serovar 4b, manufactured (fermented and dried) in a smoke chamber at 17–23°C for 15 days, and then stored at 15–17°C for 35 days. The sausages were smoked for 30 min during days 2–5. L. monocytogenes decreased more rapidly in the high-inoculum sausages produced with starter A than with starter B, but no significant difference was found between the starters in the medium-inoculated sausages. This pathogen was eliminated from the mediuminoculated sausages after 49 days (Figure 13.6). Bacteriocins have also been used in studies aimed at inactivating or preventing the attachment of L. monocytogenes to muscle surfaces. El-Khateib et al. [77] investigated the ability of lactic acid, nisin, and pediocin to inactivate L. monocytogenes on beef muscle, as well as to prevent its attachment after short-term exposure and during 48 h of refrigerated storage. The latter experiments were done to simulate secondary contamination of meat during refrigerated storage. Lactic acid (2%), nisin (4 × 104 IU/mL), and pediocin (3.2 × 103 AU/mL) decreased numbers of the organism on the meat surface by 1.7, 1.1, and 0.6 log10 CFU/6 cm2 (cubical piece), respectively. Interestingly, in comparison to the control, lactic acid treatment caused a greater percentage of listeriae cells to attach to meat surfaces. This was partially attributed to acidic conditions that can sometimes enhance adhesion of bacteria to surfaces [77]. In general, the percentage of L. monocytogenes cells attached to beef muscle in the presence of the two bacteriocins did not change significantly or slightly decreased, although after 1 h, nisin- and pediocin-treated samples had 30 and 17% cells, respectively, attached, as compared to the respective controls with 6.3 and 12%. Other work done by Cutter and Siragusa [64,65] has shown nisin to be effective in reducing (by 2.0 to 2.83 log10 CFU/cm2) numbers of L. innocua in beef. The latter study showed the combination of vacuumpackaging and nisin spraying to be effective in suppressing growth of L. innocua enough so that at the end of the 4-week incubation period at 4°C, counts were lower than the corresponding nontreated controls. In summary, bacteriocins, especially pediocin, appear to be good candidates for controlling growth of listeriae in meats, as they have proved to be effective in controlling growth of the organism in wieners and wiener exudates, semidry and dry sausages, and fresh beef and pork. The main obstacles at present to their use in foods are development of bacteriocin resistance, activity loss with time, inactivation by proteases, and adsorption to meat and fat particles resulting in inactivation, limited diffusion (especially in minced meats and poultry), and finally regulatory acceptance.

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High-inoculum starter A

1000000

Medium-inoculum starter A High-inoculum starter B Medium-inoculum starter B

100000

CFU/g

10000

1000

100

10

1 0

3

7

14

21

35

49

Time (d)

FIGURE 13.6 Survival of L. monocytogenes in dry sausage produced using starter cultures A (Flora-Carn LC) and B (Müller RM 52). Counts are given as CFU/g. High-inoculum starter A(♦), medium-inoculum starter A (•), high-inoculum starter B(
), medium-inoculum starter B(). Values of high inoculum are means of six subsamples and values of medium inoculum are means of three subsamples. The value 1 CFU/g represents counts below the enumeration limit (<3 CFU/g) but detected by enrichment. The value 0 CFU/g represents counts below enumeration limit (3 CFU/g) and not detected by enrichment. (Adapted from Lahti, E., T. Johansson, T. Honkanen-Buzalski, P. Hill, and E. Nurmi. 2001. Survival and detection of Escherichia coli O157:H7 and Listeria monocytogenes during the manufacture of dry sausage using two different starter cultures. Food Microbiol. 18: 75–85.)

REFERENCES 1. Adesiyun, A.A. 1993. Prevalence of Listeria spp., Campylobacter spp., Salmonella spp., Yersinia spp. and toxigenic Escherichia coli on meat and seafoods in Trinidad. Food Microbiol. 10: 395–403. 2. Adesiyun, A.A. and C. Krishnan. 1995. Occurrence of Yersinia enterocolitica O:3, Listeria monocytogenes O:4 and thermophilic Campylobacter spp. in slaughter pigs and carcasses in Trinidad. Food Microbiol. 12: 99–107. 3. Aguado, V., A.I. Vitas, and I. Garcia-Jalon. 2001. Random amplified polymorphic DNA typing applied to the study of cross-contamination by Listeria monocytogenes in processed food products. J. Food Prot. 64: 716–720. 4. Amézquita, A. and M.M. Brashears. 2002. Competitive inhibition of Listeria monocytogenes in readyto-eat meat products by lactic acid bacteria. J. Food Prot. 65: 316–325. 5. Amoril, J.G. and A.K. Bhunia. 1999. Immunological and cytopathogenic properties of Listeria monocytogenes isolated from naturally contaminated meats. J. Food Saf.19: 195–207. 6. Amtsberg, G. von, A. Elsner, H.A. Gabbar, and W. Winkenwerder. 1969. Die epidemiologissche und lebensmittelhygienische Bedeutung der Listerieninfektion des Rindes. Dtsch. tierärztl. Wschr. 76: 497–536. 7. Antoniollo, P.C., F. da S. Bandiera, M.M. Jantzen, E.H. Duval, and W.P. da Silva. 2003. Prevalence of listeria spp. in feces and carcasses at a lamb packing plant in Brazil. J. Food Prot. 66: 328–330. 8. Anonymous. 1988. AMI data show frankfurters most likely Listeria carrier in meat. Food Chem. News 30(16): 37–39. 9. Anonymous. 1988. Hot dog processing “borderline” for Listeria destruction: ARS. Food Chem. News 30(41): 46. 10. Anonymous. 1988. Listeria destruction in cooked meat products ineffective: Hormel. Food Chem. News 30(15): 32–34.

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11. Anonymous. 1989. Listeria in raw meat restricted in Germany. Food Chem. News 31(31): 28–29. 12. Anonymous. 1989–1994. Annual Reports. Agri-Food Safety Division, Food Production and Inspection Branch Agriculture, Canada, ON. 13. Anonymous. 1989–1996. Food recalls. Health Protection Branch, Health and Welfare Canada, ON. 14. Anonymous. 1990. The Microbiological Safety of Food. Part 1. Report of the Committee on the Microbiological Safety of Food. HMSO. p.137. 15. Anonymous. 1990. Data needed to change zero Listeria tolerance stance: FDA. Food Chem. News 32(40): 9–10. 16. Anonymous. 1991. Listeria in food. Report of the West and North Yorkshire Joint Working Group on a two year survey of the presence of Listeria in food. Environ. Health 99: 132–137. 17. Anonymous. 1991. All L. monocytogenes said to be potentially pathogenic. Food Chem. News 32(45): 18–19. 18. Anonymous. 1991. Skinless hot dogs recalled for Listeria. Food Chem. News 33(19): 39. 19. Anonymous. 1991. Ham salad recalled due to Listeria. Food Chem. News 33(40): 38. 20. Anonymous. 1991–1997. Food and Drug Administration Enforcement Reports, 1991–1997. Food and Drug Administration, Washington, DC. 21. Anonymous. 1992. Frankfurters recalled by Connecticut firm for Listeria. Food Chem. News 34(5): 51–52. 22. Anonymous. 1992. Mrs. Drake, Good ‘n’ Fresh sandwiches recalled due to Listeria. Food Chem. News 34(30): 36. 23. Anonymous. 1992. Class I recalls involve botulinum, Listeria. Food Chem. News 34(36): 42. 24. Anonymous. 1993. Listeria found in 20% of hot dogs in L.A. Times survey. Food Chem. News 35(21): 45–46. 25. Anonymous. 1995. FSIS issues snapshot of raw meat, poultry microbiological profile. Food Chem. News 37(43): 19–20. 26. Anonymous. 1996. FDA targets high risk foods in pathogen monitoring program. Food Chem. News 38(8): 15–16. 27. Anonymous. 1997. Possible L. monocytogenes contamination prompts recall of “fresh” sandwiches. Food Chem. News 38(49): 41. 28. Anonymous. 1997. Food recalls. Canadian Food Inspection Agency, Canada, ON. 29. Anonymous. 1997. Microbiological monitoring of ready-to-eat products, 1993–1996. United States Department of Agriculture, Food Safety and Inspection Service, Washington, DC. 29a. Anonymous. 1998. Gould’s smoked breakfast ham recalled from New Hampshire for Listeria. USDA–FSIS release, July 31. 30. Arumugaswamy, R.K., G.R.R. Ali, and S.N. Hamid. 1994. Prevalence of Listeria monocytogenes in foods in Malaysia. Int. J. Food Microbiol. 23: 117–121. 31. Avery, S.M., J.A. Hudson, and N. Penney. 1994. Inhibition of Listeria monocytogenes on normal ultimate pH beef (pH 5.3–5.5) at abusive storage temperatures by saturated carbon dioxide controlled atmosphere packaging. J. Food Prot. 57: 331–333. 32. Avery, A.M., A.R. Rogers, and R.G. Bell. 1995. Continued inhibitory effect of carbon dioxide packaging on Listeria monocytogenes and other microorganisms on normal pH beef during abusive retail display. Int. J. Sci. Tech. 30: 725–735. 33. Barbosa, W.B., J.N. Sofos, G.R. Schmidt, and G.C. Smith. 1995. Growth potential of individual strains of Listeria monocytogenes in fresh vacuum-packaged refrigerated ground top rounds of beef. J. Food Prot. 58: 398–403. 34. Benezet, A., J.M. De La Osa, M. Boras, N. Olmo, and F.P. Florea. 1993. Study of Listeria monocytogenes in meat products. Alimentaria 30: 19–23. 35. Berry, E.D., M.B. Liewen, R.W. Mandigo, and R.W. Hutkins. 1990. Inhibition of Listeria monocytogenes by bacteriocin-producing Pediococcus during manufacturing of fermented semidry sausage. J. Food Prot. 53: 194–197. 36. Berry, E.D., R.W. Hutkins, and R.W. Mandigo. 1991. The use of bacteriocin-producing Pediococcus acidilactici to control post processing Listeria monocytogenes contamination of frankfurters. J. Food Prot. 54: 681–686. 37. Beuchat, L.R., R.E. Brackett, D.Y.-Y. Hao, and D.E. Conner. 1986. Growth and thermal inactivation of Listeria monocytogenes in cabbage juice. Can. J. Microbiol. 32: 791–795.

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Internet-Based References 246. http://www.fsis.usda.gov/OA/background/lmfinal.htm. 247. http://www.fsis.usda.gov/OA/recalls/rec_pr.htm. 248. http://www.inspection.gc.ca/english/corpaffr/recarapp/recal2e.shtml.

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and Behavior 14 Incidence of Listeria monocytogenes in Poultry and Egg Products Elliot T. Ryser CONTENTS Introduction ....................................................................................................................................572 USDA–FSIS Listeria-Testing Program for Cooked/Ready-to-Eat Poultry Products ...................572 Listeria Contamination in Cooked/Ready-to-Eat Poultry Marketed outside the United States ............................................................................................................................574 Incidence of Listeria spp. in Raw Poultry Marketed in the United States ..................................577 Chicken .................................................................................................................................577 Turkey ...................................................................................................................................579 Incidence of Listeria spp. in Poultry Products Marketed in Europe and Elsewhere ...................580 Raw Poultry ..........................................................................................................................580 United Kingdom .......................................................................................................580 Continental Europe...................................................................................................584 Elsewhere..................................................................................................................585 Cooked/Ready-to-Eat Poultry ..............................................................................................586 Behavior of L. monocytogenes in Raw and Cooked Poultry Products.........................................587 Growth: Raw Chicken ..........................................................................................................587 Antimicrobial Additives ...........................................................................................589 Susceptibility to Gamma Radiation .........................................................................589 Growth: Cooked/Ready-to-Eat Poultry Products.................................................................590 Thermal Inactivation During Cooking .................................................................................593 USDA Guidelines for Control of Listeria in Ready-to-Eat Poultry .............................................597 Postpackaging Pasteurization ...............................................................................................598 Antimicrobial Agents for Listeria Growth Suppression......................................................598 Egg Products ..................................................................................................................................599 Incidence...............................................................................................................................600 Growth ..................................................................................................................................601 Thermal Inactivation.............................................................................................................604 References ......................................................................................................................................606

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INTRODUCTION Avian listeriosis was first recognized in 1932 when TenBroeck isolated Listeria monocytogenes (then Bacterium monocytogenes) from diseased chickens [186,187]. Chickens have remained a common avian host for L. monocytogenes since avian listeriosis was first recognized. Listeriosis also has been observed in at least 22 other avian species, including such frequently consumed fowl as turkeys [32,93,158], ducks [84,188], geese [84,162], and pheasants [84]. Large outbreaks of listeriosis in domestic fowl are relatively rare [157]; however, sporadic cases occur much more frequently and are often accompanied by asymptomatic shedding of Listeria in feces. According to one report, 4.7% of cecal samples from Danish broiler chickens harbored L. monocytogenes [162]. Hence, as was true of beef, pork, and lamb, poultry meat destined for human consumption also is at risk of becoming contaminated with L. monocytogenes from the abattoir environment during defeathering, evisceration, and further processing. Several early studies suggested that poultryand egg-processing workers could contract Listeria infections by handling contaminated birds [65,108,109]. Additionally, several reports from England during the 1970s indicated that L. monocytogenes could be isolated with some frequency from raw chickens as well as turkeys, ducks, and pheasants. An added concern with poultry relates to eggs that can become contaminated with this pathogen during collection and processing. Emergence of L. monocytogenes as a bona fide foodborne pathogen following the Mexicanstyle cheese outbreak of 1985 prompted immediate concern about the presence of Listeria in dairy products and also generated a parallel interest in the incidence and behavior of L monocytogenes in meat and poultry products; the latter is the topic of this chapter. Interest in this area has increased dramatically since 1988 as a result of four major listeriosis outbreaks that were directly linked to consumption of turkey frankfurters [83] and ready-to-eat delicatessen turkey meat [49,70,163] in the United States. In this chapter, information concerning the incidence, behavior, and control of L. monocytogenes in poultry products will be reviewed first, followed by a discussion of L. monocytogenes in egg products.

USDA–FSIS LISTERIA-TESTING PROGRAM FOR COOKED/READY-TO-EAT POULTRY PRODUCTS Public health concerns regarding Listeria-contaminated raw and, particularly, processed ready-toeat poultry products sold in the United States also stem directly from the 1985 listeriosis outbreak in California that was associated with consumption of Mexican-style cheese. Soon thereafter, U.S. Department of Agriculture-Food Safety Inspection Service (USDA–FSIS) officials announced their intention to develop a Listeria-monitoring/verification program for cooked and ready-to-eat meat as well as poultry products based on product type. Because the monitoring/verification programs for meat and poultry products developed in parallel and were both similar in terms of sampling scheme, methodology, and action taken when L. monocytogenes was found, the following discussion focuses on the various poultry products tested and pertinent results rather than on an in-depth analysis of these programs. A Listeria-monitoring/verification program for cooked/ready-to-eat poultry was first suggested in December 1985 and was to cover all such products prepared in federally inspected establishments as well as those produced by certified foreign manufacturers [94]. However, actual testing of poultry sausage, that is, the first category of ready-to-eat poultry examined, did not begin until September 1988, 1 year after the Listeria-monitoring/verification program was first instituted for cooked beef, roast beef, and cooked corned beef. Before April 1989, the USDA’s Listeria policy, which gave firms the opportunity to clean up their facility before additional verification samples were analyzed under hold-test procedures,

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was consistent with the fact that listeriosis had not yet been linked to consumption of poultry products. However, the official USDA–FSIS position regarding the presence of L. monocytogenes in cooked ready-to-eat poultry products changed radically on April 14, 1989, when Centers for Disease Control and Prevention (CDC) investigators directly linked consumption of contaminated turkey frankfurters to an instance of listerial meningitis in a breast cancer patient in Oklahoma [15]. After isolating L. monocytogenes serotype 1/2a of the same electrophoretic enzyme type from the woman and opened, as well as unopened, retail packages of turkey frankfurters, USDA–FSIS officials requested that the manufacturer issue an immediate Class I recall for approximately 600,000 pounds of Texas-produced turkey frankfurters that were marketed by retail and institutional establishments in Alaska, Arizona, Arkansas, California, Florida, Georgia, Idaho, Illinois, Indiana, Kentucky, Louisiana, Mississippi, Missouri, New Jersey, New York, Ohio, Oklahoma, Pennsylvania, Tennessee, Texas, Utah, and Washington. As expected, this recall immediately prompted an intensified monitoring/verification program to determine the extent of Listeria contamination in a far wider range of cooked/ready-to-eat poultry products marketed in the United States. Despite initial attempts by the National Turkey Association to develop tolerance levels for L. monocytogenes in cooked and ready-to-eat poultry products [10], USDA–FSIS officials maintained that because an “acceptable level” of L. monocytogenes in such products could at that time not be determined, the only acceptable alternative was to adopt a “zero tolerance” for this pathogen in cooked/ready-to-eat poultry and meat products [11]. Consequently, under this program [56], which was identical to that developed for cooked and ready-to-eat red meat products, USDA–FSIS officials requested that firms issue a Class I recall for all lots of cooked and ready-to-eat poultry products in which L. monocytogenes was detected in monitoring samples taken from intact packages of product. However, Listeria-positive lots under direct control of the manufacturer could be recalled internally, thus avoiding adverse publicity. If the pathogen was initially detected in monitoring samples from unpackaged products, USDA–FSIS officials did not request that firms immediately recall the sampled lot. Instead, government officials analyzed subsequent lots and, if necessary, initiated further steps (i.e., hold-test programs) to prevent distribution of contaminated products. Our knowledge concerning the incidence of Listeria in cooked/ready-to-eat poultry products marketed in the United States initially came from the USDA–FSIS Listeria-monitoring/verification program, with the first results from 1987 to 1990 indicating that 1.5–2.0% of all such products were contaminated with L. monocytogenes [8,19,47]. Poultry products in which L. monocytogenes had been found during 1989 and 1990 included chicken patties [8], chicken salad [8], diced poultry [8], poultry salad [19], poultry spread [19], poultry frankfurters [8], poultry bologna [8], and turkey sausage [8]. In all likelihood, the pathogen entered these products during the latter stages of manufacture and/or packaging. By 1990, USDA–FSIS established nine different regulatory programs in which ready-to-eat meat and poultry products were collected at federally inspected establishments and tested for L. monocytogenes [119,210]. Four of these nine programs were entirely meat based. The remaining programs included poultry products; however, only one of these programs, entitled “Cooked Poultry Products—Uncured,” was solely devoted to poultry. Based on results from this USDA–FSIS program, 0.95 to 3.17% of all uncured cooked poultry products tested from 1990 to 2000 yielded L. monocytogenes with an average of 1.97% of the samples positive during this 11-year period (Figure 14.1). In December 2000, USDA–FSIS ceased testing according to product type and began collecting samples based on Hazard Analysis and Critical Control Point (HACCP) processing categories as defined in the Code of Federal Regulations for ready-to-eat products, with these new categories no longer definable in terms of meat or poultry. In one remaining report available, the Oregon State Department of Agriculture [164] surveyed a wide range of meat and poultry products that included breaded chicken (22 samples), ready-to-eat

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FIGURE 14.1 USDA–FSIS monitoring program for L. monocytogenes in cooked poultry products: 1990–2000. (From Levine, P., B. Rose, S. Green, G. Ransom, and W. Hill. 2001. Pathogen testing of ready-to-eat meat and poultry products collected at federally inspected establishments in the United States, 1990 to 1999. J. Food Prot. 64: 1188–1193; USDA–FSIS. 2005. Electronic reading room: microbiological testing program. Available at: http://www.fsis.usda.gov/ophs/rtetest/rettable6.htm.)

chicken (72 samples), sliced turkey breast (118 samples), turkey ham (8 samples), turkey pastrami (7 samples), and ready to-eat turkey (4 samples). From this list of products, L. monocytogenes was only recovered from 3 samples—2 (2.8%) ready-to-eat chicken and 1 (14.3%) turkey pastrami— with all others negative for L. monocytogenes by enrichment. Formal Class I recalls for ready-to-eat poultry products were quite limited during the first few years of the USDA–FSIS Listeria-monitoring/verification program and include the aforementioned recall of turkey frankfurters [15], two recalls of chicken salad [5,7], two recalls of deli sandwiches containing turkey and /or chicken [12,18], and one additional incident involving 13,000 lb of chicken spread produced by a Virginia-based firm [16]. Beginning in 1994, recall information is more complete, with 9 recalls totaling less than 25,000 lb of product issued through September 1998 (Table 14.1). However, this situation changed dramatically on December 22, 1998 when 35 million pounds of turkey frankfurters and deli meats were recalled in response to a nationwide listeriosis outbreak involving 108 cases, including 14 deaths and 4 miscarriages/stillbirths, in 22 states [83]. This widely publicized outbreak prompted increased testing of ready-to-eat poultry which, in turn, greatly increased the number of Listeria-related Class I recalls. In 2000 and 2002, two additional recalls involving 16.9 and 27.4 million pounds of delicatessen turkey meat, respectively, were issued when both products were traced to two separate nationwide outbreaks of listeriosis [49,163]. All totaled, 76 Class I recalls have been issued from 1994 to October 2005 for 124.6 million pounds of ready-to-eat poultry products contaminated with L. monocytogenes. Of these 76 recalls, 48 (63%) involved cooked chicken products, with the remaining 14 (18%), 6 (8%), 5 (7%), and 3 (4%) traced to manufacturers of ready-to-eat turkey, turkey and chicken, duck, and unspecified poultry products, respectively.

LISTERIA CONTAMINATION IN COOKED/READY-TO-EAT POULTRY MARKETED OUTSIDE THE UNITED STATES Since 1997, several small European surveys that included samples of various ready-to-eat poultry products have been conducted. Working in Belgium, Rijpens et al. [175] reportedly recovered L. monocytogenes from 7 of 36 (29%) samples of ready-to-eat sliced turkey and

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TABLE 14.1 Class I Recalls Issued in the United States for Ready-to-Eat Poultry Products Contaminated with L. monocytogenes during 1994–2005 Product Chicken salad Chicken salad Smoked chicken sausage Chicken salad Duck confit Chicken salad Duck rillettes Chicken breast and broccoli Cooked chicken Chicken and turkey frankfurters Turkey frankfurters and deli meats Poultry hot dogs and deli meats Chicken burritos Turkey franks Chicken salad Sliced turkey and other luncheon meats Roast chicken meat, ground Chicken nuggets, patties Sliced turkey breast and other meats Chicken nuggets Sliced turkey breast and other meats Chicken enchiladas Chicken salad Chicken salad Turkey breast and other meats Chicken salad spread Chicken caesar salad Cooked turkey and chicken Duck Chicken wings Mexican-style poultry products Duck Duck Turkey Frozen chicken dinners Chicken fajitas Frozen chicken Turkey ham Fajita chicken strips Smoked turkey and turkey ham Chicken/pork hot dogs Turkey bologna Turkey frankfurters Chicken salad

Date of Recall 3/23/94 5/24/94 7/1/94 8/31/94 10/26/94 5/5/95 5/19/95 6/5/95 6/2/97 10/22/98 12/22/98 1/22/99 2/2/99 8/27/99 9/9/99 12/14/99 1/28/00 2/23/00 5/3/00 5/9/00 5/24/00 5/30/00 7/6/00 7/17/00 8/8/00 9/27/00 10/10/00 12/14/00 1/16/01 1/24/01 1/30/01 2/9/01 4/4/01 6/20/01 8/10/01 8/14/01 10/4/01 11/8/01 2/26/02 3/15/02 4/25/02 5/23/02 8/2/02 8/4/02

State of Manufacture California Tennessee California North Carolina New York North Carolina New Jersey Pennsylvania West Virginia Florida Michigan Arkansas Illinois California Pennsylvania New Jersey Michigan Alabama Michigan Puerto Rico Alabama New Mexico Alabama Massachusetts Idaho Minnesota Pennsylvania Texas Indiana New Jersey Illinois Indiana California Pennsylvania California California New Jersey Iowa Alabama Minnesota Ohio New York New York North Carolina

Quantity (lb) 278 1,643 2,894 1,953 88 105 270 50 17,434 1,734,002 35,000,000 35,000,000 26,975 33,710 240 900 4,500 160,760 215 7,083 45 45 5,376 3,300 380 80 90 16,895,000 4,400 2,100 Unknown 6,000 780 75 650 950 7,500 11,800 30,700 23,000 140,000 Unspecified 3,400 2,200 (continued)

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TABLE 14.1 (CONTINUED) Class I Recalls Issued in the United States for Ready-to-Eat Poultry Products Contaminated with L. monocytogenes during 1994–October 2006 Product Turkey and chicken breast Turkey and chicken breast Turkey and chicken breast Turkey and chicken breast Chicken frankfurters Chicken salad Turkey, ham, roast beef Cooked poultry Turkey and ham Cooked diced chicken breast Chicken salad Cooked chicken Cooked chicken Chicken products Chicken products Chicken salad Chicken dumplings Chicken products Chicken wrap sandwiches Smoked turkey and pork products Turkey and ham wraps Chicken breast wraps Chicken salad Cooked chicken meat Chicken products Chicken salad Chicken frankfurters Chicken sausages Poultry products Chicken products Chicken salad Turkey and chicken products

Date of Recall 10/9/02 10/12/02 11/2/02 11/20/02 1/3/03 5/5/03 9/10/03 9/12/03 11/18/03 11/25/03 12/11/03 7/1/04 7/21/04 11/3/04 1/31/05 3/15/05 3/16/05 3/28/05 4/5/05 4/11/05 4/26/05 4/28/05 6/15/05 7/21/05 7/28/05 8/31/05 9/20/05 10/2/05 10/21/05 11/8/05 11/10/05 12/1/05

State of Manufacture

Quantity (lb)

Pennsylvania Pennsylvania New Jersey New Jersey New York Illinois California Massachusetts Florida Georgia Florida North Carolina North Carolina Maryland New York New York California California Florida New York Texas New York New York Georgia New York Oklahoma New York Illinois Massachusetts California Pennsylvania Missouri

295,000 27,400,000 200,000 4,200,000 26,400 400 550 9,230 1,135 7,500 2,700 404,730 36,980 1,275 5,760 250 12,090 12,500 3,316 39,000 191 385 5,065 170 3,200 23,435 23,040 1,000 11,200 275 5,523 2,800,000

Source: From USDA–FSIS. 2005. FSIS Recalls. Available at: http: //www.fsis.usda.gov/Fsis Recalls/index.asp.

chicken purchased locally for sandwiches. The fact that none of seven similarly packaged samples contained L. monocytogenes suggests that contamination most likely occurred during slicing of the product at retail, as suggested in recent surveys of prepackaged and sliced deli meats. This hypothesis is further supported by the work of Vorst et al. [217] that demonstrated extensive transfer of L. monocytogenes between delicatessen slicer blades and roast turkey breast during slicing. However, if the Belgian data for unpackaged product are excluded, then the overall L. monocytogenes contamination rate of 1.1% for ready-to-eat poultry products marketed in Europe is similar to that reported by USDA–FSIS for similar products available in the United States.

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INCIDENCE OF LISTERIA SPP. IN RAW POULTRY MARKETED IN THE UNITED STATES Immediately after the 1985 listeriosis outbreak in California was announced, public concerns were raised about safety of dairy products and other potentially contaminated foods, including meat and poultry products. Interest in the safety of raw poultry soon escalated after a nationwide telecast informing the general public that 50% of all raw chicken marketed in the United States was contaminated with Salmonella. Beginning in the late 1980s, several surveys were initiated to determine the extent to which raw chicken and turkey meat are contaminated with Listeria and other foodborne pathogens.

CHICKEN The aforementioned concerns prompted USDA–FSIS officials to initiate a poultry back/neck testing program for L. monocytogenes, Salmonella, and Escherichia coli serotype O157:H7 in January of 1989. L. monocytogenes and Salmonella were detected in 508 of 2686 (18.9%) and 792 of 2739 (28.9%) samples, respectively, with E. coli O157:H7 being absent from 2696 samples [19]. About the same time, Bailey et al. [25] assessed the incidence of L. monocytogenes and Listeria spp. on the surface of broiler carcasses processed in the southeastern United States and also compared L. monocytogenes serotypes isolated from raw chicken with those associated with human cases of listeriosis. Using an enrichment procedure together with three selective plating media, Listeria spp. were detected in rinse samples from 34 of 90 (37.8%) broiler carcasses; however, recovery of Listeria varied widely with three lots of 10 birds each being reported as negative. More important, 21 of 90 (23.3%) carcasses yielded L. monocytogenes, with 64, 18, 6, and 12% of the isolates being identified as serotypes 1/2b, 1/2c, 3b, and nontypeable strains, respectively. Although only 7 of 115 (6.1%) L. monocytogenes isolates from clinical listeriosis cases in the United States were of serotype 1/2b or 1/2c, the fact that most L. monocytogenes strains isolated from chickens were pathogenic to mice suggested chicken meat as a possible vehicle of listeriosis in humans. Between June 1988 and May 1989, Genigeorgis et al. [72,73] conducted two large surveys that examined the incidence of Listeria spp. on fresh and/or semifrozen chicken and turkey parts obtained from retail sources and slaughterhouse. According to their results for chicken, 12.5% of fresh wings, 16.0% of fresh legs, and 15.0% of fresh livers purchased at three supermarkets in northern California contained detectable levels of L. monocytogenes (Table 14.2). Furthermore, with the exception of fresh chicken liver, L. innocua was generally two to three times more prevalent in the remaining samples than was L. monocytogenes. Overall, the highest incidence of Listeria spp. was observed for fresh legs (54.0%), followed by fresh wings (42.5%) and fresh livers (32.5%). In contrast to fresh products, only 10% of semifrozen chicken wings, legs, and livers contained Listeria spp. However, finding L. monocytogenes alone in 1 of 10 semifrozen legs and livers attests to the ability of this pathogen to survive in semifrozen raw chicken and turkey, as was also observed by Palumbo and Williams [166]. In addition to these efforts, Genigeorgis et al. [72] also attempted to trace the route of Listeria contamination on fresh chicken wings, legs, and livers by examining samples at various stages of production and storage. Although all chicken parts at the start of production were free of L. monocytogenes, results in Table 14.3 indicate that most contamination occurred during the latter stages of production when carcasses came in direct contact with Listeria-laden fecal material, because at the time of packaging, 70, 30, and 33% of chicken wings, legs, and livers contained L. monocytogenes, respectively. Not surprisingly, L. innocua, which was virtually absent from chicken parts at the beginning of production, also was routinely isolated from wings, livers, and particularly legs at the end of production. Despite these relatively high contamination rates, both Listeria spp. failed to grow on all three packaged products during the first 4 days of refrigerated storage. Wimpfheimer et al. [222] observed a 3- to 4-day lag phase for L. monocytogenes when inoculated

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TABLE 14.2 Incidence of Listeria spp. on Fresh and/or Semifrozen Chicken and Turkey Parts Purchased from Three California Supermarkets between June 1988 and May 1989 Number of Parts Examined

Type and Part of Poultry Chicken Wings Legs Livers Turkey Wings Legs Tails

Number (%) of Positive Parts L. monocytogenes

L. innocua

14 (35.0) 1 (10.0) 19 (38.0) 0 8 (20.0) 0

1 (2.5) 0 0 0 1 (2.5) 0

17a (42.5) 1 (10.0) 27 (54.0) 1 (10.0) 13a (32.5) 1 (10.0)

3 (5.0) 0 0

12 (20.0) 9 (15.0) 7 (11.7)

27 (45.0) 17 (28.3) 14 (23.2)

Fresh Semifrozen Fresh Semifrozen Fresh Semifrozen

40 10 50 10 40 10

5 0 8 1 6 1

(12.5)

Fresh Fresh Fresh

60 60 60

12 (20.0) 8 (13.3) 7 (11.7)

(16.0) (10.0) (15.0) (10.0)

L. welshimeri

Total Listeria spp.

a

Some chicken parts contained both L. monocytogenes and L. innocua or L. innocua and L. welshimeri.

Source: Adapted from Genigeorgis, C.A., D. Dutulescu, and J. F. Garayzabal. 1989. Prevalence of Listeria spp. in poultry meat at the supermarket and slaughterhouse level. J. Food Prot. 52: 618–624; Genigeorgis, C.A., P. Oanca, and D. Dutulescu. 1990. Prevalence of Listeria spp. in turkey meat at the supermarket and slaughterhouse level. J. Food Prot. 53: 282–288.

samples of raw minced chicken were held at 4°C. Given this information, the failure of Genigeorgis et al. [72] to detect growth of L. monocytogenes and L. innocua on naturally contaminated packaged chicken parts is not surprising. In a 1997 study by Cox et al. [55] to determine how often L. monocytogenes enters poultry processing plants on live chickens, L. monocytogenes was infrequently found in hatchery samples and on the exterior of fully grown birds. Although L. monocytogenes was not recovered from the intestinal tract of broiler chickens at the time of slaughter, 25% of postprocessing and retail-level carcasses contained L. monocytogenes. In another study involving perorally dosed chicks, Husu et al. [99] reported that L. monocytogenes was generally eliminated within 9 days, which again suggests that intestinal carriage of L. monocytogenes is most often transient. The scientific literature relating to pathogens commonly associated with processed poultry is extensive, and a review of this literature has been published [218]. In another review paper [106] covering the years 1971–1994, the prevalence of L. monocytogenes in meats was highly variable, with approximately 16% of the products being positive. In general, the highest numbers of L. monocytogenes have been found in processed meat and poultry products, with fresh meats generally containing much lower numbers. In raw chicken, serotypes 1/2a, 1/2b, and 1/2c are encountered more often than serotype 4b, with the latter most often associated with outbreaks of foodborne listeriosis [216]. Thus, chicken, in contrast to turkey, has remained an uncommon vehicle for infection. However, in 1996, Ryser et al. [182] identified Listeria spp. in 34 of 45 retail raw chicken pieces, with 11 different L. monocytogenes ribotypes, including three ribotypes associated with previous foodborne listeriosis cases, being detected among the Listeria-positive samples. These findings suggest the presence of multiple incoming sources of contamination and/or heavily contaminated single sites within the poultry processing environment, including

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floors, drains, and conveyor belts [33,50,138]. Hence, routine disinfection procedures are essential to minimize the presence of both transient and persistent strains of Listeria that may colonize the processing environment. It is important to remember that, similar to other meats, poultry products also can be used for purposes other than human consumption. Al-Sheddy and Richter [2] determined the incidence of L. monocytogenes in frozen ground meat that contained raw chicken together with chopped beef by-products. Although not conclusive, recovery of L. monocytogenes from all five samples examined, as well as the fact that this pathogen is more commonly found in chicken rather than beef products, suggests that raw chicken was the most likely source of contamination. Hence, considering the high incidence of L. monocytogenes in raw chicken, it may be prudent to eliminate raw poultry products from the diet of zoo animals to curb the number of listeriosis cases occurring in zoological parks.

TURKEY Chickens and other types of domesticated fowl are similarly processed. Hence, one would also expect to isolate various Listeria spp., including L. monocytogenes, from the surface of other raw poultry products, such as turkey [161], ducks [20], and pheasants [113]. After completing the aforementioned chicken part survey in California [72], Genigeorgis et al. [73] initiated a similar study to determine the prevalence of various Listeria spp. on fresh turkey parts obtained from retail sources and slaughterhouses. Listeria contamination rates for fresh turkey were generally similar to those previously observed for fresh chicken, with 45.0% of turkey wings, 28.3% of legs, and 23.3% of tails obtained from three northern California supermarkets harboring various Listeria spp. (see Table 14.2). Although their findings further demonstrate that L. monocytogenes is equally common on fresh chicken and turkey parts, with isolation rates of 10.0–16.0% and 11.7–20.0%, respectively, the same cannot be said of L. innocua and L. welshimeri. In fact, L. innocua, the Listeria sp. most commonly detected on fresh chicken in this study, as well as several others [85,167,225], was recovered from only 3 of 180 (1.7%) fresh turkey parts. Similarly, L. welshimeri, the dominant Listeria sp. on fresh turkey, was only rarely observed on fresh chicken. Although both surveys were confined to fresh chicken and poultry parts available from three local supermarkets, these findings still suggest that chickens and turkeys may be preferential hosts for L. innocua and L. welshimeri, respectively, with the latter most likely to harbor L. monocytogenes strains belonging to serotype 4b—the serotype that continues to be most frequently associated with foodborne listeriosis outbreaks, including those traced to turkey [83,163]. In a subsequent survey [53], 9 of 42 turkey skin samples harbored L. monocytogenes, with the contamination rate apparently unrelated to the incidence in flocks before processing or after defeathering. As was true for fresh chicken, additional testing at a local slaughterhouse once again demonstrated that fresh turkey parts are most likely to become contaminated with Listeria during the latter stages of processing (i.e., evisceration and chilling) (see Table 14.3). This scheme, mentioned earlier as the route by which fresh poultry becomes contaminated, was further confirmed by identifying various Listeria spp., including L. monocytogenes, in 4 of 15 (26.7%) samples of mechanically deboned raw turkey meat obtained from the same slaughterhouse. In a similar study, Wesley et al. [221] recovered L. monocytogenes and other Listeria spp. from 57 (38%) and 29 (19%) of 150 samples of mechanically separated turkey meat that were obtained from one midwestern turkey processor. Among the 57 isolates serotyped, 29 (51%) and 25 (44%) belonged to serotypes 1 and 4, respectively. Ryser et al. [182] further stressed the importance of postprocessing contamination during separation and grinding when they reported that 33 of 45 (73%) retail samples of ground turkey contained Listeria spp., including a diverse group of L. monocytogenes strains belonging to nine different ribotypes.

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TABLE 14.3 Incidence of Listeria spp. on Commercially Produced Fresh Chicken and Turkey Parts before and after Being Packaged and/or Stored at 4°C Production Line

Type and Part of Poultry Chicken Wings Legs Livers Turkey Wings Legs Livers

Listeria sp.

Beginning

End

L. monocytogenes L. innocua L. monocytogenes L. innocua L. monocytogenes L. innocua L. monocytogenes L. welshimeri L. monocytogenes L. welshimeri L. monocytogenes L. welshimeri

0/20a 0/20 0/20 0/20 0/31 2/31 (6.5)b 1/30 (3.3) 1/30 (3.3) 0/30 1/30 (3.3) 0/30 1/30 (3.3)

21/30 (70) 6/30 (20) c 11/30 (37) 19/30 d (67) 5/15 (33) 4/15 (27) 0/30 4/30 (13.3) 2/30 (6.7) 1/30 (3.3) 0/30 5/30 (16.7)

4-Day-Old Packaged Product Stored at 4°C 18/25 (72) 4/25 (16) 13/25 (52) 17/25 (68) 6/15 (40) 4/15 (27) ND ND ND ND ND ND

ND, not determined. a

Number of positive parts/number of parts examined. Percentage positive. cStrain of L. welshimeri also detected. b

Source: Adapted from Refs. 72 and 73.

INCIDENCE OF LISTERIA SPP. IN POULTRY PRODUCTS MARKETED IN EUROPE AND ELSEWHERE RAW POULTRY European scientists have been aware of the possible relationship between listeriosis in fowl and humans for nearly 50 years, as evidenced by several reports in which infected poultry was found in the immediate vicinity of human cases. Considering the fecal carriage rate for L. monocytogenes in domestic and wild fowl, as well as the mechanized slaughtering practices that result in fecal contamination of carcasses during defeathering, evisceration, and subsequent chilling in “spinchillers,” it is not surprising that interest in the incidence of Listeria in poultry has increased. However, given that the first European case of avian listeriosis was diagnosed in England during 1936 [169] and that no additional cases were recorded in England over the next 22 years [84], one would probably be surprised to learn that our first knowledge concerning the incidence of Listeria in European raw poultry products originates from surveys made in England and Wales during the 1970s. United Kingdom After identifying L. monocytogenes in human stool samples from 32 of 5100 (0.6%) asymptomatic individuals who resided in an area of Wales in which clinical cases of listeriosis had not been identified for 15 years, Kwantes and Isaac [112] postulated that the fecal carriage rate may be related to food consumption and began surveying both fresh and frozen chicken for L. monocytogenes. Following a 1971 preliminary report [112], Kwantes and Isaac [113] published final results of their study in 1975 when they reported detecting L. monocytogenes on the internal/external

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surface of 27 of 51 (52.9%) raw chickens obtained from a local processor (Table 14.4), with 23 (85.2%) and 4 (14.8%) L. monocytogenes isolates being identified as serotype 1 and 4b, respectively. To determine actual public exposure to contaminated poultry, these investigators went to homes of poultry consumers in Wales and swabbed the external/internal surfaces of locally purchased fresh and frozen chicken, turkey, duck, and pheasant carcasses. Overall, L. monocytogenes was isolated from 50% of fresh chickens sampled from home refrigerators, as well as from 64% of frozen chickens stored in home freezers, thus demonstrating the ability of this pathogen to persist on frozen carcasses. However, unlike chickens obtained directly from processors, L. monocytogenes isolates of serotype 4b outnumbered those of serotype 1 on fresh and, particularly, on frozen chickens obtained from consumers’ homes. Although relatively few samples were examined, isolation of L. monocytogenes from the internal/external surface of one turkey, three ducks, and one wild pheasant (see Table 14.4) indicates that improperly handled poultry products other than chicken may also pose a potential threat to consumers. One year later, Gitter [78] published results from a similar study, which examined the incidence of L. monocytogenes on surfaces of various “over-ready” poultry products purchased at 26 different shops and supermarkets in southern England. Using a combination of direct plating and cold enrichment, L. monocytogenes was identified on 7 of 56 (12.5%) frozen and 2 of 6 (33.3%) fresh chickens, as well as on 1 of 2 frozen ducks (see Table 14.4). Although the incidence of L. monocytogenes on raw poultry products was markedly lower than that previously found by Kwantes and Isaac [113], L. monocytogenes isolates identified as serotype 4 again outnumbered those of serotype 1/2. Following emergence of L. monocytogenes as a serious foodborne pathogen in June 1985, Pini and Gilbert [173] determined the prevalence of this pathogen in uncooked fresh and frozen chickens obtained from retail outlets throughout London. Unlike previous studies that relied on swab samples from carcasses, these researchers examined two different samples from each chicken carcass whenever possible—one sample consisting of edible offal (trimmings and/or viscera) and the other a composite sample of skin and carcass remnants. Using cold enrichment in conjunction with the Food and Drug Administration (FDA) procedure, L. monocytogenes was recovered from 33 of 50 (66%) fresh and 27 of 50 (54%) frozen chickens. These results are similar to those reported from other 1987–1994 surveys [77,97,125,131,174] in which L. monocytogenes was detected on 10 of 16 (62.5%), 60 of 100 (60%), and 21 of 32 (66%) fresh chicken carcasses marketed in England and Wales (see Table 14.4). According to a second report by Pini and Gilbert [172], other Listeria spp., including L. innocua, L. seeligeri, and L. welshimeri, also were detected either alone or together with L. monocytogenes in 26 and 30% of the fresh and frozen chicken samples tested, respectively. Overall, 74 L. monocytogenes strains representing serotypes 1/2, 3a, 3b, 3c, 4b, 4d, and two nontypeable strains, with serotype 1/2 predominating, were isolated from 60 edible offal and 100 composite samples. Composite samples yielded more isolates of L. monocytogenes (57%) as well as a higher percentage of other Listeria spp. (23%) than did edible offal samples (22 and 15%), respectively. In another survey from 1994, 91% of cooked poultry samples contained listeriae. Although L. monocytogenes was isolated from 59% of raw samples, all cooked samples were negative [116]. Among the chicken parts examined (e.g., drumsticks, breasts, wings, and livers), drumsticks were most frequently contaminated with listeriae and also harbored the highest populations. From January 1997 to May 1998, Uyttendaele et al. [212] surveyed fresh poultry carcasses and poultry products that were marketed in Belgium for Listeria. Among 44 samples originating from the United Kingdom, 6 and 28 yielded L. monocytogenes at levels of >1 CFU/100 cm2 or 25 g and >1 CFU/cm2, respectively, these levels being significantly higher than those detected in similar samples from Belgium and France. In the only other survey reported to date, Soultos et al. [199] recovered L. monocytogenes from 14 of 80 (17.5%) packages of chicken pieces that were purchased at various supermarkets in Northern Ireland from September to December 2002.

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TABLE 14.4 Incidence of L. monocytogenes in Raw Poultry Products Marketed in Europe and Elsewhere Country Europe Belgium Belgium and France

Denmark England/Wales

Finland France

Germany

Italy

Nordic countries Northern Ireland Norway Portugal

Type of Poultry

Fresh chicken carcasses and pieces Fresh chicken carcasses Fresh chicken parts Fresh turkey parts Fresh chicken Fresh turkey parts Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh chicken Turkey Frozen turkey Fresh turkey Duck Frozen duck Wild pheasant Fresh chicken pieces Fresh chicken carcasses and pieces Fresh chicken Fresh/frozen chicken Unspecified Unspecified Poultry Fresh chicken Fresh chicken Fresh chicken carcasses and pieces Fresh chicken Fresh chicken (supermarkets) Fresh chicken Fresh chicken pieces Fresh chicken Fresh chicken

Number of Samples Analyzed

Number of Positive Samples (%)

Reference

279

111 (39.8)

212

635 421 429 17 46 100 64 58 56 51 50 50 38 32 30 16 6 4 3 1 3 2 2 61 434

152 (23.9) 113 (26.8) 22 (5.3) 8 (47.1) 8 (17.4) 60 (60.0) 41 (64.0) 34 (58.6) 7 (12.5) 27 (52.9) 33 (66.0) 27 (54.0) 19 (50.0) 21 (65.6) 15 (50.0) 10 (62.5) 2 (33.3) 1 (25.0) 0 0 3 (100.0) 1 (50.0) 1 (50.0) 38 (62.0) 156 (65.9)

213 213 213 196 161 174 113 116 78 113 172,173 172,173 113 125 97 131 78 113 78 78 113 78 113 132 212

44 100 30 11 1,269 200 50 13

7 85 6 3 24 0 18 0

(15.9) (85.0) (20.0) (27.3) (1.9)

18 80

4 (22.2) 14 (17.5)

85 199

150 95 65 63

75 48 16 26

179 179 86 21

(36.0)

(50.0) (50.5) (24.6) (41.0)

50 185,201 165 201 40 54 71 212

(continued)

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TABLE 14.4 (CONTINUED) Incidence of L. monocytogenes in Raw Poultry Products Marketed in Europe and Elsewhere Country Spain

Sweden Switzerland Turkey United Kingdom

Type of Poultry Fresh chicken and turkey Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh minced chicken Fresh chicken carcasses and pieces

Total Elsewhere Brazil

Egypt India

Japan Korea Malaysia South Africa

United Arab Emirates Total

Number of Samples Analyzed 158 100 5 45 24 26 44 5478

Fresh and frozen chicken Fresh chicken carcasses at packaging Frozen squab Fresh chicken Poultry Fresh chicken Minced chicken Fresh chicken Fresh chicken Fresh chicken Fresh chicken carcasses (butcher) Frozen chicken carcasses (butcher) Fresh chicken carcasses (supermarket) Frozen chicken carcasses (supermarket) Fresh chicken carcasses (street vendor) Fresh chicken

90 21 50 37 50 9 46 86 30 16 15

Number of Positive Samples (%) 57 32 0 0 5 3 28

(36.1) (32.0)

(20.8) (11.5) (63.6)

Reference 216 43 198 202 38 225 212

1316 (24.8) 4 (4.4) 3 (14.3) 0 3 0 0 17 26 3 4 2

114 28

(0) (8.1) (0) (0) (37.0) (30.2) (10.0) (25.0) (13.3)

1 29 167 60 103 24 51 74 214

17

6 (35.3)

214

45

9 (20.0)

214

16

1 (6.3)

214

6

1 (16.7)

214

30

1 (3.3)

81

564

80 (14.2)

Despite wide differences in time, sample collection, sampling techniques, Listeria isolation methodologies, and types of samples analyzed in the aforementioned studies, averaging the results in Table 14.4 indicates that 196 of 447 (43.8%) fresh chickens and 75 of 170 (44.1%) frozen chickens marketed in the United Kingdom from 1971 to 2002 contained L. monocytogenes. That the 1986 findings of Pini and Gilbert [172,173] are similar to those obtained in both American and European surveys as far back as the mid-1970s underscores the continuing need for proper kitchen hygiene, cooking of raw poultry products, and continuous inspection of carcasses (e.g., identification of liver and heart lesions), along with use of good manufacturing and sanitizing practices in poultry processing facilities. Because 152 of 214 (71%) clinical L. monocytogenes isolates obtained from British patients between November 1986 and 1987 [130] were of serotype 4b, poultry products, in

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which L. monocytogenes serotype 1/2 predominates, may be a less common vehicle for listeriosis than other foods. However, pâtés, which are in essence poultry spreads prepared from chicken or goose liver, may be an exception. Continental Europe Concern about the incidence of L. monocytogenes in raw poultry products marketed outside the United Kingdom dates back to at least 1982 when two Swedish workers, Ternstrom and Molin [202], examined 45 chickens obtained from two local slaughterhouses (see Table 14.4). Although these researchers failed to isolate L. monocytogenes from any of the chickens examined, it appears that the Listeria isolation /detection methods used in this study were primarily responsible for their lack of success, because listeriosis in Swedish poultry is relatively common, with 112 cases being diagnosed in the 10-year period between 1948 and 1957 [158]. In support of this view, more recent surveys have yielded L. monocytogenes contamination rates of 47.1, 62.0, and 50.0 for fresh chickens marketed in Denmark [196], Finland [132], and Norway [179]. Given the high incidence of L. monocytogenes in raw poultry sold in the United States and the United Kingdom, inadequate isolation/detection methods of the early 1980s also were likely responsible for the inability of Comi and Cantoni [54] to recover this pathogen from approximately 200 chicken samples (i.e., carcass, skin, and entrails) obtained from slaughterhouses in northern Italy. After the 1985 cheeseborne listeriosis outbreak in California, Western European scientists began to determine the incidence of Listeria in a wide range of foods, including fresh poultry products. In the first of these studies, which was published in 1988, Skovgaard and Morgen [196] visited two large Danish poultry slaughterhouses and examined chilled chicken carcasses for evidence of Listeria contamination. According to these authors, Listeria spp. were detected in neck-skin samples from 16 of 17 (94.1%) chicken carcasses, with L. innocua being identified in all but two Listeriapositive samples. Although most of the poultry processed at these facilities was heavily contaminated with L. innocua, 8 of 17 (47.1%), 1 of 17 (5.9%), and 2 of 17 (11.8%) carcasses also contained detectable levels of L. monocytogenes (see Table 14.4), L. innocua, and other Listeria spp. (L. welshimeri, L. murrayi, and/or L. denitrificans), respectively. Thus the L. monocytogenes contamination rate for chickens processed in Denmark closely reflected the average (43.8%) for fresh chicken carcasses marketed in England and Wales. These Danish researchers also identified Listeria spp., including L. monocytogenes, in chicken feces and transport cage material, further supporting the widespread belief that poultry carcasses most likely become contaminated with Listeria during evisceration and subsequent handling. This theory was later confirmed by Ojeniyi et al. [162], who surveyed seven Danish abattoirs for L. monocytogenes. The pathogen was absent in fecal samples from over 2000 broilers representing 90 randomly selected flocks. However, L. monocytogenes was isolated from 0.3 to 18.7% of all poultry processing environmental samples examined. Based on serotyping, phage typing, pulsed-field gel electrophoresis, and ribotyping, 62 distinct L. monocytogenes strains were identified from the 247 isolates tested, several of which predominated in poultry processing samples. Interest in the incidence of Listeria in European fresh poultry again intensified following reports that 34 individuals in Switzerland died after consuming contaminated Vacherin Mont d’Or soft-ripened cheese. Breer [38] isolated L. monocytogenes, L. innocua, and L. seeligeri from 5 of 24 (20.8%), 6 of 24 (25.0%), and 1 of 24 (4.2%) raw chickens purchased in Switzerland (see Table 14.4), respectively. Using a modified version of the FDA procedure, German investigators [185,200] recovered Listeria spp. and L. monocytogenes from 94 of 100 (94%) and 85 of 100 (85%) chicken carcasses, respectively. However, results from two smaller surveys of German poultry [165,200] suggested far lower L. monocytogenes contamination rates, with only 20.0–27.3% of unspecified fresh poultry carcasses (presumably chicken) containing this pathogen (see Table 14.4). The incidence of Listeria in raw poultry products has received increased attention in Belgium and France, with L. monocytogenes recognized in French poultry since 1983 [34]. In the first large

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survey of poultry carcasses collected from 33 abattoirs in France and Belgium, Uyttendaele et at. [213] reported that the L. monocytogenes contamination rate decreased from 32.1% in 1992 to 9.2% in 1995, with 87–98% of the carcasses tested yielding L. monocytogenes at levels of <1 CFU/cm2. Contamination of poultry parts also decreased from 25.8 to 3% during this same 4-year period, with L. monocytogenes-positive samples primarily being confined to chicken legs and wings. In a follow-up study, Uyttendaele et al. [212] examined 772 poultry carcasses marketed in Belgium during January 1997 to May 1998, most of which originated from Belgium and France with a few samples also coming from Italy, The Netherlands, and the United Kingdom. Overall, 12.3 and 38.2% of these samples yielded L. monocytogenes at levels of >1 CFU/100 cm2 or 25 g and >1 CFU/cm2 or g, respectively, with generally similar contamination rates seen for samples coming from Belgium and France. However, L. monocytogenes was more prevalent in a subset of samples without skin (17.6%) rather than with skin (6.9%), suggesting contamination from the environment during processing. In one additional subset, L. monocytogenes was recovered from 34 of 78 (43.6%) and 41 of 82 (50.0%) French samples classified as free range and non–free range, respectively, with this difference not statistically significant. In 1995, Franco et al. [69] identified Listeria spp. on 96, 84, 80, and 0% of fresh chicken legs, wings, breasts, and livers, respectively, that were processed at a Spanish facility, with Listeria counts ranging from <2.0 to 2.8 log CFU/g on chicken wings. Follow-up testing of the processing environment again showed that most of the contamination occurred during the latter stages of production as carcasses entered the quartering room and exited on conveyor belts. Several additional surveys revealed L. monocytogenes contamination rates of 0 to 36.0, 24.6 to 41.0, and 11.5% for fresh chicken and turkeys marketed in Italy [40,71,126], Portugal [21,86], and Turkey [225], respectively. Overall, these findings from continental Europe (Table 14.4) show that fresh poultry is a common source of L. monocytogenes, with 1031 of 4898 (21.1%) samples taken primarily from fresh chicken carcasses testing positive. Elsewhere At least 12 small-scale surveys for Listeria in raw poultry products from the Middle East, Asia, Africa, and South America are now recorded in the scientific literature (see Table 14.4). In one of these studies, 1 of 30 domestically grown fresh chickens from the United Arab Emirates [81] reportedly contained Listeria spp., with one of these carcasses being positive for L. monocytogenes. However, Listeria spp. were detected on 32 of 39 (82%) imported frozen chickens, 18 (46%) of which contained L. monocytogenes. Similarly, Rusul et al. [74] identified L. monocytogenes in 4 of 16 (25%) poultry samples collected from six local Malaysian markets, with one of these markets subsequently yielding L. monocytogenes in 15 of 24 (62.5%) samples. Limited work with fresh poultry carcasses or pieces in India and Korea indicated L. monocytogenes contamination rates of 0 to 8.1 and 10 to 30.2%, respectively, with 37.0% of minced chicken from Japan yielding the pathogen. In the first survey of poultry processing facilities in South Africa [61], L. innocua was recovered from the rubber fingers of a defeathering machine and on all neck-skin samples after evisceration. L. monocytogenes was also detected on the rubber fingers, packaging funnel, and all neck-skin samples after chilling. Following two studies that assessed microbial contamination in six raw poultry samples collected from street vendors in Johannesburg [135,136], Van Nierop et al. [214] conducted a more extensive survey to determine the incidence of L. monocytogenes on fresh and frozen chickens available from butchers, supermarkets, and street vendors in Gauteng, a densely populated province in South Africa. Overall, 19.2, 19.2, and 32.3% of the 99 carcasses were culture positive for L. monocytogenes, Salmonella spp., and Campylobacter, respectively. However, 41 of 99 carcasses tested positive for L. monocytogenes using a PCR method. Furthermore, 12 of 66 (19.2%) fresh and 7 of 33 (21.2%) frozen carcasses rinse samples yielded L. monocytogenes, with the highest and lowest contamination rates seen on frozen carcasses from butchers (35.3%) and supermarkets (6.3%). Interestingly, only 1 of 6 chickens slaughtered and plucked by street

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vendors at the time of purchase was positive for L. monocytogenes. In the last of these Listeria surveys, Barbalho et al. [28] analyzed chicken carcass, chill water, and worker hand/glove samples from one poultry processing facility in Bahia, Brazil that were collected over a 9-month period. Listeria innocua was recovered from carcasses at bleeding, scalding, defeathering, and evisceration with 76.2% of the packaged carcasses positive for L. innocua. Only 3 of 21 carcasses at packaging yielded L. monocytogenes (3.3%), with both L. monocytogenes and L. innocua originating from the hands and gloves of the workers. Overall, the data from Table 14.4 indicate incidence rates of 24.8% and 14.2% for L. monocytogenes in fresh and frozen poultry marketed in Europe and elsewhere, respectively, with the lower incidence of L. monocytogenes in products of non-European origin most likely related to differences in sample collection methods and isolation techniques.

COOKED/READY-TO-EAT POULTRY Overwhelming evidence now points to the meat processing environment as the primary source for Listeria contamination of deli meats, including turkey and chicken. With this premise in mind, Lawrence and Gilmour [116] examined the incidence of Listeria spp., including L. monocytogenes, in one poultry processing facility, along with raw and cooked chicken at this same facility, in Northern Ireland between March and August 1992. Within the raw and cooked poultry processing environments, 36 of 79 (46%) and 51 of 173 (29%) samples harbored Listeria spp., whereas 21 of 79 (26%) and 27 of 173 (15%) yielded L. monocytogenes, respectively. Contamination rates were fairly uniform, with several environmental sites yielding L. monocytogenes throughout the study. Among raw and cooked products tested, 53 of 58 (91%) and 8 of 96 (8%) contained Listeria spp., whereas 34 of 58 (59%) and none of 96 cooked samples yielded L. monocytogenes, respectively. Although L. monocytogenes was absent from all cooked products tested, the presence of other Listeria spp. and a subsequent report by the same authors [117] attesting to isolation of identical L. monocytogenes strains from raw poultry, cooked poultry, and the processing environment (some environmental strains of which persisted for up to 1 year) confirm the importance of minimizing postprocessing contamination. The association between human listeriosis and consumption of cooked/cooked-chilled/readyto-eat poultry products that are frequently held refrigerated for at least 5 days before being consumed without further heating is now well documented as a result of four widely publicized outbreaks in the United States that were directly linked to consumption of turkey frankfurters [83] and readyto-eat delicatessen turkey meat [49,70,163]. However, interest in the incidence of Listeria in poultry originated in the United Kingdom 20 years ago. In the first survey of ready-to-eat poultry products conducted by the Public Health Laboratory Service in London [76,77,174], L. monocytogenes was present in 63 of 127 (12.0%) precooked ready-to-eat poultry products collected from London-area retail establishments between November 1988 and January 1989. Little information is available concerning actual numbers of L. monocytogenes present in cooked poultry products; however, 14 samples that were examined quantitatively contained <100 CFU/g. In addition L. monocytogenes was isolated from 13 of 74 (18%) retail chilled meals, most of which were poultry products given to hospital patients. The pathogen was also discovered in 6 of 24 (25%) cook-chilled poultry products [14], 7 cook-chilled poultry dishes at levels up to 700 L. monocytogenes CFU/g, and 2 cooked chicken products labeled “ready-to-eat” that contained up to 400 L. monocytogenes CFU/g. Working at the Cardiff Public Health Laboratory Service in Wales, Morris and Ribeiro [134] determined the potential Listeria-related risks associated with consumption of pâté, an appetizertype poultry spread that is typically prepared from chicken or goose liver. According to their report, 14 varieties of pâtés (primarily imported from Belgium) were obtained in bulk from area delicatessen counters or in unopened packages from supermarket refrigerators and examined for L. monocytogenes using established methods. Overall, this pathogen was isolated from 37 of 73 (50.4%) pâtés, with 28 (75.7%) and 9 (24.3%) of these positive samples originating from

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delicatessens and supermarket refrigerators, respectively. These pâtés were subsequently withdrawn from the market after officials discovered dangerously high L. monocytogenes populations of ≥104–105 CFU/g in seven samples [9]. L. monocytogenes serotype 4b, the serotype responsible for ~80% of all human listeriosis cases in England and Wales, was isolated from 36 of 37 positive samples, with one strain of L. monocytogenes serotype 4b matching clinical isolates from a 1987 cluster of listeriosis cases in which the exact origin of illness could never be determined. These findings prompted Public Health Laboratory officials to greatly expand their survey of pâté. By March 1990 [75,174], workers at 48 of 53 (90.6%) participating laboratories in England and Wales isolated L. monocytogenes from 187 of 1834 (10.2%) samples of imported and domestic pâté. As in the previous survey, 10% of all positive samples contained >104−106 L. monocytogenes CFU/g, with over half of all isolates belonging to serotype 4. Following this pâté-related outbreak, the number of listeriosis cases reported in England and Wales decreased to approximately half the level reported in 1988. Although some individuals expected to see a further decrease [75,174], the incidence of human listeriosis in the United Kingdom has since stabilized, with about 100–125 cases being reported annually over the last 5 years. In another study, Lieval et al. [121] isolated L. monocytogenes, L. seeligeri, and L. innocua from one of nine, two of nine, and one of nine chicken sandwiches obtained from cafes in and around Paris. Although identical efforts to recover Listeria from 20 fast-food fried chicken items failed, the ability of such foods to harbor Listeria, including L. monocytogenes, has been well established by the previously discussed surveys in England. More recently, Rijpens et al. [175] reportedly recovered Listeria spp. and L. monocytogenes from 35.5 and 15.5%, respectively, of poultry samples examined in Belgium, with the incidence of Listeria spp. markedly higher in unpackaged (41.7%) than in prepackaged (11.1%) poultry.

BEHAVIOR OF L. MONOCYTOGENES IN RAW AND COOKED POULTRY PRODUCTS Although L. monocytogenes was first detected on European raw chicken nearly 30 years ago, the behavior of Listeria in raw and processed poultry products received no attention until this organism was recognized as a bona fide foodborne pathogen in the mid-1980s. Initial research efforts provided the poultry industry with valuable information concerning growth of L. monocytogenes in raw and cooked chicken products, including levels of heat and microwave/gamma irradiation needed to destroy this pathogen in raw chicken. However, our present-day knowledge of Listeria behavior in raw and processed chicken products has greatly expanded since 1998, when the first of four poultryrelated listeriosis outbreaks in the United States was directly linked to consumption of turkey frankfurters [83] that became surface-contaminated after processing. Results from these studies should add much to our knowledge about Listeria behavior in poultry products and aid in the development of improved microbial reduction strategies that will decrease the incidence of this pathogen in raw and ready-to-eat poultry products.

GROWTH: RAW CHICKEN Wimpfheimer et al. [222] were among the first to examine the behavior of L. monocytogenes in raw chicken. In their study, raw minced chicken meat was inoculated to contain 102 L. monocytogenes CFU/g, packaged anaerobically (75% CO2:25% N2), microaerobically (72.5% CO2:22.5% N2:5% O2), or aerobically (air) and examined for numbers of L. monocytogenes as well as aerobic spoilage organisms during storage at 4, 10, or 27°C. Neither L. monocytogenes nor aerobic spoilage organisms grew in anaerobically packaged raw chicken during extended storage at any of the three temperatures, as also reported by Hart et al. [92], with both populations decreasing to <10 CFU/g

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Bacteria log10 CFU/g

588

FIGURE 14.2 Growth of L. monocytogenes and aerobic spoilage organisms in aerobically (O), microaerobically (
), and anaerobically () packaged raw chicken during incubation at 4°C. (Adapted from Wimpfheimer, L., N.S. Altaian, and J.H. Hotchkiss. 1990. Growth of Listeria monocytogenes Scott A, serotype 4 and competitive spoilage organisms in raw chicken packages under modified atmospheres and in air. Int. J. Food Microbiol. 11: 205–214.)

after 6 days of storage at 4°C (Figure 14.2). When packaged aerobically or microaerobically under conditions more closely simulating commercial practices, numbers of L. monocytogenes in raw chicken increased rapidly during extended storage at 4°C, whereas growth of aerobic spoilage organisms was strongly inhibited (see Figure 14.2). Under these conditions, L. monocytogenes can rapidly proliferate in normal unspoiled raw chicken during refrigerated storage, as also reported by two other investigators [92,115]. Zeitoun and Debevere [226] also later reported on the successful use of 10% lactic acid/sodium acetate buffer (pH 3) in conjunction with modified atmosphere packaging (90% CO2:10% O2) to prevent growth of L. monocytogenes on uncooked chicken legs and extend the product’s shelf life at 6°C to 17 days. According to Wimpfheimer et al. [222], the ability of L. monocytogenes to grow in microaerobically packaged raw chicken was not affected by initial levels of Listeria (<101 or 102 CFU/g) or aerobic spoilage organisms (104 or 108 CFU/g). However, the ratio of Listeria to spoilage organisms was strongly temperature dependent, with both organisms reaching populations of 107–108 and 109–1010 CFU/g in microaerobically packaged chicken following < 2 and 8 days of storage at 10 and 27°C, respectively. Neither L. monocytogenes nor aerobic spoilage organisms were inhibited in aerobically packaged raw chicken, with Listeria and spoilage organisms attaining populations >107 and 109 CFU/g, respectively, in products stored at 4, 10, and 27°C. Thus, except for microaerobically packaged product, raw chicken would likely become overtly spoiled before L. monocytogenes could proliferate to the point at which the pathogen might be detectable in minimally cooked chicken. Nevertheless, it is important to remember that

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consumers must take special precautions to prevent cross-contamination between raw chicken that may contain L. monocytogenes, Salmonella, and/or Campylobacter and ready-to-eat products, including cooked chicken. Antimicrobial Additives L. monocytogenes is now considered a microbiological hazard that is reasonably likely to occur in any poultry processing facility in the United States and must now be addressed in USDA-mandated HACCP programs. Various food additives have been evaluated for their ability to inactivate Listeria spp. on fresh poultry. In the first of such studies, dipping inoculated chicken wings in 10% trisodium phosphate (TSP) at 10°C for 15 sec and hot water (95°C) for 5 sec resulted in a 79.5% reduction in numbers of L. monocytogenes, with minimal changes in subcutaneous temperature [177]. Hwang and Beuchat [100] found that washing chicken skin in 1% TSP or 1% lactic acid significantly reduced viable populations of L. monocytogenes compared with washing in water. According to Shelef and Yang [191], growth of L. monocytogenes in sterile comminuted chicken was slowed during refrigerated storage by adding 4% sodium or potassium lactate. When used at levels ≥ 0.3%, sodium diacetate also was inhibitory to L. monocytogenes in poultry slurries [184], with the antilisterial activity of sodium diacetate being further enhanced by supplementing the product with 0.25% ALTA, a commercially available microbially produced shelf life extender. More recently, several investigators have shown that a 15-min treatment in 12% TSP [44,45,46,176] or chlorine-based sanitizers [82,181] just before packing can greatly minimize or suppress growth of L. monocytogenes, Salmonella, and other surface contaminants on raw poultry carcasses and pieces during refrigerated storage. Susceptibility to Gamma Radiation Survey results indicate that up to ~60% of all raw poultry products sold in retail stores may be contaminated with L. monocytogenes, Salmonella, and Campylobacter. These statistics have prompted development of various processes to eliminate such pathogens from raw poultry. Exposure to the bactericidal effects of gamma radiation appears to be among the most effective means of reducing populations of both pathogenic and spoilage organisms. Although presently allowable, gamma radiation doses of 2.5–7.0 kGy in England [138] and <3.0 kGy in the United States [17] are generally regarded as sufficient for eliminating these organisms from raw poultry [203]. Readers should be aware that exposing cooled poultry to levels >2.5 kGy may adversely affect product odor, color, and flavor [88]. In 1989, Patterson [171] examined sensitivity of Listeria to gamma radiation using radiationsterilized raw minced chicken meat that was inoculated to contain ~106 L. monocytogenes CFU/g. After exposing the product to gamma radiation doses of 0, 0.5, 1.0, 1.5, 2.0, and 2.5 kGy, L. monocytogenes exhibited a D10-value (i.e., radiation dose required to decrease the population 10fold) of 0.42 to 0.55 kGy, depending on bacterial strain and type of plating medium used to quantify the pathogen in minced poultry. In support of these findings, Huhtanen et al. [98] also reported that a gamma radiation dose of 2 kGy was sufficient to inactivate an L. monocytogenes population of ~104 CFU/g (average D-value of 0.45 kGy) in artificially contaminated, mechanically deboned chicken. Similar D-values also have been published for Salmonella spp. in fresh poultry [137]. Although all of the aforementioned studies support the use of 2.5 kGy of gamma radiation to eliminate L. monocytogenes at levels of <104 CFU/g from raw poultry meat, researchers in England [131] recovered this pathogen from 1 of 12 (8.3%) fresh chicken carcasses that had been surfaceinoculated to contain L. monocytogenes at approximately 102 or 104 CFU/cm2 and exposed to 2.5 kGy of gamma radiation. More important, after extended storage at 5–10°C, the pathogen was recovered from 1 of 18 (5.6%) and 7 of 18 (38.8%) irradiated carcasses that originally contained low and high inoculum levels, respectively. When poultry carcasses were inoculated to contain >104 L. monocytogenes CFU/g, several investigators [120,215] also confirmed that small numbers of the pathogen survived

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irradiation at 0.5 kGy and eventually grew, particularly in the absence of air, in samples stored at 4°C. However, Shamsuzzaman et al. [189] reported that combined use of 3.1 kGy irradiation and sous-vide cooking to an internal temperature of 71.1°C was sufficient to reduce L. monocytogenes populations in chicken breast meat from 106 CFU/g to undetectable levels, with no growth of the pathogen being observed during 5 weeks of storage at 8°C. Hence, in the absence of multiple treatments, these findings suggest that small numbers of Listeria may either escape sublethal injury during irradiation or undergo repair and grow on these carcasses during refrigerated storage. From this information, it appears that a gamma radiation dose of 2.5 kGy may be only marginally sufficient to inactivate levels of Listeria that one might reasonably expect to find on naturally contaminated raw poultry. Nevertheless, provided that irradiated poultry products are properly packaged to prevent recontamination (and subsequent growth) with Listeria and other foodborne pathogens, this procedure should markedly decrease the risk of contaminating ready-toeat foods (e.g., salads and raw vegetables) during consumer preparation in the kitchen. Unfortunately, although the scientific community generally contends that foods exposed to such low levels of radiation are safe for human consumption, irradiated foods have not yet gained full acceptance by consumers. Perhaps the continued outpouring of scientific evidence will eventually curb the remaining unfounded fear regarding the safety of irradiated foods in the mind of the general public.

GROWTH: COOKED/READY-TO-EAT POULTRY PRODUCTS Confirmation of cooked-chilled chicken and turkey frankfurters as vehicles of listeriosis in England and the United States during 1988 and 1989 prompted immediate international efforts to assess the potential hazards associated with growth of L. monocytogenes in a wide range of retail cooked/readyto-eat poultry products. Six of these early studies are summarized in Table 14.5. Because all these studies differ in experimental design, sampling times, and initial inoculum levels of L. monocytogenes, these findings cannot be compared directly. However, it is evident that numbers of Listeria increased 1−6 orders of magnitude in all six artificially contaminated products after 6−28 days of storage at 3−7°C. Higher populations were observed in aerobically packaged as opposed to vacuum-packaged or modified-atmosphere-packaged products as reported by Murphy et al. [145], who also found that an atmosphere containing 15−30% CO2 decreased L. monocytogenes populations about 1 log on cooked chicken-breast patties during extended storage at 8°C. Equally important, sensory acceptability of these products was not altered by growth of this pathogen. Overall, L. monocytogenes grew most abundantly in vacuum-packaged sliced chicken and in one brand of sliced turkey, both of which were similar in pH (6.3 and 6.4) and in contents of moisture (71.3 and 74.0%), protein (18.9 and 22.6%), carbohydrate (1.3 and 0.9%), and salt (1.7 and 1.4%). These authors attributed decreased growth of the pathogen in a second brand of sliced turkey to higher levels of salt (2.7%) and carbohydrate (1.7%); the latter was largely responsible for the eventual decrease in pH of this product to 4.97. These findings are in general agreement with several predictive models for growth of L. monocytogenes in different poultry products [35,59,160]. In a 1990 report by Ingham et al. [102] cooked/sterilized chicken loaf was inoculated to contain 103 L. monocytogenes and 103 Pseudomonas fragi CFU/g, packaged aerobically (air), microaerobically (10% O2), or anaerobically and examined for both organisms during 6 days of incubation at 3, 11, and 37°C. Regardless of incubation temperature, P. fragi attained maximum populations of approximately 4 × 109 CFU/g in all aerobic samples after 6 days of storage (Figure 14.3). However, growth of Listeria was totally or partially suppressed in similar samples stored at 3°C for up to 15 days [101]. Although both organisms attained lower maximum populations in cooked chicken loaf following 6 days of microaerobic or anaerobic incubation at all three temperatures, these conditions led to Listeria populations that were 1−2 orders of magnitude higher than those attained by P. fragi. Thus, as was true of raw poultry (see Figure 14.2), microaerobic and anaerobic refrigerated storage both appear to selectively favor growth of L. monocytogenes over P. fragi and

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TABLE 14.5 Populations of L. monocytogenes (log10 CFU/g) in Artificially Contaminated Cooked/Ready-to-Eat Poultry Products of Acceptable Organoleptic Quality during Extended Refrigerated Storage

Product Sliced chickena Sliced turkey (brand A)a Sliced turkey (brand B)a Chicken homogenate Breaded chicken fillets Chicken casserole Chicken nuggets Chicken nuggetsd

Incubation Temperature (°C)

Initial Inoculum

Length of Incubation (days) 3

6

8

10

4.4 4.4 4.4 4.4 4.4 4.4 4 4 5 5 3 6 3 7 3 7

2.8 0c 3.0 –1.3 2.9 –1.7 6.7 2.7 2.7 1.7 2.7 2.7 4.8 4.8 4.7 4.7

—b — — — — — — — — — 2.9 3.6 4.8 5.3 4.8 4.9

— — — — — — — — 3.6 3.6 3.5 5.3 5.2 6.0 4.8 5.1

— — — — — — — — — — 3.3 6.3 5.7 6.1 5.0 5.3

— — — — — — — — — — 3.6 7.5 6.1 7.4 5.2 5.3

14 6.9 5.9 5.0 2.4 6.7 4.8 — — — — — — 6.3 — 6.0 —

20 — — — — — — 9.4 7.9 — — — — — — — —

28 — — 6.2 3.7 — 7.7 — — — — — —

Reference 79 79 79 79 195 194 77 127 127

a

Vacuum-packaged. Not tested. c1 CFU/g. dModified atmosphere—80% CO : 20% N . 2 2 b

possibly other spoilage organisms, which, in turn, could potentially yield an organoleptically acceptable product with dangerously high numbers of Listeria. Identification of turkey frankfurters as the infectious vehicle in a 1988 case of listeriosis involving an Oklahoma breast-cancer patient eventually led to a safety assessment of poultry sausage. According to McKellar et al. [129], L. monocytogenes grew on the surface of 13 of 27 (48%) artificially contaminated retail poultry wieners, with populations on some vacuum-packaged samples increasing nearly 4 orders of magnitude during 21 days of storage at 5°C. Wederquist et al. [220] reported similar growth of L. monocytogenes when vacuum-packaged slices of turkey bologna were stored at 4°C. However, incorporating 0.5% sodium acetate, 2.0% sodium lactate, or 0.26% potassium sorbate into the product completely suppressed growth of the pathogen during the first 35 days of refrigerated storage, with populations in 3-month-old samples remaining 2−5 orders of magnitude lower than those in the controls. Two additional studies on fermented summer sausage prepared from ground chicken [23] and turkey [122] also demonstrated the importance of an active starter culture in limiting growth of L. monocytogenes during fermentation. When these sausages were prepared from a chicken or turkey batter (pH 6.6) containing a pediococcal starter culture and L. monocytogenes at a level of 104−107 CFU/g, numbers of Listeria decreased 0.9−1.8 orders of magnitude after an 11-h fermentation (pH 5). Replacing this pediococcal starter culture with a pediocin-producing strain of Pediococcus acidilactici essentially doubled the inactivation rate of L. monocytogenes during fermentation. However, regardless of the starter culture used, all remaining listeriae were subsequently inactivated during 45 min of cooking to an internal temperature

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10 L. monocytogenes P. fragi

9 8

Bacteria log10 CFU/g

7 6 5 4 3 2 1

0

1

4

6

Days

FIGURE 14.3 Growth of L. monocytogenes and Pseudomonas fragi in aerobically (O), microaerobically (
), and anaerobically () packaged cooked chicken loaf during incubation at 7°C. (Adapted from Ingham, S.C., J.M. Escude, and P. McCown. 1990. Comparative growth rates of Listeria monocytogenes and Pseudomonas fragi on cooked chicken loaf stored under air and two modified atmospheres. J. Food Prot. 53: 289–291.)

of 66.5°C. Thus, an active fermentation combined with normal thermal processing before packaging should result in a Listeria-free product. Several investigations also assessed behavior of Listeria in inoculated samples of chicken gravy and chicken broth during cooling and/or refrigerated storage. According to Huang et al. [95], L. monocytogenes populations in individual 1000-g samples of artificially contaminated chicken gravy (prepared from poultry stock, spices, waxy maize wash, and chicken base) increased by 2 orders of magnitude as the product cooled from 40 to 9°C during 24 h of storage at 7°C (Figure 14.4). Even though generation times for L. monocytogenes approximately doubled after the chicken gravy stabilized at 7°C, the pathogen still attained a maximum population of 109 CFU/g in 8-day-old gravy. Huang et al. [96] subsequently reported a reduction in L. monocytogenes when similar chicken gravy samples were held at 65°C for 1.3 min; however, such a heat treatment is clearly inadequate to inactivate higher Listeria populations that can develop in chicken gravy during prolonged refrigerated storage. Walker et al. [219] also demonstrated the ability of three L. monocytogenes strains to multiply in artificially contaminated sterile chicken broth (pH ∼ 6.4) held at near-freezing temperatures, with Listeria populations in this product increasing 100-fold during extended incubation at 0.8°C (Figure 14.5). In fact, growth of this pathogen was also evident in samples of chicken broth that were held at temperatures as low as −0.4°C, below which the broth froze and was no longer sampled. Results from these investigations stress the importance of cooling foods as rapidly as possible and show that hazardous situations can easily develop if refrigerated foods are subjected to mild temperature abuse, that is, holding at temperatures above 4°C. In an effort to increase the safety of cooked, cooked-chilled, and ready-to-eat poultry, USDA officials lowered the recommended long-term storage temperature for such products from 4.4 (40) to 1.7°C (35°F) and also have developed stricter guidelines that require faster (than previously recommended) cooling of warm products at the end of manufacture [4]. Continued attention to rapid cooling of finished products

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10 50 L. monocytogenes Temperature

9

45

8 35 30

7

25 6

20

Temperature (°C)

L. monocytogenes log10 CFU/g

40

15 5 10 0

4 0

1

2

3

4

5 Days

6

7

8

FIGURE 14.4 Behavior of L. monocytogenes in chicken gravy during cooling to 7°C and extended storage. (Adapted from Huang, D., A.E. Yousef, M.E. Matthews, and E.H. Marth. 1993. Growth and survival of Listeria monocytogenes in chicken gravy during cooling and refrigerated storage. J. Food Serv. Syst. 7: 185–192.)

and avoidance of postprocessing contamination are both essential to decrease the incidence of this psychrotrophic pathogen in cooked/ready-to-eat poultry products.

THERMAL INACTIVATION DURING COOKING Heating is the most obvious means of destroying L. monocytogenes and other foodborne pathogens in any raw food, including poultry. However, numerous early reports attesting to unusual thermal

9

Log10 CFU/mL

8 7 6 5

0

10

20

30 Days

40

50

FIGURE 14.5 Growth of L. monocytogenes in sterile chicken broth during extended incubation at 8.7 (), 3.5 (), 1.5 (), and 0.8°C (). (Adapted from Walker, S.J., P. Archer, and J.G. Banks. 1990. Growth of Listeria monocytogenes at refrigeration temperatures. J. Appl. Bacteriol. 68: 157–162.)

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tolerance of L. monocytogenes in various foods, coupled with discovery of L. monocytogenes in several cooked poultry products that were directly linked to cases of listeriosis in the late 1980s, have raised questions about the exact thermal processing times and temperatures required to eliminate this pathogen from raw poultry products. Beginning in 1989, Carpenter and Harrison reported results from three studies in which raw chicken breasts were surface-inoculated to contain ∼105−107 L. monocytogenes CFU/g and cooked to internal temperatures of 65.6, 71.1, 73.9, 76.7, or 82.2°C using dry heat [48], moist heat [90], and microwave radiation [91]. All cooked chicken breasts were then vacuum-packaged or wrapped in oxygen-permeable film and analyzed for numbers of Listeria during 4 weeks of storage at 4 and 10°C. Overall, L. monocytogenes was recovered from chicken breasts cooked to all five internal temperatures using dry heat, moist heat, and microwave radiation. As expected, the magnitude of lethality was directly related to cooking temperature. Because chicken breasts contained somewhat different levels of L. monocytogenes before heating, one cannot directly compare the effectiveness of the three cooking methods used in these studies. However, assuming that L. monocytogenes populations in these chicken breasts decreased linearly during heating (admittedly, some “tailing” of the survivor curve likely occurred at the three highest temperatures using dry heat, moist heat, and microwave irradiation), then the number of survivors in chicken breasts that contained any initial inoculum can be estimated. Thus, if Carpenter and Harrison had used an initial population of 1.0 × 106 L. monocytogenes CFU/g in all three studies, one would expect their results to have been similar to the estimated number of survivors shown in Table 14.6. Considering these approximations, it appears that L. monocytogenes was generally more tolerant of microwave radiation than dry or moist heat, with numbers of Listeria decreasing less than 4 orders of magnitude on chicken cooked to an internal temperature of 82.2°C. Of greater importance is the fact that Listeria populations decreased < 2 orders of magnitude on chicken breasts cooked to an internal temperature of 71.1°C, the minimum internal temperature to which poultry was previously required to be heated to designate the product as fully cooked in the United States [3]. Although numbers of Listeria decreased approximately 5 orders of magnitude when chicken was cooked to higher internal temperatures using either dry or moist heat, these authors [89] later demonstrated that moist heating of surface-inoculated chicken breasts to an internal temperature of 73.9°C also failed to completely inactivate more realistic L. monocytogenes populations of ~102−104 CFU/g. Overall, microwave

TABLE 14.6 Estimated Decrease in Numbers of L. monocytogenes on the Surface of Chicken Breasts Inoculated to Contain 6.00 log10 CFU/g and Cooked to Various Internal Temperatures Using Dry Heat, Moist Heat, or Microwave Radiation Decreasea in L. monocytogenes Population (log10 CFU/g) after Cooking to Various Internal Temperatures Cooking method Dry heat Moist heat Microwave radiation

65.6°C 2.42 2.08 0.82

71.1°C 2.00 1.83 1.95

73.9°C 5.05 3.46 2.50

76.7°C 5.24 5.08 3.77

82.2°C 5.04 4.96 3.26

a

Initial inoculum of 6 log10 L. monocytogenes CFU/mL.

Source: Adapted from Carpenter, S.L. and M.A. Harrison. 1989. Survival of Listeria monocytogenes on processed poultry. J. Food Sci. 54: 556–557; Harrison, M.A. and S.L. Carpenter. 1989. Survival of large populations of Listeria monocytogenes on chicken breasts processed using moist heat. J. Food Prot. 52: 376–378; Harrison, M.A. and S.L. Carpenter. 1989. Survival of Listeria monocytogenes on microwave cooked poultry. Food Microbiol. 6: 153–157.

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heating was less effective than either dry or moist heating, with Listeria populations decreasing less than 4 orders of magnitude on chicken breasts cooked to an internal temperature of 82.2°C (see Table 14.6). In 1989, researchers in England [123] also reported that microwave heating was less effective than other forms of cooking for eliminating L. monocytogenes from the surface and interior (stuffing) of whole stuffed chickens (~1.7 kg each) inoculated to contain 106 and 107 Listeria CFU/g of skin and stuffing, respectively. These findings were further substantiated by Farber et al. [64] using naturally contaminated raw whole broilers. As a result of uneven heating, which is an inherent problem in microwave cooking, 20 min of additional standing time was required after 38 min of cooking (final skin temperature of 80–99°C) to completely inactivate the pathogen on the surface of whole chickens. However, low levels of L. monocytogenes (<10 CFU/g) were still detected in stuffed samples from one of two similarly treated whole chickens that attained temperatures of 72–85°C after 20 min of standing. Thus, these findings serve as a warning to people who regularly cook large stuffed birds (particularly turkeys) in microwave ovens, and they also stress the importance of postcooking standing time for further inactivation of Listeria and other foodborne pathogens after microwave cooking. Not surprisingly, follow-up work by Carpenter and Harrison [48,90,91] demonstrated that L. monocytogenes survivors (most likely sublethally injured during heating) can persist and multiply on both oxygen-permeable film-wrapped and vacuum-packaged cooked chicken during extended storage at 4 and 10°C. As shown in Figure 14.6, growth of Listeria on chicken breasts packaged

7 Dry heat (D) Moist heat (M) Microwave heat

L. monocytogenes log10 CFU/g

6

5

4

3

2 (M) 1

(D)

(D)

0

1

2

3

4

Weeks

FIGURE 14.6 Effects of three different heating methods on behavior of L. monocytogenes on inoculated chicken breasts that were cooked to internal temperatures of 71.1 (), 76.7 (), or 82.2°C (), packaged in oxygen-permeable film, and stored at 4°C. (Adapted from Carpenter, S.L. and M.A. Harrison. 1989. Survival of Listeria monocytogenes on processed poultry. J. Food Sci. 54: 556–557; Harrison, M.A. and S.L. Carpenter. 1989. Survival of large populations of Listeria monocytogenes on chicken breasts processed using moist heat. J. Food Prot. 52: 376–378; Harrison, M.A. and S.L. Carpenter. 1989. Survival of Listeria monocytogenes on microwave cooked poultry. Food Microbiol. 6: 153–157.)

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in oxygen-permeable film was most evident after 2 weeks of refrigerated storage, with larger populations generally developing on products that were cooked using microwave radiation, followed by those given moist or dry heat. Most important, L. monocytogenes was recovered via direct plating from all 4-week-old aerobically packaged chicken breasts except those that were cooked to an internal temperature of 82.2°C using moist heat. In addition, higher numbers of Listeria also developed on aerobically packaged chicken breasts that were exposed to less severe heat treatments. As expected, increasing the incubation temperature also led to much faster growth of Listeria, with the pathogen generally attaining a level of 106–107 CFU/g on aerobically packaged chicken breasts after only 7 days at 10°C. Behavior of L. monocytogenes on chicken breasts was influenced by the heating method/treatment, temperature at which the cooked product was ultimately stored, and type of packaging material. According to these same authors, Listeria populations were generally 1–2 orders of magnitude lower in vacuum-packaged than in aerobically wrapped product following 4 weeks of storage at 4°C; however, the pathogen was present in all 4-week-old samples except those that were originally cooked to an internal temperature of 82.2°C using moist or dry heat (Figure 14.7). Although numbers of Listeria were again generally 1–2 orders of magnitude lower in vacuum-packaged than in aerobically wrapped chicken breasts following 1 week of storage at 10°C, populations were as much as 5 orders of magnitude lower in vacuum-packaged chicken breasts that were cooked to an internal temperature of 82.2°C using moist heat.

7

Dry heat (D) Moist heat (M) Microwave heat

L. monocytogenes log10 CFU/g

6

5

4

3

2 (D)

(M) (M) (D) (D)

1

0

(D)

1

3

2

4

Weeks

FIGURE 14.7 Effects of three different heating methods on behavior of L. monocytogenes on inoculated chicken breasts cooked to internal temperatures of 71.1 (), 76.7 (), or 82.2°C (), vacuum-packaged, and stored at 4°C. (Adapted from Carpenter, S.L. and M.A. Harrison. 1989. Survival of Listeria monocytogenes on processed poultry. J. Food Sci. 54: 556–557; Harrison, M.A. and S.L. Carpenter. 1989. Survival of large populations of Listeria monocytogenes on chicken breasts processed using moist heat. J. Food Prot. 52: 376–378; Harrison, M.A. and S.L. Carpenter. 1989. Survival of Listeria monocytogenes on microwave cooked poultry. Food Microbiol. 6: 153–157.)

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Because raw chicken normally contains <1000 L. monocytogenes CFU/g [131] and Listeria populations generally decrease approximately 3–5 orders of magnitude in fully cooked poultry heated to an internal temperature of 63.9°C, as previously specified in the U.S. Code of Federal Regulations, such cooking temperatures were, at best, only marginally adequate to eliminate this pathogen from raw poultry products. In fact, Harrison and Carpenter [89] reported that moist heating of inoculated chicken breasts to an internal temperature of 73.9°C failed to completely inactivate L. monocytogenes surface populations of 102–104 CFU/g, with the pathogen reestablishing itself at levels equal to or greater than the original inoculum level after 4 weeks at 4°C. Nonetheless, the adequacy of this poultry processing method was maintained by another report [6], which indicated that a turkey meat emulsion (containing salt, sodium tripolyphosphate, carrageenan, and water) inoculated to contain 5.78 L. monocytogenes log10 CFU/g was free of the pathogen after holding the product at 71.1°C for 2 min (estimated D71.1° C = 0.28 min). Following mandatory HACCP implementation by all meat and poultry processors in the United States, USDA–FSIS changed its cooking requirement in 1999 from specific end-point temperatures to minimum required reductions for pathogens. This latter lethality-based performance standard requires a minimum Salmonella reduction of 7.0 logs for fully cooked poultry [207] and other ready-to-eat poultry products [208]. FSIS has supported use of Salmonella cocktails containing relatively heat-resistant strains to verify the adequacy of various time/temperature treatments [208]. Using multistrain cocktails of Salmonella and L. monocytogenes, Murphy and colleagues at the University of Arkansas [148] reported that Salmonella was more heat resistant than L. monocytogenes in ground chicken thigh/leg meat. However, cooking chicken leg quarters in an air-impingement oven to an internal temperature of 73.9°C, as previously required by FSIS, sometimes failed to decrease L. monocytogenes 7 logs as now required by USDA–FSIS [154]. Using these same cocktails of Salmonella and L. monocytogenes, Murphy et al. [147] demonstrated greater heat resistance of L. monocytogenes in previously cooked chicken breast, turkey breast, duck muscle, and duck skin. When the same Salmonella cocktail was compared to L. innocua M1—a heat-resistant surrogate for L. monocytogenes, Listeria was somewhat more heat resistant than Salmonella in ground chicken breast meat [143,144] and ground turkey [153], with similar resistance reported for chicken patties [149] and chicken tenders [149]. Consequently, certain poultry products that meet the 7-log reduction for Salmonella may not necessarily be free of Listeria.

USDA GUIDELINES FOR CONTROL OF LISTERIA IN READY-TO-EAT POULTRY Despite the findings just discussed that suggest possible inadequacies in the lethality-based performance standards in regard to Listeria, overwhelming evidence from the more than 80 recalls to date points to postprocess contamination (rather than contamination during processing) as the root of the problem. In response to the 1998 turkey frankfurter outbreak and three others that were traced to ready-to-eat delicatessen turkey meat [49,70,163], various postcook thermal and nonthermal pasteurization strategies were investigated for packaged products along with product formulations that would suppress growth of Listeria during extended storage. The end result in October 2003 was issuance of the USDA–FSIS rule on L. monocytogenes in ready-to-eat meat and poultry products [209] that applied to any product exposed to the processing environment between cooking and packaging. This rule mandated that manufacturers of ready-to-eat meat and poultry products adopt one of the following three L. monocytogenes control alternatives: (1) use of a postlethality treatment to reduce or eliminate L. monocytogenes and an antimicrobial agent or process that suppresses or limits L. monocytogenes throughout shelf life, (2) use of a postlethality treatment that reduces or eliminates L. monocytogenes or an antimicrobial agent or process that suppresses or limits L. monocytogenes throughout shelf life, or (3) use of sanitation measures in conjunction with regular environmental sampling to prevent

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L. monocytogenes contamination. The various Listeria postprocessing pasteurization treatments and Listeria growth inhibitors that can be used to satisfy these alternatives will now be presented.

POSTPACKAGING PASTEURIZATION Alternative 1 is partially based on exposing the fully processed product to some type of postlethality treatment, typically employing steam or hot water, to inactive small numbers of Listeria that may have contaminated the product surface from the surrounding environment before packaging. Any such postprocessing pasteurization step must be verified as being effective and included as a critical control point in the HACCP plan. Working at the University of Arkansas, Murphy and colleagues conducted a series of studies to assess the ability of different steam and hot water postprocessing pasteurization treatments to decrease populations of L. monocytogenes or L. innocua M1 (a heat-resistant surrogate for L. monocytogenes) ≥7 logs on surface-inoculated fully cooked poultry products. When chicken breast strips were vacuum-packaged in 0.2-mm-thick bags and steam pasteurized at 88°C using either a continuous or batch process, a 7-log reduction was achieved after 28 and 34 min, respectively [155]. In contrast, 22 min of steaming at 96°C was required for similar inactivation of L. monocytogenes on chicken leg quarters [151]. As expected, thermal inactivation of Listeria is also dependent on the amount of product being pasteurized, with 5, 25, and 35 min of steaming at 90°C required to achieve a 7-log reduction in packages containing single cooked chicken breast fillets (120 g), and 227 and 454 g of chicken strips, respectively [156]. Similar inactivation of L. innocua M1 on vacuum-packaged cooked chicken breast fillets was obtained using hot water rather than steam [156], with the weight of product [150,142] and thickness of the packaging film [150] impacting lethality. Although both hot water and steam pasteurization led to slight decreases in expressible total moisture and product yield [141], these thermal pasteurization treatments have gained wide acceptance in the poultry industry, with at least one process lethality model now published for hot water pasteurization [146]. Hot water postpackage pasteurization is also the preferred method to inactivate Listeria on the surface of ready-to-eat delicatessen turkey products. Muriana et al. [139,140] reported Listeria reductions of 2 to 3.5 logs when commercial chubs of various ready-to-eat deli-style turkey products were surface-inoculated with a 4-strain cocktail of L. monocytogenes, vacuum-packaged, and then submerged in a 96.1°C water bath for 3 to 4 min. However, these reductions were significantly lower than those seen in inoculated purge, with these differences attributed to product surface imperfections. The impact of surface roughness (e.g., crevices, dents, cuts, folds, wrinkles, and netting marks), packaging-film thickness and pasteurizer design/water circulation velocity on heat transfer, and thermal inactivation of Listeria was later confirmed by Murphy et al. [152]. When surface-inoculated, vacuum-packaged ready-to-eat turkey breast was hot water pasteurized at 96°C, these researchers reported that 20 and 50 min of heating was required to raise the product temperature to 71°C at depths of 8 and 15 mm, respectively, with this treatment producing a 7-log reduction in L. monocytogenes. Nonthermal pasteurization strategies, including gamma radiation [68,87,197,204] and high-pressure processing [192], have thus far proven to be more costly and/or less effective than thermal pasteurization in reducing numbers of Listeria on packaged ready-toeat meat and poultry products, with gamma radiation doses of 2.5 kGy or higher also negatively impacting product quality. Thus, although time consuming, with the end result being strongly dependent on surface characteristics of the product, hot water postpackage pasteurization remains the most effective treatment and has been widely adopted by poultry processors seeking to satisfy the requirements of USDA–FSIS alternatives 1 and 2.

ANTIMICROBIAL AGENTS

FOR

LISTERIA GROWTH SUPPRESSION

In lieu of a thermal or nonthermal postlethality treatment that reduces or eliminates L. monocytogenes on the product surface, manufacturers that wish to operate under alternative 2 can incorporate

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one or more antimicrobial agents into their product to suppress or limit growth of L. monocytogenes throughout normal shelf life. Among the numerous food additives investigated, combined use of lactate and diacetate has proven to be most effective, with these two preservatives acting synergistically to minimize Listeria growth. According to Zhu et al. [227], populations of L. monocytogenes in inoculated turkey hams increased <1 log during 42 days of refrigerated storage when the product was formulated to contain 2% sodium lactate with either 0.1% sodium diacetate or 0.1% potassium benzoate. Rather than incorporating these generally-recognized-as-safe additives into the product formulation at the time of manufacture, these same antimicrobial agents can be directly applied to the product surface at the time of packaging to minimize Listeria growth. When retail chicken luncheon meat was surface-inoculated to contain about 104 L. monocytogenes CFU/g, Islam et al. [105] reported initial Listeria reductions of 1.32, 1.19, 0.41, and 0.25 logs after spraying the product with a 25% solution of sodium diacetate, sodium benzoate, potassium sorbate, or sodium propionate, respectively, with populations decreasing an additional 0.2 to 0.5 logs for all treatments during 2 weeks of subsequent storage at 4°C. At lower concentrations of 15 and 20%, growth of Listeria was also suppressed about 2–3 logs compared to the unsprayed controls, with sodium diacetate and sodium benzoate again proving to be most efficacious. These same researchers [104] reported similar results after inoculated turkey frankfurters were held in 15, 20, or 25% solutions of sodium diacetate, sodium benzoate, potassium sorbate, or sodium propionate, with Listeria populations 3.5 to 4 logs lower in treated as compared to untreated samples after 2 weeks of refrigerated storage. Despite the high antimicrobial concentrations used, uptake of these solutions by the frankfurters was only about 0.2 to 0.4%. Because all 3-day-old samples were acceptable to consumers based on a sensory evaluation, dipping and or spraying ready-to-eat poultry products in sodium benzoate appears to be a promising means for minimizing Listeria growth on these products during refrigerated storage. However, use of several bacteriocins, including enterocin [22] and sakacin P [110], and different plant extracts [88] also has been suggested. As an alternative approach to dipping and spraying, various types of antimicrobial edible films or coatings have been proposed for inactivating Listeria on the surface of ready-to-eat foods, with these films potentially functioning as antimicrobial packaging materials [41]. Cagri et al. and Capita et al. [42,43] developed a whey protein-based hot dog casing containing 1.0% p-aminobenzoic acid that successfully inhibited growth of L. monocytogenes on surface-inoculated hot dogs during 42 days of refrigerated storage, with this film also likely to be suitable for ready-to-eat poultry products. In another study, Lungu and Johnson [124] reported that L. monocytogenes generally failed to grow on surface-inoculated turkey frankfurters during 28 days of storage at 4°C if the frankfurters were first coated with a zein solution containing various combinations of nisin, sodium diacetate, or sodium lactate. Additional studies have also shown that growth of L. monocytogenes can be prevented on turkey bologna during extended refrigerated storage by packaging the product in wheat gluten films containing nisin [128] or soy-based films containing nisin and/or lauric acid [57]. Thus, antimicrobial packaging films and coatings may provide another alternative for meeting the goals of the USDA–FSIS final rule on L. monocytogenes in ready-to-eat meat and poultry products [209].

EGG PRODUCTS L. monocytogenes is most frequently isolated from the heart, liver, and spleen tissue of fowl suffering from listeriosis; however, based on early scientific literature, this pathogen also has been detected in necrotic oviduct lesions of several infected hens [84] and in follicles of one artificially infected chicken [109]. These observations prompted a large-scale survey in 1958 [107] in which 600 intact hen’s eggs were examined and found to be negative for L. monocytogenes. Similarly, Busani et al. [40] were also unable to isolate L. monocytogenes from 431 eggs collected in Italy during 2001 and 2002. In keeping with these findings, consumption of eggs and egg products also has not yet

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been linked to a single case of listeriosis. However, the possible presence of L. monocytogenes on egg shells that may contain minute cracks, along with the ability of this pathogen to survive 90 and >14 days on eggs stored at 5 [27] and 10°C [30], respectively, persist on inoculated eggs treated with sodium hypochlorite containing 100 ppm available chlorine [30], and grow in artificially contaminated eggs stored at refrigeration as well as ambient temperatures [27] suggests that although unlikely, eggs cannot be completely ignored as a possible source of listerial infection. Hence, it is not surprising that recent concerns surrounding contaminated poultry products also have prompted interest in reducing the numbers of listeriae on intact whole eggs and better defining both the incidence and behavior of L. monocytogenes in eggs and egg products, including pasteurized liquid and dried egg.

INCIDENCE As mentioned in the preceding paragraph, isolation of L. monocytogenes from the interior of intact whole eggs has not yet been documented. However, the outer eggshell surface can harbor Listeria, as suggested by Farber et al. [63], who recovered L. innocua from 6 of 119 egg wash water samples collected in Quebec and Ontario, Canada. Given these findings, several intervention strategies have been explored to reduce the likelihood of Listeria contamination during egg washing. When Park et al. [168] inoculated intact shell eggs to contain about 107 L. monocytogenes CFU/egg, populations decreased about 0.25, 4, and 5 logs after 5 min of washing in deionized water and water containing 45 and 200 ppm available chlorine, respectively, with acidic electrolyzed water yielding reductions of 3–4 logs. In support of these findings, Russell [180] concluded that spraying of electrolyzed water is likely to eliminate the normal numbers of L. monocytogenes that might be expected to contaminate the surface of shell eggs, with Brackett and Beuchat [37] reporting that L. monocytogenes populations on surface-inoculated eggs decreased 1–2 logs after 1 week of storage at 5 or 20°C. In the only other inactivation work thus far reported, Wong and Kitts [223] found that L. monocytogenes was reduced to nondetectable levels following a 3-min exposure to 3 kGy of electron beam irradiation. However, this treatment adversely affected albumin quality. Although the incidence of Listeria is very low on the surface of intact shell eggs, the same is not true of broken eggs. According to Leasor and Foegeding [118], Listeria spp. were isolated from 15 of 42 (36%) previously frozen samples of raw commercially broken solids-adjusted liquid whole egg (21 samples), natural-proportion liquid whole egg (20 samples), and yolk (1 sample) obtained on several occasions from 6 of 11 (54%) commercial manufacturers located throughout the United States. On closer examination of the data, L. innocua and L. monocytogenes were identified in 15 of 15 (100%) and 2 of 15 (13.3%) positive samples, respectively. Moreover, 12 of 15 (80%) and 3 of 15 (20%) positive samples, including 1 sample each with L. monocytogenes, were classified as solids-adjusted and natural-proportion liquid whole egg, respectively. Increased handling during manufacture and, as suggested by the authors, a higher solids content which may enhance growth and survival of Listeria are just two of many possible reasons why a higher incidence of Listeria was observed in solids-adjusted rather than natural-proportion liquid whole egg. Although results from direct plating indicated that the two L. monocytogenes-positive samples contained approximately 1 and 8 L. monocytogenes CFU/g at the time of analysis, the fact that these samples were held frozen for up to 4.5 months and subjected to two freeze-thaw cycles suggests that numbers of Listeria most likely decreased by at least 50% during storage. Hence, both samples probably contained <100 L. monocytogenes CFU/g before being frozen. Working in the United Kingdom, Moore and Madden [133] recovered Listeria from 125 of 173 (72%) inline filters used to remove shell fragments from raw blended whole egg, 78 and 47 samples of which contained L. innocua and L. monocytogenes, respectively, generally at levels <400 CFU/mL. However, because 500 pasteurized egg samples yielded negative results for listeriae, pasteurization at 64.4°C for 2.5 min, as required in the United Kingdom, appears to afford a high degree of safety. During a subsequent survey of three Australian egg factories in New South Wales,

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Desmarchelier et al. [58] identified L. innocua in 9 of 13 (69%) samples of unpasteurized liquid whole egg, with no other Listeria spp. being observed. Floors, drains, and mobile equipment in raw processing areas of these factories were later confirmed as major sources of contamination through strain-specific typing of environmental Listeria isolates. Although 7 samples of raw sugared yolk and 26 peeled boiled eggs were Listeria free, L. innocua was recovered from 1 of 51 samples of pasteurized liquid egg, with this organism also being detected in 2 of 14 peeled boiled eggs following 3 weeks of modified-atmosphere storage at 4°C. Hence, under certain conditions, Listeria can multiply to detectable levels in egg products during extended cold storage.

GROWTH The ability of Listeria to grow in hen’s eggs was first recognized in 1940 when Paterson [170] inoculated a laboratory culture of L. monocytogenes into the chorioallantoic membrane of a chicken embryo. This procedure was traditionally used to determine virulence of L. monocytogenes isolates [200]. Information concerning growth of this pathogen in nonfertile eggs and egg products is limited. A search of the scientific literature has uncovered only two studies pertaining to growth of L. monocytogenes in raw whole eggs or egg components. According to results from the first such paper published in 1955, Urbach and Schabinski [205] found that populations of L. monocytogenes in intact experimentally infected nonfertile eggs increased nearly 6 orders of magnitude during 10 days of storage at ambient temperature. Following this study, 20 years passed until viability of L. monocytogenes was again examined in artificially contaminated raw as well as cooked (121°C/15 min) whole egg, albumen, and egg yolk during extended storage at 5 and 20°C [111]. Results for raw whole and separated egg showed that growth of L. monocytogenes was primarily confined to egg yolk (Figure 14.8), with the pathogen exhibiting respective generation times of 1.7 days and 2.4 h at 5 and 20°C. Overall, Listeria populations in raw whole egg generally varied less than 1 order of magnitude from the original inoculum during extended storage at either temperature; however, numbers of Listeria in raw albumen (pH 8.9) decreased

9

L. monocytogenes log10 CFU/mL

8 7 6 5 Raw Cooked

4 3 2 0

20

10

30

Days

FIGURE 14.8 Behavior of L. monocytogenes in raw and cooked whole egg (), albumen (), and egg yolk () during extended incubation at 5°C. (Adapted from Khan, M.A., I.A. Newton, A. Seaman, and M. Woodbine. 1975. Survival of Listeria monocytogenes inside and outside its host. In M. Woodbine, Ed., Problems of Listeriosis. Surrey, U.K., Leicester University Press, pp. 75–83.)

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3 and 5 orders of magnitude during prolonged incubation at 5 and 20°C, respectively. Despite the reported ability of L. monocytogenes to grow in laboratory media having pH values as high as 10 [188], loss of Listeria viability in raw albumen was pH related, with numbers of Listeria decreasing less than 2 orders of magnitude in samples that were preadjusted to pH 7 and held at 5°C. Unlike raw whole egg, albumen, and egg yolk, Listeria grew rapidly in corresponding cooked samples. Generation times for the pathogen in cooked whole egg, egg yolk, and albumen were 1.9, 2.3, and 2.4 days, respectively, at 5°C, and 2.6, 2.6, and 3.5 h, respectively, at 20°C. These authors speculated that loss of the aforementioned antilisterial properties of raw albumen resulted from inactivation of one or more binding proteins (i.e., ovotransferrin, ovoflavoprotein, and avidin) during heating. Because L. monocytogenes can grow rapidly in cooked whole as well as separated eggs, investigators of foodborne outbreaks should not completely overlook these products as potential vehicles of infection. In 1990, Sionkowski and Shelef [193] provided the first information concerning growth of L. monocytogenes in pasteurized egg products. To simulate postpasteurization contamination, pasteurized (64.4°C/2.5 min) samples of liquid egg and reconstituted dried egg were inoculated to contain ~104–105 L. monocytogenes CFU/mL and examined for numbers of Listeria during 7 days of storage at 4°C. As shown in Figure 14.9, the pathogen grew similarly in both products, reaching populations of ~107 CFU/mL after 7 days of refrigerated storage. Although transmission of L. monocytogenes by pasteurized egg products has not yet been documented, these findings suggest that a possible

108

L. monocytogenes log10 CFU/mL

107

106

105

Liquid egg Reconstituted dried egg

104 0

1

2

3

4 Days

5

6

7

FIGURE 14.9 Growth of L. monocytogenes in liquid and reconstituted dried egg incubated at 4°C. (Adapted from Sionkowski, P.J. and L.A. Shelef. 1990. Viability of Listeria monocytogenes strain Brie-1 in the avian egg. J. Food Prot. 53: 15–17, 25.)

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public health problem could develop if L. monocytogenes enters pasteurized liquid egg, since the shelf life of some of these refrigerated products now has been extended to several months. Growth of L. monocytogenes in commercially processed, liquid whole egg was first assessed by Foegeding and Leasor [67]. In this study, commercially broken, liquid whole egg was ultrapasteurized (68°C/118 sec), homogenized, inoculated to contain one of five L. monocytogenes strains (Scott A [clinical isolate], F5069 [milk isolate], ATCC 19111 [poultry isolate], NCF-U2K3, or NCF-F1 KKr [raw liquid whole-egg isolate]) at a level of 5 × 102 to 1 × 103 CFU/mL, overlaid with mineral oil to prevent oxygen transfer, and examined for numbers of Listeria during extended incubation at 4, 10, 20, and/or 30°C. Generation times and maximum populations were generally similar to those previously observed in fluid milk products (see Chapter 11) except for strain Scott A, which failed to grow in liquid whole egg during prolonged incubation at 4 and 10°C. Although growth of strain Scott A in fluid milk, cheese, and cabbage juice during refrigeration is well documented, Buchanan et al. [39] reported that this strain failed to grow in certain meat and poultry products incubated at 4°C. As shown in Table 14.7, generation times for the five L. monocytogenes strains ranged from 24.0 to 51.0, 8.0 to 31.0, 7.5 to 26.0, and 4.3 to 15.0 h at 4, 10, 20, and 30°C, respectively. Maximum populations ranged from 5.00 to 7.00, 5.48 to 8.48, 6.85 to 8.00, and 7.00 to 8.00 L. monocytogenes log10 CFU/g in liquid whole eggs incubated at 4, 10, 20, and 30°C, respectively. However, Sheldon and Schuman [190] reported that when stored at 4°C, L. monocytogenes populations in an inoculated commercially available reduced-cholesterol liquid whole-egg product could be reduced as much as 3.9 orders of magnitude by adjusting the pH of the product to 6.6 with citric acid and adding nisin at a level of 1000 IU/mL. Considering current distribution and marketing practices, it is likely that perishable products such as liquid whole egg will occasionally encounter periods of temperature abuse. Hence, from these data, it follows that even brief exposure to temperatures ≥10°C can lead

TABLE 14.7 Generation Times and Maximum Populations of L. monocytogenes in Ultrapasteurized Liquid Whole Egg Incubated at 4, 10, 20, and 30°C Strain of L. monocytogenes

Incubation Temperature (°C) 4

F5069 Scott A ATCC 19111 NCF-U2K3 NCF-FIKK4

24 NG 51 26 25

F5069 Scott A ATCC 19111 NCF-U2K3 NCF-FIKK4

6.70 NG 5.00 7.00 6.48

10 Generation Time 12 NG 31 7.8 8.0

20

30

(h) 7.5 26 22 ND ND

4.3 7.1 12 ND ND

Maximum Population (log10 CFU/g) 7.00 8.00 ND 7.00 5.48 6.85 8.48 ND 8.30 ND

8.00 7.85 7.00 ND ND

Note: ND, not determined; NG, no growth. Source: Adapted from Foegeding, P.M. and S.B. Leasor. 1990. Heat resistance and growth of Listeria monocytogenes in liquid whole egg. J. Food Prot. 53: 9–14.

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to a dramatic increase in both growth rate (i.e., decreased generation time) and maximum populations of L. monocytogenes attained in ultrapasteurized liquid whole egg. Because most L. monocytogenes strains can proliferate in ultrapasteurized liquid egg products at refrigeration temperatures, postpasteurization contamination should be avoided and the product should be stored at temperatures at or preferably below 0°C. The behavior of L. monocytogenes has been assessed in several additional egg products. Brackett and Beuchat [36] showed that L. monocytogenes could survive throughout the normal shelf life of powdered and frozen egg products. The survival characteristics of L. monocytogenes on egg shells as well as during and after cooking raw, whole, scrambled eggs by frying were also determined by Brackett and Beuchat [37]. On the egg shell surface, L. monocytogenes populations decreased from 104 to <10 CFU/egg after 6 days of storage at 5 and 20°C. Frying scrambled eggs reduced L. monocytogenes populations >3 orders of magnitude, whereas frying eggs sunny-side up did not significantly reduce levels of L. monocytogenes. Similarly prepared fried scrambled eggs that were inoculated with L. monocytogenes failed to support growth of the pathogen during 36 h of storage at 5°C or 12 h of storage at 18–22°C [224]. However, when hard-boiled eggs were inoculated by Claire et al. [52] to contain 102 L. monocytogenes CFU/g, the pathogen increased to a maximum population of 109 CFU/g following 7, 14, and 21 days of storage at 4, 8, and 12°C, respectively, suggesting possible public health concerns if hard-boiled eggs become contaminated during subsequent peeling and handling. Looking at other egg-related products, Erickson and Jenkins [62] showed that L. monocytogenes inactivation rates in commercial mayonnaise were directly correlated with aqueous phase acetic acid concentration as follows: sandwich spread ≥ real mayonnaise > cholesterol-free reduced calorie mayonnaise dressing > reduced calorie mayonnaise dressing. Higher antilisterial activity in the cholesterol-free formulation was attributed to egg white lysozyme. Additionally, Glass, and Doyle [80] reported that L. monocytogenes populations in two types of commercially produced lowcalorie mayonnaise containing 0.7% acetic acid in the aqueous phase decreased from an initial inoculum of ~106 CFU/g to nondetectable levels following 10–14 days of ambient storage. These studies document that commercial mayonnaise products represent a negligible consumer safety risk. In the only other egg-related growth study thus far reported, Notermans et al. [159] examined the viability of several foodborne pathogens, including L. monocytogenes, in an eggnog-like product prepared from raw whole egg and sugar (25% w/v) with or without ethanol (7% v/v). When samples of ethanol-free product were inoculated to contain ~104–105 L. monocytogenes CFU/g, numbers of Listeria generally decreased 10-fold during the first 2 days of incubation at 4°C and then slowly increased to levels near or slightly above the original inoculum level after 5 additional days of refrigerated storage. Although L. monocytogenes generally exhibited similar behavior patterns in nonalcoholic samples incubated at 22°C, initial population decreases were far more abrupt, with the pathogen then increasing to populations 1 to 3 orders of magnitude lower than the original inoculum after 7 days of incubation. Unlike alcohol-free samples, L. monocytogenes was slowly inactivated in samples containing 7% ethanol, with populations typically 1–2 and more than 4 orders of magnitude lower in 7-day-old samples held at 4 and 22°C, respectively, than were present initially. Hence, given the normal 2-week refrigerated shelf life of similar commercially available nonalcoholic eggnog-like products, recontamination of these beverages during packaging could lead to potential public health problems involving Listeria and other foodborne pathogens, with Salmonella enteritidis and Salmonella Typhimurium reportedly also remaining viable in artificially contaminated samples during 63 days of refrigerated storage.

THERMAL INACTIVATION Interest in possible heat resistance of L. monocytogenes in eggs is of recent origin; however, concerns raised by European scientists regarding potential transmission of Listeria through eggs prompted a 1955 study by Urbach and Schabinski [205], which examined the ability of this pathogen to survive in artificially infected eggs that were fried. According to these authors, L. monocytogenes

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was isolated from fried eggs (congealed white and soft yolk) prepared from inoculated raw eggs in which the pathogen had previously grown to levels >5 × 105 CFU/g. Whereas the aforementioned work appears to be fairly crude by current standards and is now primarily only of historical interest, Foegeding and Leasor [67] conducted a more sophisticated study in which D-values were determined for five strains of L. monocytogenes (Scott A [clinical isolate], F5069 [milk isolate], ATCC 19111 [poultry isolate], NCF-U2K3 and NCF-F1KK4 [raw liquid wholeegg isolates]) in sterile raw egg. Inoculated samples of raw liquid whole egg were added to glass capillary tubes that were then heat-sealed and immersed in a water or oil bath at 51.0, 55.5, 60.0, and 66.0°C. After various times, tubes were removed and contents examined for survivors. Numbers of Listeria decreased linearly in raw egg during all four heat treatments, with D-values for the five L. monocytogenes strains ranging from 14.3 to 22.6, 5.3 to 8.2, 1.3 to 1.7, and 0.06 to 0.20 min at 51.0, 55.5, 60.0, and 66.0°C, respectively. Strain Scott A was generally less heat resistant than the other four strains, particularly at the two lower temperatures; however, strain F5069 and the two isolates from raw egg exhibited moderate thermal tolerance at all four temperatures. Muriana et al. [138] subsequently reported similar D-values at 60°C for L. monocytogenes strain Scott A when inoculated samples of liquid whole egg were tested using either capillary tubes (D-values of 1.8 min) or a flow injection system (D-value of 1.95 min). Although this pathogen appears to exhibit a similar degree of heat resistance in both raw whole milk (see Chapter 11) and raw liquid whole egg, survival of L. monocytogenes is significantly enhanced by supplementing liquid whole egg or egg yolk with 10% NaCl [31,133]. At 64°C, L. monocytogenes exhibited D-values of ~10 and 10.5 min in salted liquid whole egg and egg yolk, respectively, as compared with ~1.0 and 1.8 min for unsalted samples, with increased thermal tolerance attributed to a decrease in water activity. In contrast, adding 10% sucrose to unsalted samples neither increased thermal resistance of listeriae nor altered the product’s water activity. In more practical terms, USDA officials currently require that liquid whole egg be pasteurized at a minimum of 60°C for 3.5 min to effect a 9-D reduction of Salmonella spp. [206]. Although results from the study just discussed indicate that minimum pasteurization of liquid whole egg would yield only a 2.1- to 2.7-D kill of L. monocytogenes, one must remember that current estimates place L. monocytogenes populations in liquid whole egg at <100 CFU/g [67]. Hence, as is true of milk pasteurization, current minimum pasteurization requirements for raw liquid whole egg appear adequate to inactivate normal levels of Listeria that might be present in the product. However, it is important to stress that current minimum pasteurization requirements for liquid whole egg, as specified in the USDA Egg Pasteurization Manual, indicate that the margin of safety is approximately 6 orders of magnitude lower for L. monocytogenes than for most Salmonella spp. Furthermore, such pasteurization treatments appear to be inadequate for salted liquid whole egg and egg yolk. In 1987, Ball et al. [26] documented that ultrapasteurization (i.e., pasteurization at >60°C for <3.5 min) in combination with aseptic processing and packaging can be used to produce liquid whole egg with a refrigerated shelf life of 3–6 months. After results from this study were published, FDA officials issued a temporary permit allowing a North Carolina firm to market ultrapasteurized liquid whole egg [13,178]. Although two of the four objectives of the process were to render the product free of Salmonella and L. monocytogenes, the exact time/temperature requirements to completely inactivate L. monocytogenes in liquid whole egg were not specified in the FDA temporary permit. Based on extrapolations from the aforementioned survivor curves that showed no evidence of tailing, Foegeding and Leasor [67] predicted that the ultrapasteurization processes used by Ball et al. [26] would effect a 1- to 34-D (average of 14-D) kill of L. monocytogenes in liquid whole egg. Assuming that the ultrapasteurization times and temperatures used in conjunction with the temporary FDA permit are the values that were previously determined by Ball et al. [26], Foegeding and Leasor [67] went on to speculate that four of the ten thermal treatments used by Ball and coworkers may not conform to the definition of ultrapasteurization in the temporary permit, depending on how one views the necessity for a 9-D reduction in numbers of Listeria. However, it appears that

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Listeria-free ultrapasteurized liquid whole egg having a refrigerated shelf life of 1 to several months can be produced, provided that the raw product is processed using one of the six more severe time/temperature treatments proposed by Ball et al. [26] and then is aseptically packaged to eliminate postpasteurization contamination. Foegeding and Stanley [66] verified the previous predictions concerning heat resistance of Listeria by determining the thermal death time (F-value) for L. monocytogenes strain F5069 in liquid whole egg. Using their previously described submerged capillary tube method [62], they found that L. monocytogenes was eliminated from inoculated samples (1.0 × 108 to 4.0 × 108 CFU/mL) of sterile liquid whole egg after processing at 62, 64, 66, 69, and 72°C for 16.0, 8.0, 4.5, 1.6, and 0.6 min, respectively. Although results from this study confirm that minimum pasteurization (60°C/3.5 min) will not result in a Listeria-free product if initial populations are large, the need for a 9-D kill as currently required by the USDA may not be appropriate for L. monocytogenes, because present estimates place the population of this pathogen at <100 CFU/g in raw liquid whole egg. Hence, based on maximum expected L. monocytogenes levels in raw liquid whole egg, pasteurization by current standards should render such products free of Listeria. The situation regarding ultrapasteurization appears to be somewhat different because the thermal death-time data obtained by Foegeding and Stanley [66] indicate that 4 of the 10 ultrapasteurization processes proposed by Ball et al. [26] (63.7°C/26.2 sec, 63.8°C/92.0 sec, 67.7°C/9.2 sec, and 71.5°C/2.7 sec) would likely fail to produce a 9-D kill in raw liquid whole egg. Nonetheless, the 9-D kill effected by the remaining six ultrapasteurization treatments proposed by Ball et al. [26] indicates that ultrapasteurization processes can be designed to produce Listeria-free liquid whole egg with an anticipated refrigerated shelf life of 3–6 months.

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180. Russell, S.M. 2003. The effect of electrolyzed oxidative water applied using electrostatic spraying on pathogenic and indicator bacteria on the surface of eggs. Poul. Sci. 82: 158–162. 181. Russell, S.M. and S.P. Axtell. 2005. Monochloramine versus CTP as antimicrobial agents for reducing populations of bacteria on broiler chicken carcasses. J. Food Prot. 68: 758–763. 182. Ryser, E.T., S.M. Arimi, M.M.-C. Bunduki, and C.W. Donnelly. 1996. Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media. Appl. Environ. Microbiol. 62: 1781–1787. 183. Samelis, J. and J. Metaxopoulos. 1999. Incidence and principal sources of Listeria spp. and Listeria monocytogenes contamination in processed meats and a meat processing plant. Food Microbiol. 16: 465–477. 184. Schlyter, J.H., A.J. Degnan, J. Loefelholz, K.A. Glass, and J.B. Luchansky. 1993. Evaluation of sodium diacetate and ALTATM 2341 on viability of Listeria monocytogenes in turkey slurries. J. Food Prot. 56: 808–810. 185. Schonberg, A., P. Teufel, and E. Weise. 1988. Isolates of Listeria monocytogenes and Listeria innocua. 10th International Symposium on Listeriosis, Pecs, Hungary, Aug. 22–26, Abstr. 45. 186. Seastone, C.V. 1935. Pathogenic organisms of the genus Listerella. J. Exp. Med. 62: 203–212. 187. Seeliger, H.P.R. 1961. Listeriosis. New York: Hafner. 188. Seeliger, H.P.R. and D. Jones. 1987. Listeria. In Bergy’s Manual of Systematic Bacteriology, 9th ed. P.H.A. Sneeth, N.S. Mair, N.E. Sharpe, and J.G. Holt, Eds., Williams and Wilkins, Baltimore, MD, pp. 1235–1245. 189. Shamsuzzaman, K., L. Lucht, and N. Chuaqui-Offermanns. 1995. Effects of combined electron-beam irradiation and sous-vide treatments on microbiological and other qualities of chicken breast meat. J. Food Prot. 58: 497–501. 190. Sheldon, B.W. and J.D. Schuman. 1996. Thermal and biological treatments to control psychrotrophic pathogens. Poul. Sci. 75: 1126–1132. 191. Shelef, L.A. and Q. Yang. 1991. Growth suppression of Listeria monocytogenes by lactates in broth, chicken, and beef. J. Food Prot. 54: 283–287. 192. Simpson, R.K. and A. Gilmour. 1997. The resistance of Listeria monocytogenes to high hydrostatic pressure in foods. Food Microbiol. 14: 567–573. 193. Sionkowski, P.J. and L.A. Shelef. 1990. Viability of Listeria monocytogenes strain Brie-1 in the avian egg. J. Food Prot. 53: 15–17, 25. 194. Siragusa, G.R. and M.G. Johnson. 1988. Detection by conventional culture methods and a commercial ELISA test of Listeria monocytogenes added to cooked chicken (abstr). Poul. Sci. 67(Suppl. 1): 157. 195. Siragusa, G.R., K.J. Moore, and M.G. Johnson. 1988. Persistence on and recovery of Listeria from refrigerated processed poultry. J. Food Prot. 51: 831–832. 196. Skovgaard, N. and C.-A. Morgen. 1988. Detection of Listeria spp. in faeces from animals, in feeds, and in raw foods of animal origin. Int. J. Food Microbiol. 6: 229–242. 197. Sommers, C.H. and D.W. Thayer. 2000. Survival of surface-inoculated Listeria monocytogenes on commercially available frankfurters following gamma irradiation. J. Food Saf. 24: 127–137. 198. Soriano, J.M., H. Rico, J.C. Moltó, and J. Mañes. 2001. Listeria species in raw and ready-to-eat foods from restaurants. J. Food Prot. 64: 551–553. 199. Soultos, N., P. Koidid, and R.H. Madden. 2003. Presence of Listeria and Salmonella spp. in retail chicken in Northern Ireland. Lett. Appl. Microbiol. 37: 421–423. 200. Steinmeyer, S. von, and G. Terplan. 1990. Listerien in Lebensmitteln—eine aktuelle Übersicht zu Vorkommen, Bedeutung als Krankheitserreger, Nachweis und Bewertung. DMZ Lebensmittelindustrie und Milchwirtschaft 11: 150–155. 201. Steinmeyer, S. von, R. Schoen, and G. Terplan. 1987. Zum Nachweis der Pathogenität von aus Lebensmitteln isolierten Listerien am bebrüteren Hühnerei. Arch. Lebensmittelhyg. 38: 95–99. 202. Ternstrom, A. and G. Molin. 1987. Incidence of potential pathogens on raw pork, beef and chicken in Sweden, with special reference to Erysipelothrix rhusiopathiae. J. Food Prot. 50: 141–146,149. 203. Thayer, D.W. 1995. Use of irradiation to kill enteric pathogens on meat and poultry. J. Food Saf. 15: 181–192. 204. Thayer, D.W., G. Boyd, A. Kim, J.B. Fox, Jr., and H.M. Farrell, Jr. 1998. Fate of gamma-irradiated Listeria monocytogenes during refrigerated storage on raw or cooked turkey breast meat. J. Food Prot. 61: 979–987.

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205. Urbach, H. and G.L. Schabinski. 1955. Zur Listeriose des Menschen. Z. Hyg. 141: 239–248. 206. USDA. 1969. Egg Pasteurization Manual. ARS 74–48. Poultry Laboratory, Agriculture Research Service, USDA, Albany, CA. 207. USDA–FSIS. 1999. Performance standards for the production of certain meat and poultry products. Fed. Regist. 64: 732–749. 208. USDA–FSIS. 2001. Performance standards for the production of processed meat and poultry products. Fed. Regist. 66: 12590–12636. 209. USDA–FSIS. 2003. Control of Listeria monocytogenes in ready-to-eat meat and poultry products; Final rule. Fed. Regist. 68: 34207–34254. 210. USDA–FSIS. 2005. Electronic reading room: microbiological testing program. Available at: http: //www.fsis.usda.gov/ophs/rtetest/rettable6.htm. Last accessed December 13, 2005. 211. USDA–FSIS. 2005. FSIS Recalls. Available at: http: //www.fsis.usda.gov/Fsis Recalls/index.asp. Last accessed December 13, 2005. 212. Uyttendaele, M., P. de Troy, and J. Debevere. 1999. Incidence of Salmonella. Campylobacter jejuni, Campylobacter coli, and Listeria monocytogenes in poultry carcasses and different types of poultry products for sale on the Belgian market. J. Food Prot. 62: 735–740. 213. Uyttendaele, M.R., K.D. Neyts, R.M. Lips, and J.M. Debevere. 1997. Incidence of Listeria monocytogenes in poultry products obtained from Belgian and French abbatoirs. Food Microbiol. 14: 339–345. 214. Van Nierop, W., A.G. Duse, E. Marais, N. Aithma, N. Thothobolo, M. Kassel, R. Stewart, A. Potgieter, B. Fernandes, J.S. Galpin, and S.F. Bloomfield. 2005. Contamination of chicken carcasses in Gauteng, South Africa, by Salmonella, Listeria monocytogenes and Campylobacter. Int. J. Food Microbiol. 99: 1–6. 215. Varabioff, Y., G.E. Mitchell, and S.M. Nottingham. 1992. Effects of irradiation on bacterial load and Listeria monocytogenes in raw chicken. J. Food Prot. 55: 389–391. 216. Vitas, A.I., V. Aguado, and I. Garcia Jalon. 2004. Occurrence of Listeria monocytogenes in fresh and processed foods in Navarra (Spain). Int. J. Food Microbiol. 90: 349–356. 217. Vorst, K.L., E.C.D. Todd, and E.T. Ryser. 2006. Transfer of Listeria monocytogenes during mechanical slicing of turkey breast, bologna, and salami. J. Food Prot. 69: 619–626. 218. Waldroup, A.L. 1996. Contamination of raw poultry with pathogens (abstr). World’s Poul. Sci. 52: 7–25. 219. Walker, S.J., P. Archer, and J.G. Banks. 1990. Growth of Listeria monocytogenes at refrigeration temperatures. J. Appl. Bacteriol. 68: 157–162. 220. Wederquist, H.J., J.N. Sofos, and G.R. Schmidt. 1994. Listeria monocytogenes inhibition in refrigerated vacuum packaged turkey bologna by chemical additives. J. Food Sci. 59: 498–500, 516. 221. Wesley, I.V., K.M. Harmon, J.S. Dickson, and A.R. Schwartz. 2002. Application of a multiplex polymerase chain reaction assay for the simultaneous confirmation of Listeria monocytogenes and other Listeria species in turkey sample surveillance. J. Food Prot. 65: 780–785. 222. Wimpfheimer, L., N.S. Altaian, and J.H. Hotchkiss. 1990. Growth of Listeria monocytogenes Scott A, serotype 4 and competitive spoilage organisms in raw chicken packages under modified atmospheres and in air. Int. J. Food Microbiol. 11: 205–214. 223. Wong, P.Y.Y. and D.D. Kitts. 2003. Physicochemical and functional properties of shell eggs following electron beam irradiation. J. Sci. Food Agric. 83: 44–52. 224. Yang, S.-E. and C.-C. Chou. 2000. Growth and survival of Escherichia coli O157: H7 and Listeria monocytogenes in egg products held at different temperatures. J. Food Prot. 63: 907–911. 225. Yucel, N., S. Citak, and M. Onder. 2005. Prevalence and antibiotic resistance of Listeria species in meat products in Ankara, Turkey. Food Microbiol. 22: 241–245. 226. Zeitoun, A.A.W. and J.M. Debevere. 1991. Inhibition of Listeria monocytogenes on poultry as influenced by buffered lactic acid treatment and modified atmosphere packaging. Int. J. Food Microbiol. 14: 161–169. 227. Zhu, M.J., A. Mendonca, H.A. Ismail, M. Du, E.J. Lee, and D.U. Ann. 2005. Impact of antimicrobial ingredients and irradiation on the survival of Listeria monocytogenes and quality of ready-to-eat turkey ham. Poul. Sci. 84: 613–620.

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and Behavior 15 Incidence of Listeria monocytogenes in Fish and Seafood* Karen C. Jinneman, Marleen M. Wekell, and Mel W. Eklund CONTENTS Introduction ....................................................................................................................................617 FDA Surveys of L. monocytogenes in Domestic and Imported Seafood .....................................618 Other Surveys for L. monocytogenes in Fish and Seafood Products ...........................................626 Crustaceans ...........................................................................................................................626 Shellfish ................................................................................................................................630 Finfish ...................................................................................................................................630 Smoked Fish Products..........................................................................................................630 Lightly Processed Fish Products..........................................................................................631 Human Listeriosis Associated with Fish and Seafood Products...................................................632 Regulatory Aspects of L. monocytogenes in Fish and Seafood....................................................634 Risk Assessment.............................................................................................................................635 Hazard Identification ............................................................................................................635 Exposure Assessment ...........................................................................................................635 Hazard Characterization .......................................................................................................635 Risk Characterization ...........................................................................................................635 Behavior of Listeria in Fish and Seafood .....................................................................................636 Modes of Transmission ........................................................................................................636 Growth and Survival.............................................................................................................638 Inhibition...............................................................................................................................640 Inactivation ...........................................................................................................................643 Summary ........................................................................................................................................646 Acknowledgments ..........................................................................................................................647 References ......................................................................................................................................647

INTRODUCTION Listeria monocytogenes is ubiquitous in nature. Therefore, the many aquatic creatures including finfish, oysters, shrimp, crabs, lobsters, squid, and scallops harvested from natural environments can be potential sources of Listeria in the human diet. Many of these products are also subjected to a variety of processing methods that can inactivate Listeria on the raw product. Listeria can also * The views expressed here are those of the authors and are not necessarily endorsed by the U.S. Food and Drug Administration, National Marine Fisheries, or the Government of the United States.

617

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be introduced during processing by poor sanitation conditions or manufacturing practices or by postprocess contamination. The psychrotrophic nature of L. monocytogenes allows its survival or even multiplication during refrigerated storage or temperature abuse situations. This is of special concern for those products receiving minimal or no heat treatment before consumption. Since L. monocytogenes was first isolated from imported cooked crabmeat in 1987, at least 126 Class I recalls (where reasonable probability exists that the use of or exposure to a violative product would cause serious adverse health consequences or death) have been issued by the U.S. Food and Drug Administration (FDA) for more than 266,070 lb of ready-to-eat (RTE) domestic or imported fish and seafood; this pathogen is routinely found in 7.6% of all such products marketed in the United States. The first of several cases of listeriosis positively linked to consumption of fish or seafood was not reported until 1989, when a 54-year-old woman in Italy contracted listeric meningitis 4 days after consuming steamed fish from which L. monocytogenes was later isolated [43]. This case and the potential hazard associated with consumption of other Listeria-contaminated RTE food such as cooked crabmeat, cooked shrimp, and smoked salmon have prompted studies on determining the incidence and control of Listeria in various seafoods. In this chapter, data reviewed are from a series of FDA surveys from 1987 to 2003. These were designed to determine the incidence of L. monocytogenes in domestic and imported shrimp, crab, smoked seafood, and various other fish and seafood products. As in previous chapters, Class I recalls that have been issued for Listeria-contaminated fish and seafoods also will be mentioned. The following topics will also be included: (1) international surveys of fish and seafood products for Listeria, (2) an overview of human listeriosis cases and outbreaks associated with fish and seafood, (3) discussion of regulatory aspects of L. monocytogenes in fish and seafoods and use of risk assessments, (4) behavior of L. monocytogenes in these foods, including growth and thermal resistance data for L. monocytogenes in seafoods, and (5) mitigation strategies such as the application of lactic acid for controlling growth of Listeria in seafood.

FDA SURVEYS OF L. MONOCYTOGENES IN DOMESTIC AND IMPORTED SEAFOOD Immediately after the June 1985 outbreak of cheeseborne listeriosis in California, FDA officials focused their attention on immediate problems that confronted the entire dairy industry. Despite a lack of evidence linking consumption of meat and poultry products to cases of human listeriosis before 1988, as early as December 1985 USDA–FSIS officials began taking an active interest in determining the incidence of L. monocytogenes in meat and poultry products. Increased concern about the potential hazard of Listeria-contaminated seafood to public health began in the spring of 1987 after a private testing laboratory in the United States isolated L. monocytogenes from frozen cooked crabmeat obtained from a Mexican supplier [3]. L. monocytogenes was confirmed in this product by the FDA in Baltimore, MD, in May 1987. In maintaining FDA’s “zero tolerance policy” for L. monocytogenes in RTE foods, the first in a series of Class I recalls for nearly 4 tons of tainted crabmeat marketed in four states was issued. These events also prompted an import alert on June 17, 1987 [4], calling for automatic detention and testing for Listeria and Escherichia coli in all frozen crabmeat shipped to the United States from Mexico. Less than 1 month after this product was recalled, the FDA in Seattle, WA, detected L. monocytogenes in samples of imported frozen raw shrimp [2] and lobster tails [14]. Although no recalls were issued for these products, which are almost invariably cooked before consumption, confirmation of Listeria in these seafoods together with the recall of cooked crabmeat noted previously prompted the FDA to initiate two surveys in July 1987. In the first of these surveys, every month six imported samples of frozen raw shrimp were collected and examined at each FDA district laboratory for Listeria. The samples represented as many different countries as possible (Table 15.1) [2]. Additionally, each district also was requested to collect three domestic samples of frozen raw shrimp per month at the wholesale or retail level.

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TABLE 15.1 Results from an FDA Survey of Imported Frozen Raw Shrimp, July to October 1987 Country of Origin

Number of Samples Analyzed

Number of Positive Samples (%) L. monocytogenes

Brazil Ecuador Guyana Honduras Hong Kong India Indonesia Macau Mexico Nigeria Norway Pakistan Panama People’s Republic of China Peru Philippines Taiwan Thailand Venezuela

4 8 1 5 1 4 1 1 10 3 1 4 7 4 1 3 9 4 3

1 (25) 1 (12.5) 1 (100) 1a (20) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Total

74

4 (5.4)

Other Listeria spp. 1 2 0 2 0 1 0 0 1 1 0 0 0 1 0 0 4 2 0 15

(25) (25) (40) (25)

(10) (33.3)

(25)

(44.4) (50) (20.3)

a

One sample contained L. monocytogenes and other Listeria spp.

Source: Adapted from FDA. 1987. Program 7303.030 Pathogen monitoring of selected high risk foods (FY 88/89). In Department of Health and Human Services. Public Health Service. FDA, U.S. Food and Drug Administration Compliance Program Guidance Manual. U.S. Government Printing Office, Pittsburgh, PA.

Listeria spp. were detected in 18 of 74 (24.3%) samples of frozen raw shrimp imported from 10 different countries between July and October 1987 (Table 15.1) using the original FDA method [89]. L. monocytogenes was also isolated from 4 of 74 (5.4%) imported samples of frozen raw shrimp, with all positive samples originating from Central or South American countries. Subsequently, three lots of raw shrimp imported from Ecuador and Honduras also were found to contain 103–105 L. monocytogenes or L. innocua CFU/g [95]. However, because shrimp are normally not consumed raw in the United States, FDA officials did not request the recall of any of these contaminated lots. The FDA conducted a 2-year nationwide survey in fiscal years 1989 and 1990 to determine the incidence of filth and microbiological (Salmonella and Listeria spp.) contamination in domestic and imported fresh and frozen shrimp [63]. Listeria spp. were detected in 14 samples (6.8%), and L. monocytogenes was recovered from 9 (4.4%) of the 205 samples examined [63]. L. monocytogenes and Salmonella were isolated from 1 of 11 samples in which decomposition was also detected by sensory evaluation [63]. In the second FDA survey, domestic and imported samples of cooked, frozen, and refrigerated crabmeat (i.e., picked or extracted) were examined for presence of L. monocytogenes, Staphylococcus aureus, Vibrio cholera, V. parahaemolyticus, V. vulnificus, and Yersinia enterocolitica and numbers of E. coli [2]. Again, samples of imported crabmeat from as many different countries as possible were collected. As of January 1988, 6 of 98 (6.1%) domestic samples of cooked crabmeat contained Listeria, with L. monocytogenes and L. innocua being recovered from 4 and 2 samples, respectively (Table 15.2). Similarly, Listeria spp. were detected in 3 of 24 (12.5%) imported samples of cooked crabmeat, with L. monocytogenes in 2 of 24 (8.3%) samples of product marketed in the United States.

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TABLE 15.2 Results from an FDA Survey of Domestic/Imported Refrigerated or Frozen Cooked Crabmeat, July 1987 to January 1988 Country of Origin United States Canada Chile Korea Japan Mexico Venezuela Total (imported)

Number of Samples Analyzed 98 3 2 11 2 3 3 24

Number of Positive Samples (%) L. monocytogenes 4 (4.1) 0 0 2 (18.2) 0 0 0 2 (8.3)

Other Listeria spp. 2 (2.0) 1 (33.3) 0 2a (18.2) 0 0 0 3 (12.5)

a

One sample contained L. monocytogenes and L. innocua.

Source: Adapted from Archer, D.L. 1988. Review of the latest FDA information on the presence of Listeria in foods. WHO Working Group on Foodborne Listeriosis, Geneva, Switzerland, February 15–19.

Weagant et al. [138] formally published the first results of a survey dealing with the incidence of Listeria spp. in imported and domestic frozen seafood products analyzed at the FDA District Laboratory in Seattle, WA, during the second half of 1987; 31 of 50 (62%) imported and 4 of 7 (57%) domestic samples of frozen seafood tested positive for Listeria spp. using the FDA method [89]. The only Listeria spp. detected were L. monocytogenes (15 of 57, 26.3%) and L. innocua (26 of 57, 45.6%); both L. monocytogenes and L. innocua were isolated from several samples. Although the number of samples examined from various product categories was limited, results suggested that frozen seafood more frequently contains L. innocua than L. monocytogenes. Hence, as was true for raw milk, meat, and poultry products, it appears that both organisms may also occupy similar niches in seafood processing environments. Therefore, presence of L. innocua in raw, and particularly in cooked, seafood should not be ignored but rather should be viewed as an indicator of possible contamination with L. monocytogenes. Discovery of Listeria in raw shrimp, crabmeat, and other seafood products, coupled with an increased concern about the general safety of seafood, prompted FDA officials in October 1987 to include analysis for L. monocytogenes in a compliance program for domestic and imported shrimp [6] and to increase testing of many other domestically produced seafoods for Listeria spp. under the pathogen monitoring program for select high-risk foods (CPGM 7303.030) [10,14,50]. This increased sampling effort was intended to determine the geographic distribution of Listeria in domestic and imported seafood and to identify the incidence of Listeria spp. in such products. In March 1988, a processed seafood assignment was issued [53]. The purpose of the Processed Seafood Compliance Program (CPGM 7303.036) was to conduct microbiological analyses for several bacterial pathogens, including Listeria, of imported and domestic processed seafood that is minimally processed or consumed raw. Products selected for Listeria analyses under the CPGM 7303.036 program included the following: crabmeat (cooked or pasteurized), crayfish/crawfish, lobster, langostinos (cooked, parboiled), molluscan shellfish, processed imitation seafood (surimi), seafood salads, shrimp (cooked), smoked or salted fish, and other processed seafood. In addition, the National Advisory Committee on Microbiological Criteria for Foods (NACMCF) in April 1988 began the laborious task of developing microbiological criteria for cooked shrimp and crabmeat [7]. During the FDA surveys from October 1988 through September 1990, L. monocytogenes was recovered from domestic samples of crabmeat,

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lobster, shrimp, smoked salmon, and surimi. Imported fish, lobster, shellfish, shrimp, smoked fish, squid, and surimi also tested positive for L. monocytogenes during the same time period [53]. Three FDA compliance programs in effect since 1991–1996 have surveyed the incidence of Listeria in fish and seafood products and reported analytical data into the FDA Microbiological Information System. These data have been reported into the FDA Field Accomplishments and Compliance Tracking System (FACTS) since late 1999. The Domestic Fish and Fisheries Products Compliance Program (CPGM 7303.842) provided coverage for investigations and sampling of domestic fish and fishery products [56]. Imported seafood and seafood products were surveyed for the presence of Listeria under the Import Seafood Products Compliance Program (CPGM 7303.844) [55]. The CPGM 7303.036 program was in existence through 1994 and covered both domestic and imported processed seafood and seafood products [53,54]. Since 1994, the items covered by this program have been incorporated into the CPGM 7303.842 and CPGM 7303.844 programs for domestic and imported products, respectively. RTE foods that require no further or minimal processing by the consumer or products collected as a follow-up to a suspected case of foodborne illness are identified for Listeria analyses in each of these compliance programs. The CPGM 7303.842 program also includes Listeria analyses of in-line and swab samples collected during processor establishment investigation reviews. Some domestic and imported RTE products containing seafood are also collected under the Food Safety/Microbiological Program (CPGM 7303.803) and the Imported Foods Program (CPGM 7303.819), respectively [57, 58]. Between these three compliance programs a total of 7158 samples of fish, seafood products, or seafood processing in-line samples were analyzed by the FDA between 1991 and 1996. L. monocytogenes was detected in 622 of these samples, for an overall incidence of 8.7%. The overall incidence between 2000 and February 2003 was 5.07% with L. monocytogenes isolated from 157 of 3101 samples. The breakdown of samples analyzed and those in which L. monocytogenes was detected by year and origin (domestic or import) are presented (Table 15.3 and Table 15.4). The incidence found by FDA is comparable to the 4–12% incidence of L. monocytogenes in seafood

TABLE 15.3 Seafood Product and Seafood Processing In-Line Samples Analyzed for Listeria monocytogenes by the FDA from 1991 through 1996

Domestic product (CPGM) Positive Negative Total Positive (%) Imported product (CPGM) Positive Negative Total Positive (%) Overall Positive Negative Total Positive (%)

1991

1992

1993

1994

1995

1996

[1,3] 20 403 423 4.7 [3] 32 362 394 8.1

[1,3] 63 501 564 11.2 [2,3] 51 643 694 7.3

[1,3] 89 558 647 13.8 [2,3] 65 801 866 7.5

[1,3] 94 623 717 13.1 [2,3] 42 737 779 5.4

[1] 67 481 548 12.2 [2] 41 589 630 6.5

[1] 41 382 423 9.7 [2] 17 456 473 3.6

52 765 817 6.4%

114 1144 1258 9.1%

154 1359 1513 10.2%

136 1360 1496 9.1%

108 1070 1178 9.2%

58 838 896 6.5%

Overall

374 2948 3322 11.3 248 3588 3836 6.5 622 6536 7158 8.7%

Note: Data compiled from the FDA Microbiological Information System. Samples collected from the following compliance programs (CPGM) [40,41,42]: (1) CPGM 7303.842 Domestic Fish and Fisheries Products (1991–1996), (2) CPGM 7303.844 Import Seafood Products (1992–1996), (3) CPGM 7303.036 Processed Seafood (1991–1994).

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TABLE 15.4 Seafood Product and Seafood Processing In-Line Samples Analyzed for Listeria monocytogenes by the FDA from October 1999 through February 2003 2000

2001

2002

2003

Overall

Domestic product (CPGM) Positive Negative Total Positive (%)

40 434 474 8.44

31 467 498 6.22

18 246 264 6.82

10 65 75 13.33

99 1212 1311 7.55

Imported product (CPGM) Positive Negative Total Positive (%)

13 298 311 4.18

18 430 448 4.02

18 749 767 2.35

9 255 264 3.41

58 1732 1790 3.24

Overall Positive Negative Total Positive (%)

53 732 785 6.75

49 897 946 5.18

36 995 1031 3.49

19 320 339 5.60

157 2944 3101 5.06

Note: Data compiled from the FDA FACTS System. Samples collected from the following compliance programs (CPGM) [40,41,42]: (1) CPGM 7303.842 Domestic Fish and Fisheries Products and CPGM 7303.803 Food Safety/Microbiological samples and (2) CPGM 7303.844 Import Seafood Products and CPGM 7303.819 Imported Foods.

and seafood products from temperate areas as reported by Embarek [38]. Surveys of other food products have indicated a 4–60% incidence in raw meat, 23–60% in fresh poultry, and 2.2% in raw milk [38,49,80]. Samples found positive for L. monocytogenes in FDA compliance programs represent a wide range of fish and seafood products. During the 1991–1996 period a total of 218 crustacean products (crab, shrimp/prawns, lobster, and crawfish) were positive, with crab accounting for the largest number with 142 positive samples. Fifteen samples were positive for L. monocytogenes from the shellfish category, which includes mussels, oysters, clams, scallops, and snails. The finfish category had 231 samples positive for L. monocytogenes, with 164 of these from smoked seafood products. The remaining positive L. monocytogenes samples represent a diverse group of products including the following: squid/calamari, 3 samples; eel, 9 samples; roe/caviar, 19 samples; imitation seafood, 17 samples; seafood salad/spread/pâté or mousse, 13 samples; and processing in-line or swabs, 94 samples. Many of the seafood products and related samples analyzed by the FDA with results reported into FACTS from 2000 to February 2003 are included among the food categories identified in the FDA/FSIS/CDC 2003 Quantitative Assessment of Relative Risk to Public Health from Foodborne Listeria monocytogenes among Selected Categories of Ready-to-Eat Foods [133,134] (Table 15.5). These include food categories for smoked seafood, raw seafood, preserved fish, cooked RTE crustaceans, and sometimes, deli salads. The food categories with greater than 50 samples analyzed and in which L. monocytogenes was recovered more frequently than the overall average (5.07%) included the following: smoked seafood–cold-smoked fish, 14.92%; smoked fish (smoke process not determined), 11.69%; prepared fish–roe/caviar, 8.62%; combination foods (containing seafood)–deli salads, 5.79%; and other deli items containing seafood, 11.32% (Table 15.5). Smoked seafood ranked fifth for predicted relative risk among the 23 food categories in the FDA/FSIS/CDC 2003 Quantitative Assessment of Relative Risk to Public Health from Foodborne

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TABLE 15.5 Seafood Product and Seafood Processing In-Line Samples Analyzed for Listeria monocytogenes by the FDA and Reported into FACTS from October 1999 through February 2003

2000

Smoked Seafood 2002

2001

2003

Overall

Total Positive Total Positive Total Positive Total Positive Total Positive Positive (%) Fish—Hot Fish—Cold Fish—N/A Shellfish and other—hot Shellfish and other—cold Shellfish and other—N/A

109 68 21 5

4 15 3 0

113 80 27 2

3 11 3 0

62 73 24 5

2 5 2 0

24 27 5 1

308 248 77 13

308 248 77 13

9 37 9 0

2.92 14.92 11.69 0.00

0

0

0

0

1

0

0

1

1

0

0.00

2

0

1

0

1

0

0

4

4

0

0.00

2000

Raw Seafood 2002

2001

2003

Overall

Total Positive Total Positive Total Positive Total Positive Total Positive Positive ( %) Raw

4

1

1

1

2

1

2

0

9

Preserved Seafood Products 2001 2002 2003

2000

3

33.33

Overall

Total Positive Total Positive Total Positive Total Positive Total Positive Positive (%) Marinated/cultured Dried Roe/caviar Mollusks Prepared

7 35 25 47 49

0 1 2 2 5

6 11 13 62 56

2000

0 0 2 1 3

4 36 15 72 98

0 0 1 2 2

4 14 5 34 36

RTE Crustaceans 2002

2001

1 0 0 0 0

21 96 58 215 239

2003

1 1 5 5 10

4.76 1.04 8.62 2.33 4.18

Overall

Total Positive Total Positive Total Positive Total Positive Total Positive Positive (%) Crab Shrimp Crawfish Lobster Spiny lobster Langostinos Other

168 61 10 9 1 1 0

6 1 0 2 0 0 0

211 114 18 11 4 7 1

1 15 0 1 0 1 0

206 173 39 7 2 5 1

5 2 3 0 0 1 0

47 39 12 3 1 0 1

3 0 0 0 0 0 0

Combination Foods Containing Seafood 2001 2002 2003

2000

632 387 79 30 8 13 3

15 18 3 3 0 2 0

2.37 4.65 3.80 10.00 0.00 15.38 0.00

Overall

Total Positive Total Positive Total Positive Total Positive Total Positive Positive (%) Deli salads Deli sandwiches

43 13

4 0

44 21

3 0

31 7

0 0

3 5

0 0

121 46

7 0

5.79 0.00 (continued)

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TABLE 15.5 (CONTINUED) Seafood Product and Seafood Processing In-Line Samples Analyzed for Listeria monocytogenes by the FDA and Reported into FACTS from October 1999 through February 2003 Deli spreads/pâté/dips Other deli items (i.e., sushi) Imitation seafood Other combination foods

15

1

15

1

13

0

3

0

46

2

4.35

12

0

14

0

17

1

10

5

53

6

11.32

25 39

0 1

25 69

0 0

54 71

0 2

22 38

0 1

126 217

0 4

0.00 1.84

2000

Other 2002

2001

2003

Overall

Total Positive Total Positive Total Positive Total Positive Total Positive Positive (%) Environ. Overall

16 785

5 53

20 946

3 49

12 1031

7 36

3 339

2 19

51 3101

17 157

33.33 5.06

Listeria monocytogenes Among Selected Categories of Ready-to-Eat Foods on a per-serving basis for all subpopulations, intermediate age, elderly, and perinatal [133]. In the FDA studies, a total of 1210 smoked finfish products were analyzed for Listeria, with 164 (13.6%) samples positive for L. monocytogenes between 1991 and 1995. Of those smoked seafood samples for which the smoke process was known, the incidence of L. monocytogenes was higher in cold-smoked (21.3%, 51 of 240) compared to hot-smoked (8.8%, 19 of 215) samples [70]. Between FY 2000 and February 2003, the overall incidence of L. monocytogenes in smoked seafood was 8.45%, with 55 positive samples among 651 samples analyzed (Table 15.5). Cold-smoked fish had the highest L. monocytogenes incidence at 14.92% (37 of 248 samples), compared to hot-smoked fish, which had an incidence of 2.92% (9 of 308 samples). Smoked fish in which the type of smoking process was not identified yielded L. monocytogenes from 9 of 77 samples (11.69%). Several other investigations have evaluated prevalence of L. monocytogenes in smoked finfish products, with incidences ranging from 0 to 75 % [38]. In those surveys with greater than 100 samples, the incidence of L. monocytogenes also tended to be greater in cold-smoked finfish products (11.3 to 24%) [76–78] compared to hot-smoked finfish products (8.4 to 8.9%) [76–78]. Postprocess contamination most likely accounts for the presence of L. monocytogenes in hot-smoked finfish products. However, with cold-smoked finfish, while the process may not eliminate L. monocytogenes present on the raw product, contamination could also occur during or after processing of the product [35]. Cooked RTE crustaceans, preserved fish, and combination foods–deli salads ranked 6th, 12th, and 19th, respectively, among the 23 food categories for relative risk based on a per-serving basis [133]. During 1991–1996, 1886 RTE crab samples were analyzed for Listeria and 142 (7.5%) tested positive for L. monocytogenes. The incidence rate for RTE crab fell to 2.37% (15 of 632 samples) during October 1999 to February 2003 (Table 15.5). The overall L. monocytogenes incidence rate for all RTE crustaceans was 3.56% (41 of 1152 samples) during this same period. Several other surveys [22,37,44,105,120,138] have reported L. monocytogenes incidence rates of 0 to 29.2%. It is highly likely that the presence of L. monocytogenes in RTE crab is the result of postprocess contamination. The preserved fish category had an overall L. monocytogenes incidence of 3.5% (22 of 629 samples), with roe/caviar having the highest positive rate in this category. Among the combination foods containing seafood, the overall incidence of L. monocytogenes was 3.12% (19 of 609 samples). Products in this category with the highest L. monocytogenes incidence were other deli items, i.e., sushi, and deli salads (11.32%).

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Since 1987, at least 126 Class I recalls have been issued in the United States for domestic and domestic/imported RTE seafood products contaminated with L. monocytogenes [13,51,52]. Recalls have only been issued when L. monocytogenes was found in RTE seafood or seafood products that would receive no subsequent or minimal heat treatment by the consumer before consumption. The number of recalls by product category is shown (Table 15.6). The products with the greatest number

TABLE 15.6 Class I Recalls Issued in the United States for Domestic and Domestic/Imported Ready-to-Eat Seafood Products Contaminated with L. monocytogenes since 1987a Product Crustacean Crab Shrimp Lobster, langostinos Shellfish Mussels (marinated) Mussels (smoked) Finfish (Hot-smoked) (Cold-smoked) (Smokedb) (Salted) Other Imitation seafood Seafood salad or spread a

Number of Class I Recalls since 1987

Pounds (lb) Affected

Location of Manufacturer

46

>114457

7 3

>31332.5 >264

AL, FL, GA, ME, NC, OR, TX, VA, WA, Chile, Mexico FL, GA, MA, NY, WA Canada, Chile

1 1

Unknown Unknown

MA New Zealand

7 22

>253 >93722

22

>10142 Unknown

CA, KY, MD, ME, NY, WA CA, MA, ME, NJ, NY, OR, WA, United Kingdom FL, IL, MD, ME, MN, NC, NY, VA, WA, Denmark Canada

>11793 >4107

VA, WA, Japan, Korea FL, ID, MA, ME, NY, VA, WA

1 4 12

Data compiled from FDA enforcement reports: Anonymous. 1995. Misdeclaration and Recall of Smoked Salmon from the United Kingdom. U.S. FDA Import Bulletin 16-B86; FDA. Recalls and Field Corrections. In FDA Enforcement Reports for June 10, 1987; December 16,1987; December 23, 1987; July 20, 1988; August 10, 1988; October 5, 1988; October 20, 1988; December 28, 1988; January 18, 1989; May 24, 1989; October 4, 1989; November 1, 1989; November 8, 1989; January 24, 1990; July 11, 1990; August 8, 1990; October 31, 1990; November 28, 1990; December 19, 1990; January 9, 1991; March 27, 1991; August 14, 1991; September 4, 1991; November 6, 1991; December 4, 1991; December 11, 1991; February 19, 1992; June 10, 1992; July 2, 1992; July 29, 1992; August 5, 1992; August 19, 1992; September 2. 1992; September 9, 1992; September 23, 1992; September 30, 1992; October 7, 1992; October 28, 1992; November 25, 1992; January 27, 1993; February 17, 1993; March 10, 1993; May 19, 1993; June 9, 1993; August 25, 1993; July 28, 1993; August 4, 1993; August 25, 1993; October 27, 1993; November 10, 1993; November 17, 1993; December 15, 1993; January 19, 1994; April 13, 1994; June 1, 1994; July 20, 1994; July 27, 1994; September 21, 1994; October 5, 1994; December 7, 1994; February 22, 1995; March 8, 1995; March 15, 1995; April 5, 1995; April 12, 1995; April 26, 1995; August 9, 1995; August 30, 1995; September 27, 1995; December 13, 1995; May 8, 1996; June 12, 1996; July 24, 1996; September 4, 1996; October 23, 1996; January 2, 1997. U.S. Government Printing Office, Pittsburgh, PA; FDA. Recalls and Field Corrections. In FDA Enforcement Reports for April 30, 1997; August 6, 1997; September 17, 1997; July 29, 1998; September 2, 1998; December 23, 1998; November 5, 1999; January 10, 2000, March 10, 2000; April 5, 2000; August 9, 2000; August 29, 2000; August 31, 2000; January 4, 2001; January 10, 2001; September 6, 2001; May 22, 2002; July 3, 2002; July 30, 3002; September 4, 2002; September 17, 2002. www.fda.gov/OC/po/firmrecalls/archive.html. b Hot- or cold-smoking process not identified.

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of recalls reflected the types of products most frequently identified as positive for L. monocytogenes in compliance program surveys. Crab accounted for 46 and smoked finfish accounted for 51 of the 126 recalls.

OTHER SURVEYS FOR L. MONOCYTOGENES IN FISH AND SEAFOOD PRODUCTS The documented presence of Listeria in fish and seafood and subsequent product recalls prompted several studies to determine the incidence of Listeria spp. in a variety of products from many geographic locations (Table 15.7). Results of many of these studies have been extensively reviewed [12,32,38,78]. Sampling strategies, number of samples analyzed, and detection methods varied, so that although it is useful to note the results from these studies, the data from them cannot always be directly compared.

CRUSTACEANS Listeria spp. including L. monocytogenes, have been recovered from cooked and picked RTE crabmeat, and as noted earlier this product has been among those most frequently recalled. Because this product is thermally processed, which should eliminate or reduce microorganisms, presence of L. monocytogenes on the finished product most likely represents postprocessing contamination. Several studies have included a limited number of crab samples. L. monocytogenes was detected in 7 and L. innocua in 12 of 24 cooked imported crab products [138]. Although no L. monocytogenes was detected in another study, L. welshimeri was found in 1 of 2 cooked crab samples [22]. Listeria spp. were recovered in 2 of 5 crab samples collected as part of a survey in Alexandria, Egypt [37], and L. monocytogenes was isolated from 1 of 7 crab samples from the United States and China [44]. Two larger U.S. studies of cooked and processed crab have also been published. Although 31 of 138 (22.5%) processed crab samples contained Listeria spp., no attempt was made for further speciation [105]. In a second study, 126 cooked and picked blue crab samples were analyzed; 10 samples (7.9%) were positive for L. monocytogenes and 3 samples (2.4%) positive for L. innocua [120]. No L. monocytogenes was detected in 154 crab samples analyzed in Canada [47]. Very few studies have quantified L. monocytogenes in naturally contaminated cooked and processed crab. Among the 10 samples positive for L. monocytogenes in one study [120], 1 sample contained 1100 Listeria/g but in the remaining samples <100 Listeria/g were detected, indicating a generally low level of contamination. Listeria spp. have been detected in fresh or raw shrimp with two of seven samples containing L. monocytogenes [138], and one of four samples containing L. innocua [22] in two U.S. studies. In a survey of fresh shrimp in Japan, Listeria spp. were recovered from 6 of 70 samples (8.6%) with one (1.4%) of these positive for L. monocytogenes [94]. In France, L. monocytogenes was isolated from 2 of 17 (11.8%) uncooked shrimp samples and Listeria spp. from 4 samples (23.5%) [119]. In Trinidad, where shrimp are consumed in a nearly raw state, Listeria spp. were recovered from 2 of 41 (5%) fresh, uncooked shrimp samples [1]. L. monocytogenes also has been detected in 6.7% of raw shrimp from China, Ecuador, and Mexico [16]. Despite cooking and other heat processing steps, which should eliminate Listeria on raw product, Listeria spp. have been recovered from cooked and RTE shrimp/prawns by several investigators. L. monocytogenes was detected in 2 of 8 (25%) cooked and processed shrimp in a U.S. study [138], 4 of 49 (8.2%) RTE shrimp, 4 of 287 (1.5%) cooked shrimp, 0 of 4 cooked shrimp in Canada [44,47], and 1 of 38 (2.6%) [128] RTE shrimp products in Japan. No L. monocytogenes was recovered from 40 retail samples of cooked prawns, shrimp, and cockles sold in England and Wales between 1987 and 1989 [122]. Brined RTE shrimp had L. monocytogenes in 3 of 18 (16.7%) samples in Norway [126].

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TABLE 15.7 Incidence of Listeria spp. in Fresh, Frozen, and Processed Seafood Product (country) Crab Crab (c) (multiple countries) Crab (c) (U.S.A.) Crab (r) (China) Crab (c or p) (U.S.A.) Crab (Egypt) Blue crab (c) (U.S.A.) Crab (c) (multiple countries) Shrimp/prawns Shrimp (f), (multiple countries) Shrimp (f and fr) (U.S.A.) Shrimp (f) (Japan) Shrimp (f) (Trinidad) Shrimp (raw, fr) (France) Shrimp (c and p) (multiple countries) Shrimp (r) (multiple countries) Shrimp (f and p) (India) Shrimp (Canada) Shrimp in brine (r) (Norway) Shrimp (c and f) (Iceland) Prawn (r) (Japan) Shrimp (Egypt) Prawns/shrimp/cockles (c) (U.K.) Shrimp (c) (multiple countries) Shrimp (peeled) (multiple countries) Shrimp (Brazil) Shrimp (r) (China, Ecuador, Mexico) Lobster Lobster tail (fr) (multiple countries) Lobster (c) (Canada) Mussels Mussels (sm) (New Zealand) Mussels (f) (Spain) Oysters Oysters (fr) (multiple countries) Oysters (p) (U.S.A.) Oysters (f) (Japan) Oysters (f) (Egypt) Clams Clam (f) (India) Clam (f) (U.S.A.) Clam (surf and c) (multiple countries) Scallops Scallops (fr) (multiple countries) Scallops (raw) (U.S.A.) Scallop (Canada)

Number of Samples

24 2 7 138 5 126 154 7 4 70 41 17 8 49 19 20 16 11 38 5 40 287 4 178

(%) of Positive Samples Listeria spp.

50 22.5 40 10.3

25 8.6 5 23.5

10.5

9 15.8 40

50

2 70

L. monocytogenes

29.2 0 14 0 7.9 0 28.6 0 1.4

Reference

138 22 44 105 37 120 47

11.8 25 8.2 0 20 18 9 2.6 20 0 1.5 0 18 6.7

138 22 94 1 119 138 44 93 44 126 69 128 37 122 47 47 28 16

50 0

138 47

14 40

22.5

35.7 7.5

73 130

1 2 84 2

0 0 0 0

0 0 0 0

138 22 94 37

1 1 59

0 0

0 0 0

62 22 47

2 1 1

50 0

0 0 0

139 22 47 (continued)

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TABLE 15.7 (CONTINUED) Incidence of Listeria spp. in Fresh, Frozen, and Processed Seafood Product (country) Other shellfish and invertebrates Shellfish (c) (Iceland) Non-oyster shellfish (f) (Japan) Shellfisha Donax spp. (coquina) (f) (Egypt) Ruditapes spp. (clam) (f) (Egypt) Crayfish (c) (multiple countries) Finfish Fish (f) (U.S.A.) Catfish (f) (U.S.A.) Fish (f) (Trinidad) Minced fish (f) (Norway) Fish (f) (Japan) Fish (f) (India) Fish (f) (Egypt) Fish (fr) (Egypt) Fish (f) (India) Fish (fr) (India) Fish (r) (New Zealand) Minced fish (raw, r) (Japan) Fish (trout) (f) (Iceland) Fish (dried haddock) (Iceland) Fish (fr) (multiple countries) Fish (ceviche) (Peru) Fish (preserved not heat-treated) (Denmark) Fish (preserved not heat-treated) (Denmark) Fish (r) (Denmark) Fish (lightly pickled) (Switzerland) Fish (Gravad) (Iceland) Fish (cold sm, salmon) (Switzerland) Fish (cold sm, salmon) (Switzerland) Fish (cold sm, fish) (Switzerland) Fish (cold sm, fish) (Switzerland) Fish (cold sm, salmon) (Norway) Fish (sm, salmon) (Iceland) Fish (cold sm, salmon) (Canada) Fish (cold sm, salmon) (Italy) Fish (cold sm, salmon) (New Zealand) Fish (cold sm, salmon) (U.S.A.) Fish (sm) (New Zealand)b Fish (smoked and cold-salted) (Finland) Fish (smoked) Fish (cold sm, salmon) (Denmark) Fish (cold sm, salmon) (Denmark) Fish (sm, salmon) (Japan) Fish (gravad or sm, salmon/rainbow trout) Fish (gravad or sm, salmon/rainbow trout)

Number of Samples

(%) of Positive Samples Listeria spp.

L. monocytogenes

Reference

11 147 25 6 4 6

0 11.6 44 16.7 50

0 1.4 25 16.7 25 0

69 94 73 37 37 47

4 1 61 8 382 51 39 17 4 10 25 37 2 5 4 32 335 282 232 89 22 100 64 324 434 33 31 32 37 12 61 12 110 380 340 190 92 51 32

50 100 14.8

50 0 2 12 2.4 0 12.8 5.9

22 22 1 126 94 93 37 37 62 62 73 128 69 69 138 61 108 108 108 77 69 76 65 77 78 126 69 44 136 73 35 73 81 23 74 82 75 88 88

12.6 3.9 25.6 17.6 25 20 52 43.2 0 0 75

63.6

29 0–80

32 8.1 0 0 25 9 10.8 22.3 14.2 25.8 22.7 24 6.3 13.6 11.3 9 3.2 31.2 0 75 78.7 66.7 20 7.1 20.9 33.7 5.4 39 9.4

(continued)

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TABLE 15.7 (CONTINUED) Incidence of Listeria spp. in Fresh, Frozen, and Processed Seafood Product (country) Fish (gravad or sm, salmon/rainbow trout) Fish (gravad or sm, salmon/rainbow trout) Fish (gravad or sm, salmon/rainbow trout) Fish (gravad or sm, salmon/rainbow trout) Fish (sm fish) (Canada) Fish (sm fish) (Canada) Fish (hot sm fish) (Switzerland) Fish (hot sm fish) (Switzerland) Fish (sm and/or salted) (Egypt) Fish (Canada import) Other fish and seafood products Seafood (smk) (Canada import) Seafood (smk) (Canada) Seafood (eel) (Canada import) Seafood (squid), langostinos) (multiple countries) Seafood (f and fr) (Taiwan) Seafood (f and p) (Iceland) Seafood (India) Seafood (f) (U.S.A.) Seafood (p) (U.S.A.) Seafood (other) (Iceland) Seafood raw (r) (Japan) Seafood (r) (New Zealand) Seafood (c) (Japan) Seafood (other) (Japan) Seafood (r) (Japan) Seafood salad (p) (U.S.A.) Fish salads (r) (Iceland) Seafood (Pasta with minced fish) (Iceland) Seafood (surimi) (multiple countries) Seafood (surimi) (U.S.A.) Seafood (surimi) (Canada) Kamaboko (surimi) (Canada import) Pâté (Canada import) Seafood salad (Canada) Processed seafood (Canada)

Number of Samples 29 103 94 35 71 496 691 11 3 64 130 14 2 57 26 200 59 14 5 28 50 5 6 213 2 37 3 7 1 46 335 20 4 11

(%) of Positive Samples Listeria spp.

L. monocytogenes 10.3 16.5 17 22.9

11.3 20.4

18.2

4.4 8.9 8.4 5.6 33.3

88 88 88 88 33 31 77 78 37 47

1.6 0 0 0

64 47 47 138

10.5 3.9 0

139 69 83 105 105 69 128 73 128 128 75 22 69 69 139 22 44 47 47 47 47

100

3.9 8 49.2 0 20 7.1 48 0 0 0 32 0 0

Reference

20 10.7 26 0 0 3.3 0 16 0 28.6 0 2 0 0 0 0

Note: (c) = cooked; (f) = fresh; (fr) = frozen; (p) = processed; (r) = ready-to-eat. a

Includes the 14 smoked mussel samples listed in mussels category. Included in the 25 ready-to-eat fish samples in the New Zealand study list given earlier.

b

Source: Adapted in part from Embarek, P.K.B. 1994. Presence, detection and growth of Listeria monocytogenes in seafoods: a review. Int. J. Food Microbiol. 23: 17–34.

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As with the surveys of cooked and processed crab, few studies have attempted to quantify L. monocytogenes in cooked shrimp. However, low levels of L. monocytogenes in three lots of naturally contaminated RTE shrimp (0.54, 5.5, and 0.04 MPN/g) and lobster (2.0, 0.23, and 0.4 MPN/g) were reported in one study [44].

SHELLFISH Smoked mussels have been associated with several listeriosis cases in Australia and New Zealand [42,97] and the subject of recalls in the United States. L. monocytogenes was isolated from 5 of 14 smoked mussel samples in a survey [73] in New Zealand, whereas for fresh mussels from Spain, L. monocytogenes was recovered from 3 and other Listeria spp. from 9 of 40 samples [130]. The incidence of Listeria in other shellfish products, including oysters, clams, and scallops has been remarkably low. No Listeria spp. were isolated from oysters in two studies in the United States and one in Egypt that included one or two samples [22,37,138]. In a Japanese study, no Listeria spp. were recovered from 84 oyster samples [94]. No Listeria spp. were found in clams in two studies with a single sample in each [22,62]; however, in Egypt, Listeria spp. were found in two of four Ruditapes spp. (clam) samples, one of which yielded L. monocytogenes [37]. Scallops are another category for which only a limited number of samples have been analyzed. L. innocua was isolated from one of two scallop samples [138]. No Listeria spp. were recovered from a single sample in a later study [22]. Similarly, no listeriae were found in 11 cooked shellfish samples from Iceland [69]. In a survey of fresh seafood purchased from markets in Alexandria, Egypt, one of six Donax spp. (coquina) samples was positive for L. monocytogenes [37]. In Japan, L. monocytogenes was isolated from 2 samples and Listeria spp. were recovered from 17 samples of a total of 147 non-oyster shellfish samples [94,101].

FINFISH In the United States raw fresh or frozen fish are generally not consumed without further processing; therefore, surveys there have included very few fresh or frozen fish samples. L. monocytogenes was isolated from two of four fresh fish samples, L. innocua from one catfish sample [22], and L. monocytogenes from one of four frozen fish samples [138] in surveys conducted in the United States. In India, Fuchs and Surendran [62] reported Listeria spp. in 1 of 4 fresh and 2 of 10 frozen fish samples. In a larger survey of fresh fish in India, Listeria spp. were isolated from 2 of 51 (3.9%), samples but no L. monocytogenes was recovered [93]. In Alexandria, Egypt, Listeria spp. were recovered from 10 of 39 (25.6%) fresh and 3 of 17 (17.6%) frozen fish samples, with L. monocytogenes isolated from 5 of 39 (12.8%) and 1 of 17 (5.9%) of these fresh and frozen fish samples, respectively [37]. In Norway, L. monocytogenes was isolated from 1 of 8 (12.5%) minced fresh fish samples [126]. The practice of consuming fresh seafood in an almost raw state is common in Trinidad, where Listeria spp. were detected in 9 of 61 (14.8%) fresh fish samples [1]. In Japan, 3 of 37 (8.1%) raw RTE minced fish samples yielded L. monocytogenes with 16 of 37 (43.2%) containing Listeria spp. [128]. In another study in Japan, L. monocytogenes was found in 9 (2.4%) and Listeria spp. in 48 of 382 (12.6%) fresh fish samples [94]. It is not clear if all these samples were RTE. A more recent study in Japan reported that 7 of 213 (3.3%) raw seafood samples were positive for L. monocytogenes [75]. However, only one of the seven positive samples contained >100 L. monocytogenes CFU/g.

SMOKED FISH PRODUCTS The prevalence of Listeria in smoked and lightly processed fish products is often a concern because many of these products are commonly eaten without further heating. The cold-smoking

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631

process does not generate sufficient heat to inactivate Listeria organisms that may be present on fish [35,66,91]. In Switzerland, L. monocytogenes was isolated from 24 and 6% of coldsmoked salmon [65,76] and 13.5 and 11.3% of cold-smoked fish samples [77,78]. In studies in Norway [123], Canada [44], and New Zealand [73], the organism was isolated from 9, 31, and 66% of the cold-smoked salmon samples examined (Table 15.7). The level of L. monocytogenes in smoked fish tends to be low. In a Swedish survey, 12 of 17 samples positive for L. monocytogenes had less than 10 L. monocytogenes CFU/g; 4 samples had levels between 10 and 1000 CFU/g, and only one had more than 10,000 CFU/g [88]. A second Scandinavian study reported 6 of 16 positive samples had less than 10 L. monocytogenes CFU/g, 6 samples with 10–1000 CFU/g, 2 samples with 1000–10,000 L. monocytogenes CFU/g, and 2 samples with more than 10,000 L. monocytogenes CFU/g [88]. In a third study 19 of 27 positive samples had less than 100 L. monocytogenes CFU/g, 4 samples had between 100 and 10,000 L. monocytogenes CFU/g, and more than 10,000 L. monocytogenes CFU/g was recovered from the remaining 4 positive samples [23]. In comprehensive studies of one processing plant, Eklund et al. [35] identified incoming raw salmon (36% combined incidence and 65% skins from a frozen storage warehouse) as the initial source of L. monocytogenes. During filleting and brining, the pathogen was transferred to other fish fillets with exposed flesh. As the product moved through the different stages of processing, equipment, personnel, and other contact surfaces became contaminated and served as additional sources of contamination. If these secondary sources of contamination are not properly eradicated, they can become a continuous source of contamination. In more recent studies by Hoffman et al. [71], a 29.5% incidence of L. monocytogenes in U.S. West Coast salmon and a lower incidence in other fish species were reported. Autio et al. [15], Rørvik et al. [125], and Vogel et al. [137] also reported a low incidence of L. monocytogenes in raw fish and concluded that the primary source for L. monocytogenes contamination was from equipment and the processing environment. Differences in the reported incidence of L. monocytogenes in raw fish could in part be related to methodology. Because of the low incidence of L. monocytogenes and possible harboring of the bacterium under scales of the fish, Eklund et al. [35] removed and enriched the entire skin of raw fish in both Listeria enrichment broth (EB) and University of Vermont modified (UVM) broth. In comparison, other investigators have used smaller samples (sections of fish, swabbed areas, and scrapings). In many instances, the incidence in raw fish may be low; however, even if only a small number of raw fish are initially contaminated, they can easily contaminate the hundreds of other fish in a day’s production. As an example, Autio et al. [15] reported that only 1 out of a total of 60 fish contained L. monocytogenes. They also reported that the water, salt, and sugar used in the brine were not contaminated. However, the frequency of fish containing L. monocytogenes clearly increased after brining, which was the most critical point of L. monocytogenes contamination. In addition, gloves of employees working on the product after brining were positive for L. monocytogenes. Recent studies using molecular subtyping also indicate that raw materials rarely seem to be responsible for finished product contamination in the production of cold-smoked seafood. Instead, the processing environment seemed to be responsible for most contamination of smoked fish [15,81,109,125,137]. In these instances, L. monocytogenes appeared to have become established on equipment and in processing areas with sanitation procedures inadequate to eliminate Listeria. In those studies that specifically identified hot-smoked fish samples, L. monocytogenes was also recovered despite the heat processing that these products received, with the pathogen present in 8.9 and 8.4% of samples [77,78].

LIGHTLY PROCESSED FISH PRODUCTS Other lightly processed RTE fish products have also harbored L. monocytogenes. These include lightly pickled fish from which L. monocytogenes was isolated from 25.8% of samples surveyed

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in Switzerland [77]. Ceviche is a lightly acidified RTE fish product, which is popular in several South American countries. Listeria spp. were recovered from 75% and L. monocytogenes from 9.4% of ceviche samples in Peru [61]. In Iceland, seafood and fish salads yielded Listeria spp. in 32.4% and L. monocytogenes in 16.2% of the samples [69]. No Listeria spp. were recovered from two seafood salad samples included in a U.S. survey [22] or from three pasta salads containing minced fish in the Iceland study [69]. Imitation seafoods made from surimi had an L. monocytogenes incidence of 28.6% in the United States [138] and 2.2% in Canada. No Listeria were recovered from a single sample in a second U.S. survey [22].

HUMAN LISTERIOSIS ASSOCIATED WITH FISH AND SEAFOOD PRODUCTS There have been several reported human listeriosis outbreaks associated with consumption of fish and seafoods. As early as 1980, an epidemic of 22 perinatal L. monocytogenes infections during an 11-month period was reported in Auckland, New Zealand [86]. Although the cause was not confirmed, an epidemiological survey suggested that shellfish and raw fish consumption may have played a role [86]. The first case of listeriosis positively linked to consumption of fish or seafood was not reported until 1989, when a 54-year-old woman in Italy contracted listeric meningitis 4 days after consuming steamed fish from which L. monocytogenes was later isolated [43]. Two L. monocytogenes isolates, one from the patient’s cerebrospinal fluid and the other from a leftover portion of the fish, were both of serotype 4 and were identical in terms of phage type and restriction analysis of chromosomal DNA, indicating that the fish was the most likely vehicle of infection in this case of listeric meningitis. However, how this fish became contaminated remains a mystery. In 1991, three healthy people, aged 83, 37, and 10, became ill in two separate incidents in the state of Tasmania, Australia, after consuming smoked mussels [97]. Symptoms included malaise, chills, fever, and headache followed by diarrhea. Samples of the implicated mussels contained over one million L. monocytogenes CFU/g with this pathogen also recovered from stool samples. The implicated mussels were exported to Australia, repackaged illegally by a retail outlet, and labeled with a code date that overestimated their shelf life by 3 months or more. Patient isolates and those from associated refrigerated packages of mussels were identified as L. monocytogenes serovar 1/2b and were indistinguishable by both phage typing and restriction fragment length polymorphism (RFLP) analysis [21]. Newborn twins died as a result of an L. monocytogenes infection in Auckland, New Zealand, in 1992. Their deaths were attributed to consumption of contaminated smoked mussels by their mother. Reports from New Zealand indicated that the company producing the smoked mussels had Listeria in their product several months before the twins died. In 1993, the company owner and a consultant to the company were charged with manslaughter by New Zealand police [42]. The two patient isolates of L. monocytogenes were indistinguishable by serotype and pulsed-field gel electrophoresis (PFGE) analysis. In addition, 2 more clinical isolates of the same serotype and PFGE pattern were identified among 15 clinical listeriosis cases in New Zealand during 1991 and 1992. These 4 patient isolates along with 26 isolates from 15 retail packages of the implicated mussels, the processing factory, the refrigerator of one patient, and a wholesaler in the United Kingdom and 7 isolates from environmental swabs taken in the processing factory environment were analyzed by various subtyping methods [21]. These included serotyping, phage typing, DNA-RFLP analysis, cadmium and arsenic sensitivity testing, and PFGE analysis. Isolates from 3 of the patients as well as 26 from the product and 4 from factory environmental swabs were all indistinguishable by all of the subtyping systems [21]. This linked the patient isolates with the implicated smoked mussels and the processing environment. These isolates were also distinct from the patient cultures in the previously mentioned Tasmanian outbreak [21].

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Since these outbreaks, the Australian National Health and Medical Research Council [102,103] has issued two bulletins recommending special dietary advice for pregnant women, transplant patients, and other immunocompromised patients and providing information for medical practitioners on the diagnosis, treatment, and other advice for patients. Shrimp was identified as a significant risk factor for illness during an epidemiological investigation in Connecticut [121]. The investigation was initiated after two pregnant women who attended a common party developed Listeria bacteremia. The case definition of Listeria infection, including isolation of L. monocytogenes from a blood or stool sample, or the occurrence of two or more symptoms of mild listeriosis including fever, musculoskeletal problems, nausea, vomiting, or diarrhea was used to evaluate the possibility of mild disease and foods associated with the illnesses. The case definition of listeric infection was met for 10 (28%) of the 36 party attendees. Consumption of shrimp, nonalcoholic beverages, Camembert cheese, and cauliflower were identified as being associated with illness, but only shrimp remained after controlling for consumption of other foods among the party attendees. Two cases of listeriosis in previously healthy adults were linked to consumption of imitation crabmeat in Ontario, Canada [48]. L. monocytogenes was isolated from several items from the patient’s refrigerator, including imitation crabmeat, canned black olives, macaroni and vegetable salad, spaghetti sauce with meatballs, and mayonnaise. The imitation crabmeat contained L. monocytogenes at a level of 2.1 × 109 CFU/g. In addition, L. monocytogenes was also isolated from unopened packages of the imitation crabmeat. The isolates from the patients’ opened and unopened imitation crabmeat were serotype 1/2b, indistinguishable by random amplified polymorphic DNA (RAPD) and PFGE analyses. The ability of imitation crabmeat to support growth of L. monocytogenes in challenge studies emphasizes the concern about refrigerated products with a long shelf life and no additional control factors to prevent growth of L. monocytogenes. Gravad or cold-smoked rainbow trout was identified as the source of a listeriosis outbreak in Värmland, Sweden [41,132]. Nine people became ill, with two deaths due to listeriosis in this small province between August 1994 and June 1995. L. monocytogenes isolated from six of the patients, a package of gravad rainbow trout in one patient’s refrigerator, and an unopened package of gravad rainbow trout from the same manufacturer were indistinguishable from each other using PFGE. The level of L. monocytogenes in the fish from the patient’s refrigerator was reported to be 6200 CFU/g. The levels in four unopened packages from the same manufacturer were less than 100 CFU/g for three samples and 120 CFU/g for the fourth sample. A fifth sample was delayed during shipment to the laboratory, resulting in a level of 2.5 × 106 CFU/g when analyzed, indicating that this product does support growth of L. monocytogenes to high levels when temperature-abused. Gravad fish is prepared from raw fillets rubbed with sugar, salt, and pepper, covered with dill, refrigerated 2 days, and then vacuum packaged either whole or sliced in oxygen-impermeable film. The cold-smoked fish products from this same manufacturer were prepared by either rubbing raw fillets with salt, or injecting the cure followed by smoking at 25–30°C for 2–3 h, and then packaging either whole or sliced under vacuum. L. monocytogenes of the same clonal group associated with the outbreak was isolated from unopened packages of gravad and cold-smoked rainbow trout and from the packaging machine in the processing plant. This observation, and the fact that the packaging machine was difficult to clean, led investigators to hypothesize that the packaging machine was the likely source of contamination. Consumption of cold-smoked rainbow trout was also implicated in an outbreak involving five previously healthy adults who developed febrile gastroenteritis in Finland [96]. They consumed the same cold-smoked fish at a meal and all experienced nausea, abdominal cramps, and diarrhea within 27 h. An unopened package from the same lot of implicated product obtained from the same retail store contained 1.9 × 105 L. monocytogenes CFU/g. The temperature in the retail store display cabinet varied from 4.6°C at the bottom to 11.6°C at the top, whereas the manufacturer recommended a storage temperature of 0–3°C. The elevated number of L. monocytogenes organisms in this product was likely due to the higher storage temperature. The L. monocytogenes isolates from the five patients and the cold-smoked rainbow trout were serotype 1/2a and indistinguishable by PFGE analysis.

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REGULATORY ASPECTS OF L. MONOCYTOGENES IN FISH AND SEAFOOD Although a detailed discussion of proposed criteria for L. monocytogenes and other pathogens in foods has been reserved for Chapter 18, the reader should be aware that the NACMCF has recommended a “zero-tolerance” for L. monocytogenes in cooked shrimp and crabmeat [9,11]. The policies of different countries vary for the regulation of L. monocytogenes in food products. In 1994, Madden stated “The policy of the (U.S.) FDA remains what has been commonly referred to as the ‘zero tolerance’ policy, which is very conservative. No L. monocytogenes organisms are permitted in a food which was not intended for further heat treatment.” [92]. The detectable presence of L. monocytogenes in RTE foods is considered to be a hazard to health under current FDA policy [36]. Elliot and Kvenberg state, “Currently, FDA requests recall of any ready-to-eat food in which L. monocytogenes is detected using present methodology, if it is determined that the cooking or heating instruction would not provide for lethality. A Class I recall [CFR 7.3 (m) (1)] is initiated when there is a reasonable probability that the use of, or exposure to, a violative product will cause serious adverse health consequences or death.” They noted a quantitative L. monocytogenes risk assessment was underway in the United States as a joint effort between HHS and USDA and concluded that a thorough review of this risk assessment should be made before making further risk management decisions about L. monocytogenes policy. Canadian regulations regarding the presence of L. monocytogenes in RTE foods have been based on the ability of L. monocytogenes to grow on a given food product [45]. Three categories of foods have been established based on assessed health risk. Category 1 includes foods linked to outbreaks and receives the highest regulatory priority. The second level of regulatory priority includes RTE foods that can support growth of L. monocytogenes and have a shelf life greater than 10 days. RTE foods that either do not support growth of L. monocytogenes or have a shelf life of less than 10 days are classified as category 3 and receive the lowest priority in terms of inspection and compliance actions [47]. Complete agreement among the countries that make up the European Economic Community (EEC) for the criteria of L. monocytogenes in all foods [131] has not to date been achieved. Criteria established for L. monocytogenes by the EEC are only within the Milk Hygiene Directive. Several approaches have been used by individual European countries, including addressing industry groups about HACCP-based hygiene plans and education of the most susceptible groups, such as pregnant women and immunocompromised individuals. A quantitative approach setting limits at the point of sale or end of product shelf life has also been explored by several countries. For example, German regulations categorize foods into four risk-level categories and set specific L. monocytogenes action levels based on the risk category. Group I foods have the most restrictive limit (absence of L. monocytogenes in 25 g or mL of food). Under German regulations, seafood products such as heat-treated shrimp or prawns would be categorized as Group III. For products in this category, low-level contamination (<100 L. monocytogenes CFU/g) would require a food establishment hygiene check. Higher levels of contamination would cause the product to be classified as “unfit for human consumption” or a health hazard in substantiated cases [131]. Danish policy, introduced in 1998, is based on inspection and testing of RTE foods to ensure that levels of L. monocytogenes in food do not exceed 100 CFU/g at the point of consumption [108]. Foods are placed into one of six categories. No L. monocytogenes is permitted in a 25-g sample for foods in three of the categories: those that receive a heat treatment in the final package, those that are heat-treated and handled after heat treatment and could support growth during the shelf life of the product, and those foods that are lightly preserved but not heat-treated. The final three categories are treated similarly and include products that are handled after heat treatment but are stabilized again to prevent L. monocytogenes growth; lightly preserved, non-heat-treated RTE foods that are stabilized; and raw RTE foods. For these latter three categories, L. monocytogenes at a level of ≤10 CFU/g is acceptable, whereas those between 10 and 100 CFU/g

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are unsatisfactory. Action may be taken based on consideration of possible growth within the product shelf-life time, and an evaluation of the processor’s HACCP system may be requested. The authorities would consider the prohibition of sale and recalls for products that contain more than 100 CFU/g.

RISK ASSESSMENT Risk assessment is likely to be used more often as the basis for regulatory risk management and policy decisions. Application of risk assessment was one of the elements reviewed at the May 1999 Food and Agriculture Organization (FAO) of the United Nations expert consultation on the trade impact of Listeria in fishery products [46]. A risk assessment is composed of the following four parts: (1) hazard identification, (2) exposure assessment, (3) hazard characterization/dose–response, and (4) risk characterization.

HAZARD IDENTIFICATION The presence of L. monocytogenes in foods is generally well established. Infections caused by L. monocytogenes are infrequent, fewer than 10 cases/million, and the population at greatest risk is the immunocompromised; however, the severe consequences, 20–30% mortality rate that increases to 75% for the most highly susceptible individuals, make this organism an important hazard and its presence in foods a significant public health concern [107,110].

EXPOSURE ASSESSMENT Hazard exposure is more difficult to specify. The ability of L monocytogenes to grow over a wide range of temperatures, 0–45°C, at pH 4.5–9.2, at water activity values > 0.92, and as a facultative aerobe contributes to the ubiquitous nature of this organism in the environment and many foods and suggests that human exposure is common [107]. In addition, L. monocytogenes has found an ecological niche for growth in refrigerated RTE foods having an extended shelf life [110]. Foods that do not have a listericidal step or could become contaminated before consumption are of particular concern [127]. Hazard exposure is also directly related to consumption patterns for RTE foods, which can be difficult to assess and vary with cultural dietary preferences in different countries. Consumption patterns of 23 different RTE foods in the 2003 FDA/FSIS/CDC Listeria monocytogenes Risk Assessment [133] were based on The Continuing Survey of Food Intakes by Individuals (CSFII 1994–1996) and the Third National Health and Nutrition Examination Survey (NHANES III) (1988–1994) and reflect U.S. dietary patterns.

HAZARD CHARACTERIZATION Although three main serotypes of L. monocytogenes (4b, 1/2a, and 1/2b) are associated with most human epidemics and sporadic cases, there are no extensive data demonstrating systematic differences between food and clinical isolates. Therefore, at this time all L. monocytogenes isolates are generally considered pathogenic [107]. Dose–response data for humans are not well established. Epidemiological investigations usually suggest that elevated levels of organisms and consumption of foods that support growth of L. monocytogenes are associated with illness [107]. Although several attempts have been made to establish dose–response curves, large variation in dose–response data has limited their usefulness [110].

RISK CHARACTERIZATION Risk assessment studies for L. monocytogenes are being undertaken by authorities from several countries, the European Union, WHO, and FAO [110]. Development of a model to characterize

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risk is complicated by lack of complete information and differences in how the variables are weighted. There is a need for more quantitative information about survival and growth of L. monocytogenes in fish and seafood during handling and processing to more fully assess health risk [127] from these products. Ideally, a risk assessment model would include all the variables that may affect growth, survival, and inactivation of L. monocytogenes in fish products, such as pH, organic acids, a w , gaseous atmosphere, smoke components, and the ability of L. monocytogenes to compete among bacterial flora present on the product (Jameson effect) [127]. The FDA/FSIS/CDC 2003 Quantitative Assessment of Relative Risk to Public Health from Foodborne Listeria monocytogenes Among Selected Categories of Ready-to-Eat Foods [133] used exposure and dose–response models to predict the relative risk of illness attributable to each of 23 food categories. Although a substantial amount of uncertainty exists around these models, several categories were identified as part of the FDA/FSIS/CDC Quantitative Risk Assessment. Food categories containing seafood that were designated “high risk” and warranted identification of new approaches for reducing potential L. monocytogenes contamination included smoked seafoods. Food categories containing seafood designated “moderate risk” included cooked RTE crustaceans and deli salads. These foods generally use bactericidal treatments during manufacture or limit growth of L. monocytogenes; therefore, consistent application of proven control measures for these foods is recommended. Foods with low predicted relative risk due to inherent characteristics less likely to be associated with listeriosis cases or outbreaks include preserved and raw seafood.

BEHAVIOR OF LISTERIA IN FISH AND SEAFOOD Before 1987, very little was known about the incidence and behavior of Listeria spp. in fish and seafoods. Since this time, a number of studies have focused on growth, inhibition, and thermal resistance of L. monocytogenes in different products. More recent studies have incorporated molecular typing methods to trace dissemination of L. monocytogenes strains in seafood processing facilities. The results from these studies and the means by which this pathogen may be transmitted to various forms of aquatic life are discussed in this section.

MODES

OF

TRANSMISSION

Current data point to cross-contamination as the major source of Listeria in cooked or otherwise processed seafood, as evidenced by recovery of healthy, noninjured cells of L. monocytogenes from the surface of many heat-processed/RTE seafood products. Raw fish contaminated in the natural environment cannot be completely ruled out as the source of contamination in a seafood processing environment that could subsequently affect the final product. Even if initial contamination of the processing environment is due to incoming raw product, often only a few strains become established in the processing environment and are recovered from final products, indicating that adherence to proper sanitation in the processing environment is necessary for control of L. monocytogenes in the final product. The salt-tolerant nature of L. monocytogenes and presence of this pathogen in sewage effluent entering the North Sea [30] and also from crustaceans that were harvested from stream water in which L. monocytogenes was previously identified [112] indicate that it is present in the marine environment and can contaminate fish and other aquatic animals. In 1989, Fuad et al. [60] evaluated the ability of L. monocytogenes to survive in the estuarine environment. Because there appears to be a higher incidence of Listeria in chitinous seafood (i.e., shrimp, crab, and lobster), samples of filtered and unfiltered seawater with and without chitin and chitin-free filtered and unfiltered stream water were inoculated with various strains of L. monocytogenes, many of which possessed chitinase activity. Although preliminary results indicate that Listeria populations decreased in chitin-free filtered and unfiltered seawater, addition of chitin to both

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types of seawater stimulated growth of Listeria. Moreover, the pathogen also grew in filtered stream water. These findings, along with those from a report in which L. monocytogenes was found on the exoskeleton but not in the digestive tract of shrimp that were exposed to high levels of L. monocytogenes in aquaculture tanks [136], suggest that this pathogen may be ecologically adapted to chitin. If this is true, then it is imperative that holding tanks for chitinous marine animals be properly set up and maintained to avoid potential microbiological problems involving L. monocytogenes and other foodborne pathogens, including Vibrio spp. and Aeromonas hydrophila. It is well established that Listeria spp. are often associated with wild animals and birds, both of which can serve as reservoir hosts. Fenlon et al. [59] demonstrated the role that scavenging birds can play in the Listeria cycle. In that study a definite association was seen between gulls feeding on sewage and fecal carriage of Listeria (26.3% positive), which compared to a Listeria carriage rate of only 8.0% for gulls feeding in less polluted areas. This higher carrier rate could lead to the conclusion that L. monocytogenes is part of the normal microflora of the near estuarine environment [12]. Two studies of estuarine waters, shrimp, and oysters along the northern Gulf of Mexico [100] and of freshwater tributaries, sediments, bay water, and oysters in the Humboldt– Arcata Bay of Northern California [25] reached similar conclusions. In one study [99], 5% of 78 salt water samples were positive for Listeria spp. In comparison, 11% of 74 shrimp samples were positive for L. monocytogenes and no Listeria were isolated from the oysters. Listeria species and L. monocytogenes were found in 81 and 62% (37 samples), respectively, of fresh or low-salinity waters in tributaries draining into Humboldt–Arcata Bay. The incidence of Listeria spp. and L. monocytogenes in sediment (46 samples) from the same tributaries was 30.4 and 17.4%, respectively. One of three bay water samples contained Listeria spp. (including L. monocytogenes), whereas L. innocua was recovered from only 1 of 35 oyster samples [25]. Both these studies indicated that estuarine environments are continuously subjected to potential contamination with Listeria spp. from processing effluents, agricultural runoff, and sewage effluents. Listeria spp. can be recovered from nonpolluted environments, and the source of this bacterium may very well be from avian species, especially seagulls [99]. The issue of whether the same strains recovered from seafood products are also isolated from human clinical cases has been studied by several groups. An investigation by Rørvik et al. [125] in a Norwegian smoked salmon processing plant identified a single L. monocytogenes strain that had colonized the processing environment and contaminated the smoked fish. This strain was also one of the most frequently recovered from patients in Norway, indicating that smoked fish could be a source of illness. In this study, L. monocytogenes was infrequently isolated from the raw product, and these strains were indistinguishable from those found in the processing environment or final product. An additional study by Rørvik et al. [124] characterized 305 L. monocytogenes isolates from patients, seafood products, seafood processing plants, seawater, and a transport terminal by MLEE and REA. Patient isolates belonged to 11 electrophoretic groups, with 4 groups accounting for 77% of the isolates. There was much greater genetic diversity among isolates from seafood products which also included strains isolated from patients. Despite the greater diversity of strain patterns among seafood and environmental isolates, as a whole individual processing plants tended to be colonized by a specific L. monocytogenes strain. These observations were further supported by Norton et al. [109] who used several molecular typing methods (ribotyping, PCR-RFLP typing of hylA and actA genes) to characterize 392 isolates from patients, smoked fish, raw materials, fish during processing, and the processing environment. Significantly more human clinical isolates (69%) belonged to lineage group 1 than industrial isolates (36.8%). All epidemic strains of L. monocyotgenes belong to lineage group 1, indicating that strains in this lineage may possess enhanced virulence characteristics. Boerlin et al. [17] concluded that isolates from human illness and fish products imported into Switzerland belonged to separate subpopulations of the species. It is generally recognized that contamination of finished seafood can occur within the processing environment. One of the first comprehensive studies of L. monocyotgenes contamination

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throughout a cold-smoked rainbow trout (Oncorhynchus mykiss) processing plant was conducted by Autio et al. [15]. The incidence of L. monocytogenes on raw fish was very low, and the frequency of fish contamination clearly increased after the brining step. In addition, the brining and postbrining areas of the plant were the most contaminated. PFGE was used to characterize 303 isolates from raw fish, finished product, and the environment. The predominant L. monocytogenes PFGE patterns associated with isolates from the finished product were associated with brining and slicing operations but not isolates from the raw fish. Adoption of a rigorous eradication program using hot steam, hot air, and hot water eliminated the presence of L. monocytogenes in the processing environment and for the remainder of the study (5 months). Isolates from a Finnish survey of an RTE vacuum-packed smoked and cold-salted fish manufacturer and fish farms providing raw materials were characterized by serotyping and PFGE [81]. No L. monocytogenes strains were isolated from raw product, but a single predominant PFGE clone of L. monocytogenes was isolated from the salting, skinning, and slicing machines and the finished product. Implementation of a HACCP program at this plant resulted in samples negative for L. monocytogenes for at least 9 months. A total of 3585 samples were analyzed over a 4-year period from products and the processing environments of two Danish cold-smoked salmon processing plants [137]. RAPD was used to subtype 429 isolates. Strains isolated from the final product were indistinguishable from strains from processing equipment and the processing environment. The brining and slicing areas were identified as the most likely areas of product contamination. This suggested that contamination of final product was due to processing rather than from incoming raw fish, but raw fish as a source of contamination could not be excluded. There were several differences noted between the two processing plants. L. monocytogenes was isolated more frequently from finished product from plant 1 than from plant 2. The same strain of L. monocytogenes was isolated over a 4-year period from plant 1, indicating that it had become established in the processing area and was not eliminated by routine cleaning. In contrast, no predominant single strain of L. monocytogenes persisted for a long period of time in plant 2. The authors of this study concluded that a rigorous cleaning and sanitation program in plant 2 reduced the incidence of persistent strains, but sporadic contamination could still occur. A study by Hoffman et al. [71] sought to determine the prevalence of L. monocytogenes in raw fish and in the processing environments of two US cold-smoked fish processors. They characterized 512 environmental isolates and 315 isolates from raw fish by automated ribotyping. Consistent with the previous studies [15,137], they concluded that contamination of the smoked-fish processor environmental was separate from incoming raw materials and that some strains could become established and persist in the plant environment for long periods of time. Both plants in this study had similar L. monocytogenes incidence rates on incoming raw materials but different prevalence rates for the environmental sites, indicating that operational and sanitation procedures can have a significant impact on contamination in processing environments. Molecular subtyping methods, including RAPD and PFGE, were used to trace L. monocytogenes contamination in a Santos, SP, Brazil, shrimp processing plant [29]. Many different sources and subtyping group profiles of L. monocytogenes were identified within the plant. Only two strain profile groups were observed on frozen finished product, and these were among nine different profile groups recovered from processing areas and food handlers. Interestingly, none of the profile groups recovered from the environment, water, or utensils were observed on shrimp.

GROWTH

AND

SURVIVAL

Raw and processed seafoods have long been regarded as excellent substrates for growth of most common agents of foodborne disease, particularly if seafoods are held at improper temperatures; the behavior of L. monocytogenes specifically in the products has also been studied [38]. According to data gathered by Lovett et al. [90] in 1988, L. monocytogenes grew readily (generation time ≅ 12 h) in inoculated samples of raw shrimp, crab, surimi, and whitefish, with the pathogen attaining maximum populations greater

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than 108 CFU/g in all four products following 14 days of storage at 7°C. Two years later, Brackett and Beuchat [18] also reported that L. monocytogenes grew and retained similar levels of pathogenicity on artificially contaminated crabmeat during 14 days of storage at 5 to 10°C. In similar studies, Rawles et al. [120] determined both the incidence and growth of L. monocytogenes in blue crabmeat during refrigeration. Of the 126 samples analyzed, 10 were positive for L. monocytogenes and 3 were positive for L. innocua. The levels of Listeria cells found in fresh picked blue crabmeat were usually less than 100 CFU/g. Based on these data, commercially pasteurized crabmeat was inoculated to contain 50 CFU/g after which the growth rate was determined at 1.1, 2.2, and 5°C. Generation times were calculated as 68.7 h at 1.1°C, 31.4 h at 2.2°C, and 21.8 h at 5°C. At 5°C there was a 7 log10 increase in L. monocytogenes populations and only a 2.5 log10 increase at 1.1°C after 21 days. The authors therefore concluded that blue crabmeat should be maintained below 1.1°C. However, in contrast to what Lovett et al. [90] observed for shrimp and whitefish, Harrison et al. [67] and Shineman and Harrison [129] found that L. monocytogenes failed to grow in overwrapped/ vacuum-packaged raw shrimp and finfish, with numbers of Listeria generally decreasing by approximately 1 log10 after 21 days of storage in an ice chest. When catfish were stored at 4°C, L. monocytogenes populations increased slowly (1–1.5 log10) during the first 12 days and then decreased 1.5 log10 by day 16 [87]. During storage, psychrotrophic populations increased from 103 to >107 CFU/g, thus reinforcing the notion that L. monocytogenes can readily survive in refrigerated raw foods, even when greatly outnumbered by other natural contaminants. Because L. monocytogenes was recovered from laboratory-contaminated shrimp (initial inoculum ≅ 105 CFU/g) after 90 days of storage at −20°C [71], it is evident that this pathogen also is fairly resistant to subfreezing temperatures. Unlike the aforementioned products, preliminary results from Kaysner et al. [84] suggest that L. monocytogenes was unable to grow in artificially contaminated oysters, with Listeria populations remaining constant in shucked oysters after 21 days at 4°C. The apparent inability of Listeria to grow in raw oysters may partially explain the difficulties in isolating Listeria from retail raw oysters. According to Farber [44], L. monocytogenes (inoculum level of 2 × 103 CFU/mL) grew fairly well on cooked lobster, shrimp, crab, and smoked fish and in most instances increased about 2–3 log10 within 7 days at 4°C. When these same products were temperature-abused for a short time (6 h) at room temperatures, the levels of L. monocytogenes increased by 1 log on shrimp, crab, and lobster, and only 0.2 log on smoked salmon. During a survey of the incidence of this pathogen on shrimp and lobster meat at the wholesale level, 13 of 113 samples were positive and were contaminated at a level of less than 10 MPN/g. Storage of these naturally contaminated products at 4°C resulted in L. monocytogenes populations below 100 MPN/g after 1 week with numbers increasing to 2.5 × 103 MPN/g after 2 weeks [44]. In these studies, the growth rate of different L. monocytogenes strains was comparable in shrimp, crab, and cooked lobster. The difference in growth rate of two strains in smoked salmon, however, probably reflected the greater tolerance of one strain to sodium chloride or possibly to the antimicrobial compounds present in smoke. The behavior of L. monocytogenes in cold-smoked salmon has been studied at several different laboratories. Guyer and Jemmi [65] and Jemmi [78] determined the growth of L. monocytogenes during fabrication and storage of cold-smoked salmon. Populations increased to 2.3 × 106 MPN/g at 10°C and up to 1.5 × 105 at 4°C during 20 days of storage. After 30 days, L. monocytogenes populations increased to 107 MPN/g in samples stored at both temperatures. A pH of 5.8 and aw of 0.93 had no influence on growth of L. monocytogenes. During fabrication of cold-smoked salmon, the numbers of L. monocytogenes inoculated onto the surfaces did not change. In contrast, Eklund, et al. [35] demonstrated that during injection of recirculated brines into the interior of cold-smoked salmon, L. monocytogenes was also inoculated into these sites. During processing at temperatures between 17.2 and 21.1°C, L. monocytogenes increased 2- to 6-fold; when the processing temperature was increased to 22.2–30.6°C, this pathogen increased up to 100-fold. These authors therefore emphasized the importance of eliminating or reducing the L. monocytogenes population on the outside of the fish before they are filleted. In addition, they also recommended that brines draining off fillets during the injection process not be collected, recirculated, and injected into the product.

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The incidence and behavior of L. monocytogenes in three lots of naturally contaminated vacuumpacked sliced smoked salmon from different processors and stored at 2 and 10°C were studied by Cortesi et al. [26]. L. monocytogenes was isolated from 20 of 100 packages stored at 2°C and 12 of 65 packages stored at 10°C [26]. In a similar study by Hudson and Mott [72], L. monocytogenes increased rapidly on cold-smoked salmon within the shelf life of the product. At 10°C, L. monocytogenes growth was comparable in samples packaged in either oxygen-permeable or oxygen-impermeable films. However, at 5°C the lag phase was longer in the vacuum-packaged sample, but once growth was established, the generation times were again comparable. The effect of inoculum level on growth of L. monocytogenes in cold-smoked salmon stored at 4°C was reported by Rørvik et al. [123]. Starting with an inoculation level of 6 or 600 CFU/g, levels increased about 4.8 log10 for the higher inoculum and 2.1 log10 for the 6 CFU/g inoculum. In these same studies, the growth rate of L. monocytogenes was compared in products with different initial bacterial counts. When the inoculum level was 6 CFU/g, L. monocytogenes populations increased faster in samples with the lower initial total bacterial populations.

INHIBITION Of the processes used to prepare smoked fishery products, the cold-smoking operation has been of special interest because the temperatures used are not lethal to L. monocytogenes. The following interventions therefore have been recommended to reduce the risks from L. monocytogenes in these products: (1) elimination or reduction of L. monocytogenes on the outside surfaces of frozen or fresh fish before filleting, (2) prevention of recontamination and growth of L. monocytogenes during all stages of processing, and (3) inhibition of any possible survivors or recontaminants during processing and distribution [35]. Several papers have been published on the inhibition of L. monocytogenes in cold-smoked fish processed with sodium chloride, sodium nitrite, and sodium lactate. In these studies, smoke was not applied to the products, so that the efficacy of the different inhibition treatments could be addressed. Peterson et al. [116] studied the behavior of L. monocytogenes (150 CFU/15 g) in coldprocessed salmon containing 3.5 or 6% water-phase sodium chloride. The products were packaged in either oxygen-permeable film or vacuum-sealed in impermeable film and stored at 5 and 10°C. By the second week at 10°C, L. monocytogenes increased to the range of 106–108 CFU/g, with no difference attributed to the sodium chloride concentration. Vacuum packaging suppressed growth of L. monocytogenes by 10- to 100-fold in samples with 3 or 5% sodium chloride. Inhibition related to salt concentration was most apparent at 5°C, and L. monocytogenes populations were held below 102 CFU/g by 6% water-phase salt, but increased to 104 CFU/g in products with 5% water-phase salt and to 105 CFU/g with 3% water-phase salt. Brown sugar is often used in processing coldsmoked salmon. The use of sugar in the product, however, did not influence growth of L. monocytogenes. In these same studies, growth of the clinical isolate Scott A and two L. monocytogenes strains isolated from salmon were found to be comparable in cold-smoked salmon at 5 and 10°C. Because of the salt tolerance of L. monocytogenes and the consumer unacceptability of smokedfish products with water-phase NaCl concentrations much above 3 or 4%, it was concluded that other inhibitors in addition to NaCl were needed to control the growth of this bacterium. Pelroy et al. [115] therefore studied the behavior of L. monocytogenes (150 or 4900 CFU/15 g sample) in relation to sodium nitrite (190–200 ppm) in combination with sodium chloride in cold-processed salmon during storage at 5 and 10°C. The combination of NaCl and NaNO2 was most effective at 5°C. L. monocytogenes with an initial inoculum of 150 CFU/15 g was held below 15 CFU/g by a combination of 190–200 ppm NaNO2 and 3% water-phase salt and below 20 CFU/g with NaNO2 × 5% NaCl. Packaging in oxygen-permeable or vacuum-sealed in oxygen-impermeable films had little effect on the growth of L. monocytogenes when NaNO2 was included in the process. Increasing the storage temperatures to 10°C markedly reduced the efficacy of both NaCl and NaCl + NaNO2 treatments. There was little difference in inhibition between 3 or 5% water-phase NaCl at 10°C,

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with the combination of NaCl and NaNO2 only slightly more effective than NaCl alone. The packaging method, however, had the most pronounced effect on growth of L. monocytogenes at 10°C. Growth was consistently higher in samples packaged in oxygen-permeable film as compared to vacuum-sealed impermeable film. L. monocytogenes populations increased from a 10 CFU/g inoculum to the range of 108 CFU/g in samples packaged in oxygen-permeable and 106 CFU/g in oxygen-impermeable films. Differences in the inhibition attributable to inoculum level and NaCl + NaNO2 concentrations were obvious in products stored at 5°C. In comparison, when the initial inoculum level was increased to 327 CFU/g, L. monocytogenes reached a population of 106 to 107 CFU/g after 20 days of storage. Of the different inhibitors studied by Pelroy et al. [114], sodium lactate used in combination with salt or salt plus sodium nitrite was most effective in controlling the growth of L. monocytogenes (150 CFU/15 g) on vacuum-packaged cold-smoked salmon stored at 5 and 10°C [114]. A concentration of 3% lactate in combination with 3% salt or 3% salt + 125 ppm nitrite prevented any increase in the L. monocytogenes population during 40 to 50 days storage at 5 or 10°C (Figure 15.1).

FIGURE 15.1 Growth of L. monocytogenes (150 CFU of Scott A/15-g sample) in comminuted salmon containing 0, 2, or 3% water-phase NaCl, with or without 125 ppm NaNO2, during storage at 10° or 5°C in vacuum-sealed impermeable film packages. Baseline symbols indicate that L. monocytogenes was detected by enrichment only. (Adapted from Pelroy, G.A., M.E. Peterson, P.J. Holland, and M.W. Eklund. 1994. Inhibition of Listeria monocytogenes in cold-process (smoked) salmon by sodium lactate. J. Food Prot. 57: 108–113.)

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Addition of 2% sodium lactate prevented L. monocytogenes growth in all samples stored at 5°C. At 10°C storage, however, a combination of sodium lactate (2%) and NaCl (3%) inhibited growth for 14 days, after which L. monocytogenes reached populations within 1 to 2 logs of those in the control samples with NaCl only. When the products contained NaNO2 (125 ppm), sodium lactate (2%), and NaCl (3%), growth of L. monocytogenes was totally suppressed at 10°C except for one of four samples in which populations reached 9.3 × 102 CFU/g. The inhibitory effects of nisin, sodium lactate, and their combination on L. monocytogenes in smoked rainbow trout were studied by Nykanen et al. [111]. Although both nisin and lactate were inhibitory, the combination had a synergistic inhibitory effect. Nisin reduced growth of L. monocytogenes by 1.1 log compared to the control, lactate prevented the growth of L. monocytogenes, and the combination resulted in a decrease from 3.33 to 1.8 log10 CFU/g in vacuum-packed smoked rainbow trout after 17 days of storage at 8°C [111]. Nilsson et al. [104] studied inhibition of L. monocytogenes on cold-smoked salmon using a combination of nisin, carbon dioxide, NaCl, and low temperature. At 10°C nisin resulted in various degrees of inhibition; however, the effect was more pronounced in the presence of 100% CO2 and increasing salt concentration (0.5–5%) [104]. Lactic acid bacterium fermentation products have been studied to determine their efficacy in controlling L. monocytogenes in blue crabmeat [27]. In these studies crabmeat, sterilized by steam, was inoculated with 5.5 log 10 CFU/g of a three-strain mixture of L. monocytogenes and then washed with various lactic acid bacterium fermentation products at levels of 2,000 to 20,000 arbitrary units (AU) per mL of wash. L. monocytogenes populations remained relatively constant in control samples stored for 6 days at 4°C. In comparison, crabmeat washed with Perlac 1911 or Micro-Gard (10,000 to 20,000 AU) initially decreased (0.5 to 1.0 log10 units/g) but recovered to original levels within 6 days. When crabmeat was washed with 10,000 to 20,000 AU of Alta 2341, enterocin 1083 or nisin per mL, the L. monocytogenes population initially decreased by 1.5 to 2.7 log10 units/g. Thereafter, counts increased by 0.5 to 1.6 log10 units within 6 days. Degnan et al. [27] also showed that when crabmeat was washed with food-grade sodium acetate (4 M), sodium diacetate (0.5 to 1 M), and sodium lactate (1 M) or sodium nitrate (1.5 M), there was only a modest reduction in L. monocytogenes population (0.4 to 0.8 log10 CFU/g). However, Listeria counts decreased 2.6 log10 CFU/g within 6 days when the sodium diacetate concentration was increased to 2 M. In addition, trisodium phosphate (1 M) reduced L. monocytogenes counts from 1.7 to greater than 4.6 log10 CFU/g within 6 days. These results demonstrated that L. monocytogenes populations on crabmeat may be reduced by washing with selected antimicrobial agents. The combination of lactic acid and various gas environments on the growth of L. monocyotgenes in crayfish tail meat during refrigerated storage was studied by Pothuri et al. [117]. Growth of L. monocytogenes was more inhibited using 0 or 1% lactic acid in the presence of modified atmosphere packaging than air or vacuum packaging. When the crayfish tail meat was treated with 2% lactic acid, L. monocytogenes levels decreased regardless of the packaging atmosphere. The potential use of strains of Enterococcus faecium to control L. monocytogenes was suggested by Embarek et al. [40]. E. faecium isolates from sous-vide cooked fish fillets were tested on different strains of L. monocytogenes and other pathogenic bacteria in brain heart infusion broth with CaCO3 to avoid decreases in pH during the growth of E. faecuim. Of the 19 isolates tested, 14 produced proteinaceous substances inhibitory to the different strains of L. monocytogenes. An inoculum of 107 CFU E. faecium reduced L. monocytogenes populations of 102 CFU per mL to 10 per mL after 14 days at 3°C and to 1 CFU/mL after 35 days. Using a lower E. faecium inoculum of 104 CFU/mL, L. monocytogenes was only slightly inhibited at 15°C and not at 3 or 5°C. However, after 11 days at 15°C a spontaneous resistance phenomenon was observed, and L. monocytogenes reached 1 × 108 CFU/mL with an inhibitory effect no longer seen. When tested for sensitivity to E. faecium, all of these L. monocytogenes isolates were resistant. The antibacterial effects of 209 psychrotrophic Pseudomonas spp. strains isolated from spoiled iced fish and newly caught fish were assessed by screening L. monocytogenes and other organisms

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using the agar diffusion assay [64]. Only eight strains inhibited the growth of L. monocytogenes. Inhibitory action was most pronounced among Pseudomonas strains producing siderophores (an iron chelator), with the addition of iron eliminating the antibacterial effect of some strains. However, some strains of Pseudomonas enhanced the growth of L. monocytogenes; dense growth was observed around the wells containing these Pseudomonas strains, and these strains may have created a more advantageous nutritional environment either by supplying of iron or by increasing the availability of low-molecular-weight nutrients.

INACTIVATION As was true for dairy, meat, and poultry products, the rash of Class I recalls during the past decade involving RTE seafoods has prompted concerns about the adequacy of thermal processing treatments currently used for raw seafood. In early 1988 Pace et al. [113] reported results from a study in which freshly shucked oysters were exposed to 150 ppm chlorine for 30 min, pasteurized at an internal temperature of 72–74°C for 8 min, and then periodically examined for major bacterial groups during 5 months of refrigerated storage. According to these authors, chlorination reduced initial aerobic plate counts of 4.5 × 105 CFU/g by 40–90%, with pasteurization reducing the population by an additional 99.9%. Despite these large reductions in microbial flora, survivors classified in eight different genera of aerobic or facultatively anaerobic bacteria were present in the product. However, at this point the oysters were unfit for consumption, as evidenced by profuse gas production and swelling of plastic pouches in which the product was pasteurized. In response to public and industry concerns, FDA [95] also examined the thermal resistance of Listeria in raw shrimp tails that were inoculated internally to contain approximately 104–105 L. monocytogenes CFU/g. Using a combination of cold (with/without broth) and warm (selective) enrichment, these investigators failed to recover the pathogen from shrimp tails that were boiled longer than 5 min. Although appreciable numbers of heat-stressed cells were detected in inoculated shrimp tails that were boiled for 3 min, frozen storage of the product at −20°C eventually led to complete inactivation of the pathogen. More important, when this study was repeated using naturally contaminated frozen shrimp from Ecuador and Honduras in which 103–105 L. monocytogenes or L. innocua CFU/g were presumably present only on the chitinous exoskeleton, all listeriae were eliminated after 1 min of boiling. Hence, because shrimp are more likely to be contaminated externally than internally with relatively low levels of Listeria, these preliminary findings suggest that current cooking methods are adequate to eliminate these organisms from raw shrimp. Since these initial studies, the thermal death time of L. monocytogenes has been determined in different seafoods and fish. Results of these studies are summarized in Table 15.8. In an effort to determine whether presence of L. monocytogenes in processed lobster could result from undercooking or postcooking contamination, Budu-Amoako et al. [24] determined the thermal death time of L. monocytogenes in 25-g samples inoculated to contain 107 CFU/g. The observed decimal reduction times (D-values) at 51.6, 54.4, 57.2, 60.0, and 62.7°C were 97.0, 55.0, 8.3, 2.39, and 1.06 min, respectively, with a z-value of 5.0°C (Table 15.8). After isolating this pathogen from facilities that used good manufacturing practices, the authors speculated that the presence of L. monocytogenes in the product was probably due to underprocessing. Blue crab is a popular seafood item with more than 50% of the picked meat being pasteurized to offset seasonal fluctuations in some regions. Several recalls of unpasteurized picked blue crabmeat have been issued due to contamination with L. monocytogenes. Based on the lack of information about growth and survival of this pathogen in blue crabmeat, Harrison and Huang [68] determined the heat resistance of L. monocytogenes strain Scott A in this product. The crabmeat was inoculated to contain 107 CFU/g after which 7.5-g samples were distributed into sausage casings (1.6 cm × 4 cm). D-values were 40.43, 12.00, and 2.61 min at 50, 55 and 60°C, respectively, with a z-value of 8.40°C as determined by using trypticase soy agar (Table 15.8). The D-values

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were less, 34.48, 9.18, and 1.31 min, respectively, with a z-value of 6.99°C for the same heating temperatures when Vogel Johnson agar was used. Heightened interest in foodborne listeriosis has also led to intense efforts toward determining whether current industry practices of cooking crawfish are adequate to inactivate L. monocytogenes. In 1993, Dorsa et al. [34] first determined the growth rate of L. monocytogenes in crawfish tail meat at 0.6 and 12°C; exponential growth began with no apparent lag phase and 109 CFU/g were observed after 10 and 4 days, respectively. Rapid growth of the bacterial pathogen at these temperatures further emphasized the need for additional information on thermal resistance. In crawfish tail meat observed D-values at 55, 60, and 65°C were 10.23, 1.98, and 0.19 minutes, respectively, with a z-value of 5.5°C (Table 15.8). Dorsa et al. [34] suggested that industry consider the use of postpackaging heat treatments based on their thermal inactivation data. Based on the psychrotrophic characteristic of L. monocytogenes and the lack of information on minimum effective dose, the New Zealand Department of Health has taken the conservative approach and recommended that for RTE seafood, L. monocytogenes should not be present in a 25-g sample. Bremer and Osborne [20] therefore determined the thermal death time of 7 strains of L. monocytogenes in green shell mussels that were brined in preparation for hot smoking. The brined mussels were blended and inoculated to contain 106 CFU/g with heat resistance determined in plastic pouches. The D-values at 56, 58, 59, 60, and 62°C were 48.09, 16.25, 9.45, 5.49, and 1.85 minutes, respectively, with a z-value of 4.25°C (Table 15.8). When D-values for the different seafoods were compared at 60°C, values for lobster meat, crawfish tail meat, and crabmeat were similar. The D-values for brined mussels, however, were two to three times higher (Table 15.8). The authors indicated that the addition of salt and brown sugar used in the smoked products may have enhanced the thermal resistance of L. monocytogenes in mussels. Based on these differences, product form and composition should be considered when determining the heat resistance of a pathogen.

TABLE 15.8 Thermal Death Times of L. monocytogenes in Different Seafoods and Fish Product Temperature (°C)

Lobster Meat (19)

Blue Crab Meat (51)

Crawfish Tail Meat (26)

Mussels Brine Soaked (17)

Salmon (30)

Cod (30)

D-value (min) 50.0 51.6 54.4 55.0 56.0 57.2 58.0 59.0 60.0 62.0 62.7 65.0 68.0 70.0

40.43 97.0 55.0 12.00

10.23 48.09

8.3

2.39

2.61

1.98

16.25 9.45 5.49 1.85

10.73

7.28

4.48 2.07

1.98 0.87

0.87 0.15 0.07

0.28 0.15 0.03

1.06 0.19

Note: Z-values = lobster meat 5.0°C; blue crabmeat 8.40°C in trypticase soy agar; crawfish tail meat 5.5°C; mussels 4.25°C; salmon 5.6°C; cod 5.7°C.

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In addition, these data emphasize the importance of determining the D-value for each product experimentally. Minimally processed foods, also called refrigerated pasteurized foods, of extended durability [98] using sous-vide technology are relatively new, and concern has been expressed about survival of L. monocytogenes. Embarek and Huss [39] studied the heat resistance of two L. monocytogenes strains (062 and 057) in a fatty fish (salmon) and a nonfatty fish (cod). The strain 062 was slightly more heat resistant. In the processing of sous-vide cooked fish, the raw ingredients were vacuum-packaged before heating at 58, 60, 62, 65, 68, and 70°C. For the cod, D-values were 7.28, 1.98, 0.87 0.28, 0.15, and 0.03 min, respectively, with a z-value of 5.7°C for L. monocytogenes strain 062 (Table 15.8). The two strains of L. monocytogenes were one to four times more heat resistant in salmon than in cod. The D-values for strain 062 in salmon were 10.73, 4.48, 2.07, 0.87, 0.2, and 0.07 min, respectively, with a z-value of 5.6°C (Table 15.8). The authors attributed the protective effect in salmon to the higher fat content. They also emphasized the importance of product form and ingredients in determining the heat resistance of L. monocytogenes. If the time and temperatures previously described in this section for different seafoods had been followed, then the recent Class I recalls likely resulted from postprocessing contamination, as has already been implied by Kvenburg [8,85] and other FDA officials [5,136]. However, considering the possibility of an error during thermal processing, members of the seafood working group of NACMCF agreed it would be prudent to consider certifying or licensing persons who are directly involved in thermal processing of different seafoods, as has been done for many years in the canned food industry [8]. Recognizing that L. monocytogenes is more likely to contaminate the surface rather than the interior of most fish products, several studies have assessed the use of different treatments to inhibit or inactivate L. monocytogenes on the surface of processed products. Noel et al. [106] investigated the possibility of using various lactic acid treatments to inactivate L. monocytogenes on processed peeled and unpeeled shrimp. The shrimp were immersed in a broth culture of L. monocytogenes, drained, immersed in an aqueous solution of 1.5, 3.0, and 6.0% lactic acid for 1, 10, or 120 min, and then examined for survivors during 28 days of storage at −20°C. Although all lactic acid treatments decreased the numbers of Listeria, exposure to 1.5% lactic acid for 10 min was deemed most appropriate because the treatment did not adversely affect the product’s organoleptic quality. Overall, initial L. monocytogenes populations of 2.6 × 103 CFU/g on inoculated shrimp decreased to less than 3.2 × 102 CFU/g following 10 min of exposure to 1.5% lactic acid. Although Listeria populations continued to decrease in all samples, fewer Listeria were observed in lactic acid treated (~10 CFU/g) as compared to the untreated shrimp (~40 CFU/g) after 28 days of frozen storage. In a similar investigation, D-values were determined for a mixture of seven strains of L. monocytogenes exposed to marinades in the presence and absence of green shell mussels [19]. With an acetic acid marinade (1.5% W/V), calculated D-values in the presence and absence of mussels were 77.3 and 33.3 h, respectively. When acetic acid–glucono-delta-lactone (1.5 and 0.2%, respectively)-based marinades were used, D-values increased to 86.3 h in the presence of mussels and decreased to 19.3 h without the mussels. Likewise, for acetic acid (0.75%) and lactic acid (0.75%) marinade, D-values increased to 125.5 h in the presence of mussels and 26.9 h in their absence. Through enrichment experiments, L. monocytogenes was detected after 26 days for acetic acid (1.5%) and after 53 days for the acetic acid–lactic acid (0.75% each) combination, but no survivors were found after 29 days with the acetic acid–glucono-delta-lactone (1.5 and 0.2%, respectively) combination. Based on these findings, the authors emphasized that care must be taken in extrapolating from the results of a model broth system to a “real food” system. Thus, despite the inability of organic acids to completely eliminate L. monocytogenes from seafoods, storage of marinated products before release is an effective method for further decreasing the possibility of L. monocytogenes being associated with these products. These studies also indicate that dipping

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products such as shrimp, mussels, lobster, crab, and scallops in organic acids could prove useful in decreasing the levels of Listeria before and during frozen storage. During the past decade there have been several recalls of smoked fish products because of L. monocytogenes contamination. To estimate the potential health hazard associated with consumption of such products, Jemmi and Keusch [79] determined the heat resistance and growth of L. monocytogenes in artificially inoculated heat-smoked trout. In these experiments, two strains of L. monocytogenes were surface-inoculated (106 MPN/g) onto raw trout and kept in a salt and spice marinade (10% NaCl and 0.7% spices, pH 6.4) for 12 h prior to smoking in a kiln. The trout were surface-dried at 60°C for 30 min; then the kiln was heated to 110°C and the fish were held at this temperature for 20 min after reaching an internal temperature of 65°C. The product was then smoked for 45 min, cooled, and stored at 8–10°C for up to 20 days. After the hot-smoking process and during storage, L. monocytogenes could no longer be detected. In these same studies, smoked trout was inoculated to contain 30 or 45 MPN/g after processing and stored at 4 and 8–10°C; L. monocytogenes increased to 107 MPN/g. It has generally been assumed that presence of L. monocytogenes on hot-smoked fishery products is the result of postprocessing contamination. Poysky et al. [118] therefore focused their studies on defining the processing parameters required to inactivate L. monocytogenes during the hot-smoked process. Based on previous research [35] that L. monocytogenes contamination is most likely to occur on the surface of raw fish fillets or steaks, the effectiveness of using a combination of heat and smoke to inactivate L. monocytogenes on the surface of brined salmon steaks was investigated. When the brined salmon steaks were heat processed without smoke, L. monocytogenes survived on steaks processed to an internal temperature of 181°F (82.8°C) for 30 min. Application of generated smoke reduced the minimum lethal temperature to 153°F (67.2°C). In contrast, when smoke was applied during the last half of the process after surface drying and formation of the surface pellicle, L. monocytogenes was recovered from steaks heated to an internal temperature of 176°F (80.0°C) for 30 min. Poysky et al. [118] also reported that use of undiluted liquid smoke applied at the beginning of the process lowered the inactivation temperature to as low as 138°F (58.9°C). Diluting liquid smoke to 50% reduced the effectiveness, and the lethal temperature was increased to 150°F (65.5°C). The oil-soluble fraction of CarSol C-10, CharOil, was less effective, and L. monocytogenes survived in samples processed to an internal temperature of 166°F (74.4°C), the highest temperature tested with this liquid smoke fraction. In these studies, a pre-enrichment technique was used for enhanced recovery of injured cells. The processed product was stored at 5°C for 4 days and then pre-enriched in trypticase soy broth for 6 h at 30°C before the addition of selective UVM and selective EB. Incubation time in EB also played an important role in the detection of survivors. Of the 245 samples tested, 57 were negative for L. monocytogenes after 1 day at 30°C, but were positive after extended incubation for 6 and 9 days. These studies demonstrated the interaction of heat and smoke in reactivation of L. monocytogenes and that smoke must be applied before the pellicle is formed on the surface of the fish. Otherwise, fewer smoke components are absorbed and the pellicle also serves as a protection blanket for L. monocytogenes from the smoke.

SUMMARY The topic of the incidence and behavior of L. monocytogenes in fish and seafood has also been addressed in several other reviews [12,32,38,49]. Risk assessment and, ultimately, implementation of risk management and identification of appropriate measures to control L. monocytogenes in fish and seafood products will benefit from a more complete understanding of the growth behaviors as well as the effectiveness of inhibition and inactivation of L. monocytogenes in fish and seafood products.

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ACKNOWLEDGMENTS The authors thank Cecilia Wolyniak (FDA, CFSAN), Pat Pinkerton (FDA, Seattle District), Stephanie Dalgliesh (FDA, Seattle District), and Daryl Thompson (FDA, Atlanta Regional Office, retired) for retrieval and assistance with the FDA recall information. Maxine Heinitz (FDA, Midwest Laboratory for Microbiological Investigations, FDA Microbiological Information Systems Manager, retired) and Jan Johnson (FDA, PRL/NW) provided invaluable assistance with accessing data from the FDA Microbiological Information System and Betsy Bower assisted in accessing the data in the FDA FACTS System. Literature searches were conducted by Walter E. Hill (now with USDA, FSIS) and Jim Hungerford (FDA, Seattle District) for which we are grateful. We would also like to thank Walter E. Hill and Nancy Hill (FDA, Seattle District) for assistance with building and finalizing a database for the reference list.

REFERENCES 1. Adesiyun, A. A. 1993. Prevalence of Listeria spp., Campylobacter spp., Salmonella spp., Yersinia sp. and toxigenic Escherichia coli on meat and seafoods in Trinidad. Food Microbiol. 10: 395–403. 2. Anonymous. 1987. FDA checking imported, domestic shrimp, crabmeat for Listeria. Food Chem. News 29: 15–17. 3. Anonymous. 1987. First Listeria finding in crabmeat confirmed by FDA. Food Chem. News 29: 38. 4. Anonymous. 1987. Mexican crabmeat, Greek pasta automatically detained. Food Chem. News 29: 43. 5. Anonymous. 1988. FDA regional workshops to discuss microbial concerns in seafoods. Food Chem. News 30: 27–31. 6. Anonymous. 1988. FDA to sample shrimp for Salmonella, Listeria. Food Chem. News 30: 9–10. 7. Anonymous. 1988. Hot dogs, shrimp, crab targeted for microbiological criteria. Food Chem. News 30: 43–45. 8. Anonymous. 1988. Micro criteria for crabmeat, shrimp would have 3-class attributes. Food Chem. News 30: 25–27. 9. Anonymous. 1989. Listeria, Salmonella zero tolerance in cooked crab, shrimp proposed. Food Chem. News 31: 11–14. 10. Anonymous. 1989. Monitoring of changes to listeriosis problem urged. Food Chem. News 31: 13–17. 11. Anonymous. 1989. Seafood micro group publishes pathogen criteria. Food Chem. News 31: 31–34. 12. Anonymous. 1991. Recommendations by the National Advisory Committee on Microbiological Criteria for Foods. Int. J. Food Microbiol. 14: 185–246. 13. Anonymous. 1995. Misdeclaration and Recall of Smoked Salmon from the United Kingdom. U.S. FDA Import Bulletin 16-B86. 14. Archer, D.L. 1988. Review of the latest FDA information on the presence of Listeria in foods. WHO Working Group on Foodborne Listeriosis, Geneva, Switzerland, February 15–19. 15. Autio, T., S. Hielm, M. Miettinen, A.M. Sjoberg, K. Aarnisalo, J. Bjorkroth, T. Mattial-Sanholm, and J. Korkeala. 1999. Sources of Listeria monocytogenes contamination in a cold-smoked rainbow trout processing plant detected by pulsed-field gel electrophoresis typing. Appl. Environ. Microbiol. 65: 150–155. 16. Berry, T.M., D.L. Park, and D.V. Lightner. 1994. Comparison of the microbial quality of raw shrimp from China, Ecuador, or Mexico at both the wholesale and retail levels. J. Food Prot. 57: 150–153. 17. Boerlin, P., F. Boerlin-Petzold, E. Bannerman, J. Bille, and T. Jemmi. 1997. Typing Listeria monocytogenes isolates from fish products and human listeriosis cases. Appl. Environ. Microbiol. 63: 1338–1343. 18. Brackett, R.E. and L.R. Beuchat. 1990. Changes in the pathogenicity of Listeria monocytogenes grown in crabmeat. Abstr. Annu. Mtg. Am. Soc. Microbiol. P-60. 19. Bremer, P.J. and C.M. Osborne. 1995. Efficacy of marinades against Listeria monocytogenes cells in suspension or associated with green shell mussels (Perna canaliculus). Appl. Environ. Microbiol. 61: 1514–1519. 20. Bremer, P.J. and C.M. Osborne. 1995. Thermal-death times of Listeria monocytogenes in green shell mussels (Perna canaliculus) prepared for hot smoking. J. Food Prot. 58: 604–608.

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and Behavior 16 Incidence of Listeria monocytogenes in Products of Plant Origin Robert E. Brackett CONTENTS Introduction ....................................................................................................................................655 Risk of Listeriosis from Products of Plant Origin ........................................................................656 Incidence of Listeria in Raw Vegetables.......................................................................................657 United States.........................................................................................................................658 Canada ..................................................................................................................................660 Western Europe ....................................................................................................................660 Africa ....................................................................................................................................662 Asia and the Middle East.....................................................................................................662 Behavior of L. monocytogenes in Vegetables................................................................................663 Growth and Survival.............................................................................................................663 Modified Atmosphere Storage..............................................................................................666 Inactivation ...........................................................................................................................667 Heat...........................................................................................................................667 Chemical Sanitizers ..................................................................................................668 Plant Components.....................................................................................................669 Processing Techniques..............................................................................................671 Incidence of Listeria in Fruits .......................................................................................................671 Behavior and Inactivation of L. monocytogenes in Fruit and Fruit Juices...................................672 Behavior of L. monocytogenes in Other Products of Plant Origin...............................................675 References ......................................................................................................................................676

INTRODUCTION Use of adequate isolation procedures, enough time, and a little perseverance by investigators make it possible to isolate Listeria spp., including Listeria monocytogenes, from most forms of animal life. A similar situation also exists with products of plant origin. An apparent association between consumption of silage and occurrence of an illness resembling listeriosis in ruminants was observed as early as 1922; however, this link between silage consumption and listeriosis in domestic livestock was not confirmed until 1960 [54]. Although several papers published during the next 15-year period documented the presence of L. monocytogenes in vegetation grown primarily for consumption by animals [103,104,105], scientists at the time were generally unconcerned about the incidence of listeriae in produce destined for human consumption, primarily because such products had not been positively linked to human listeriosis. In fact, the only instance in which listeriae were

655

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reportedly recovered from raw retail produce before 1981 occurred in 1975, when investigators isolated three untypeable Listeria strains from lettuce marketed in Brazil [62]. Although 18 of 41 Canadians died of listeriosis in 1981 after consuming coleslaw from which L. monocytogenes was isolated and positively identified [92], it was not until 1985—when a listeriosis outbreak in California was associated with cheese—that the research community began to understand the public health significance of L. monocytogenes in foods including vegetables, fruits, and other products of plant origin. Nevertheless, with the exception of one isolated listeriosis case in Finland involving homemade salted mushrooms [66] and a cluster of five cases traced to frozen broccoli and cauliflower in Texas [75], no additional cases of listeriosis have been positively linked to consumption of plant products produced in North America or elsewhere. Despite the lack of confirmed cases of listeriosis arising from consumption of contaminated fruits and vegetables, the potential for such outbreaks has prompted significant attention to be paid to the presence of L. monocytogenes in these products since the previous edition of this book was published. Indeed, fruits and vegetables were chosen for evaluation of their relative risk of contribution to foodborne listeriosis in the “United States Food and Drug Administration (FDA)/United States Department of Agriculture (USDA) Listeria monocytogenes Risk Assessment” [7], the results of which will be discussed later in this chapter. This chapter will specifically address the incidence of Listeria species in raw retail vegetables and fruits. As in earlier chapters, information concerning behavior of L. monocytogenes in fresh produce and other plant-based products (orange juice/serum, soy milk, pasta, and beet pigment) will also be presented, along with some possible means by which listeriae can be inactivated in some of these products.

RISK OF LISTERIOSIS FROM PRODUCTS OF PLANT ORIGIN Raw fruits and vegetables would normally be expected to be free of most human and animal enteric pathogens unless somehow contaminated by human or animal waste. Although few cases of foodborne disease had traditionally been associated with consumption of fresh produce, such outbreaks have been recognized in recent years to occur with greater frequency than previously thought. Although not among the organisms most often associated with illness resulting from consumption of produce, L. monocytogenes is among the foodborne pathogens most often associated with these foods. This may result, in part, from the variety of ways in which L. monocytogenes can contaminate fresh vegetables, as shown in Figure 16.1 [19,20]. Consequently, the fact that L. monocytogenes can find its way into fresh produce makes its potential presence on such products a public health issue. In 2003, the FDA and USDA published a quantitative risk assessment (LMRA) [7] to determine the public health impact of L. monocytogenes in various foods, including fruits and vegetables; other plant-based products discussed in this chapter were not addressed as such. The LMRA determined both qualitatively and quantitatively the relative likelihood that various foods would result in listeriosis, taking into consideration such factors as (1) the frequency with which a food is likely to be contaminated with L. monocytogenes, (2) potential for growth or survival of L. monocytogenes in the food, (3) historical involvement of particular foods in cases of listeriosis, and (4) the frequency and quantity of foods consumed (and therefore the probability that a consumer would be exposed to a contaminated product). Among the most sought-after results of the assessment were the relative risk of various categories of foods with respect to one another, and the estimated number of listeriosis cases associated with the food on a per-serving and on a per-annum basis. Information published on the number of listeriosis outbreaks generally supports the predictions of the LMRA in that relatively few outbreaks of the disease have been associated with consumption of plant products. Nevertheless, there have been some cases. As mentioned earlier, the first case of

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FIGURE 16.1 Potential pathways for transmission of L. monocytogenes to humans via vegetables. (From Beuchat, L.R. and M.P. Doyle. 1995. Survival and growth of Listeria monocytogenes in foods treated or supplemented with carrot juice. Food Microbiol. 12: 73–80.)

listeriosis actually traced to a food was associated with cabbage [92]. Subsequently, other outbreaks have come to light. One outbreak of febrile listeriosis in Italy has been cited as having been associated with consumption of corn; however, the actual food involved was a corn salad [13]. The investigation of this outbreak was important in establishing unequivocally that L. monocytogenes is capable of causing a febrile gastrointestinal form of listeriosis. However, it failed to demonstrate that corn was the source of the organism. Rather, the illness was associated with consumption of a cold salad prepared with canned corn and canned tuna in which the two ingredients were allowed to drain in trays before preparation. Samples of the corn salad yielded very high populations of L. monocytogenes (>106 CFU/g), but corn from unopened cans was reported by the authors to be virtually sterile. Hence, L. monocytogenes likely came from environmental sources or food handlers, although none of the workers reported symptoms of classic listeriosis or febrile gastroenteritis.

INCIDENCE OF LISTERIA IN RAW VEGETABLES Despite less attention being paid to fruits and vegetables as a source of listeriosis, the original Canadian outbreak [92], which resulted in 18 fatalities, prompted increased surveillance and regulatory programs focused on these commodities. This increased surveillance has resulted in numerous recalls of fresh produce. During 1997 and the first half of 1998, six Class I recalls were issued in the United States for fresh frozen coconut [10], hummus with red peppers and vegetables [4,6,9], sprouts [5], and potato salad [3], the last of which involved over 5.5 million lb of product. From 1999 to 2003, the FDA was made aware of at least 86 Class I recalls for the presence of L. monocytogenes. Among the types of products of plant origin recalled were cut fruits and vegetables, salad mixes, sprouts, bay leaves, lettuce, and bell peppers [107]. Although the presence of L. monocytogenes in fresh produce has been a concern, the LMRA revealed that foods in the vegetable category had a low predicted relative risk of causing listeriosis in the United States on a per-serving basis and only a slightly higher risk on a per-annum basis. The increase in the per-annum risk over the per-serving risk reflects the large volume of vegetables

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TABLE 16.1 Vegetables from Which L. monocytogenes Has Been Isolated Country

Food

Australia Brazil Canada

Lettuce Cabbage, lettuce, parsley, watercress Broccoli florets, cabbage salad, celery, coleslaw mix, green peppers, lettuce, chopped lettuce, radishes, salad mix, tomatoes, vegetables Cabbage salad Vegetables Legumes, mushrooms, minimally processed fresh vegetables, salads, vegetable foods Beetroot, cabbage, capsicum, carrot, coriander, cucumber, lettuce, vegetable leaves and roots, radish roots, ready-to-eat salads Celery, lettuce, fennel Vegetables Raw vegetables Fresh and cut vegetables Cabbage, carrot, cucumber, lettuce Celery, cilantro, fresh vegetables, laurel, lettuce, onion, parsley, thyme, spinach, winter sweet Salads, vegetables Vegetables Individual salad ingredients (bean sprouts, cabbage, carrot, celery, cress, cucumber, lettuce, mushroom, peppers, radish, spring onions, tomato, vegetables, watercress), mixed vegetables, mixed and prepacked salads, raw salad vegetables, vegetables and salads Broccoli, cabbage, celery, cilantro, cucumbers, green beans, jalapeño, kelp, kidney beans, lettuce, mixed vegetable salads, mung beans, mushrooms, onion, pea, potato, radish, spinach, sprouts, tomato, vegetable, vegetable salads, yam Salad (leafy bagged and precut)

Costa Rica Czech Republic Germany India Italy Japan Korea Netherlands Saudi Arabia Spain Switzerland Taiwan United Kingdom

United States

United States (California) United States (Maryland)

Salad (leafy bagged and precut)

Source: Adapted from Anonymous. 2003. Quantitative Assessment of Relative Risk to Public Health from Foodborne Listeria monocytogenes Among Selected Categories of Ready-to-Eat Foods. http: //www.foodsafety.gov/~dms/lmr2toc.html, pp. 146–151.

eaten per year. It estimated that the median number of cases of listeriosis from vegetables likely occurring in the United States was less than one case per year. In response to heightened concern about foodborne listeriosis, many surveys have been initiated to determine the extent of Listeria contamination in raw fresh and frozen vegetables destined for human consumption. Table 16.1, adapted from the LMRA [7], lists the many types of vegetable products and the geographical regions from which L. monocytogenes has reportedly been isolated. The discussions that follow provide more details on some of the more pertinent reports.

UNITED STATES During 1986 and 1987, Petran et al. [85] attempted to isolate Listeria spp. from 23 retail samples of vegetables, including fresh beet peels, broccoli, cabbage (outer leaves), carrot peels, cauliflower

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stems, corn husks, head lettuce, leaf lettuce, mushroom stems, potato peels, and spinach as well as frozen green beans, pea pods, green peas, and spinach. Using FDA and Centers for Disease Control and Prevention (CDC) procedures along with direct plating, officials at the CDC [11] tried to isolate listeriae from 22 samples of broccoli, carrots, celery, lettuce, green peppers, and potatoes in conjunction with several clusters of listeriosis cases in Los Angeles County, California, and Philadelphia, Pennsylvania. Finally, as part of a much larger survey dealing with the incidence of Listeria spp. in retail meat, poultry, and seafood products, Buchanan et al. [32] used an MPN (most probable number) method to examine two samples of potato salad for listeriae. As already implied, no Listeria spp. were recovered from any samples examined in the three surveys just described. However, when one considers the small number of samples examined, these findings should not indicate that these products will always be free of listeriae. As a result of an extensive review of both published and unpublished surveys, the LMRA reported that the percentage of vegetable samples with detectable L. monocytogenes contamination was about 3.6% [7]. In the first truly definitive survey reported, Heisick et al. [60] determined the incidence of various Listeria spp., including L. monocytogenes, in 10 different varieties of raw unwashed vegetables (total of 1000 samples) obtained from two Minneapolis-area supermarkets between October 1987 and August 1988. Listeria spp. were detected in one or more samples of cabbage, cucumbers, lettuce, mushrooms, potatoes, and radishes, but were never found in broccoli, carrots, cauliflower, or tomatoes. Listeria monocytogenes, L. innocua, L. welshimeri, and L. seeligeri were recovered from 5.0, 2.6, 0.8, and 1.3% of all raw produce examined, respectively, with 41 of 50 (82%) and 9 of 50 (18%) L. monocytogenes strains classified as serotypes la and 4a/4ab, respectively. However, the overall incidence of Listeria spp. as well as L. monocytogenes was markedly higher in radishes and potatoes than in other types of vegetables. Given that carrots possess some inherent antilisterial activity [15,21,23,77,78], it appears that root crops such as potatoes and radishes more frequently carry viable listeriae than other vegetables, ostensibly because of their close association with soil. Interestingly, contamination rates for most raw vegetables were fairly consistent throughout the year, and this reinforces the belief that listeriae populations remain relatively constant in soil. These findings are supported by the study of Heisick et al. [60], who used four different procedures to ultimately identify Listeria spp. in 19 of 70 (27.1%) and 25 of 68 (36.8%) potato and radish samples, respectively. No listeriae were detected in mushrooms, carrots, cabbage, broccoli, cauliflower, lettuce, tomatoes, or cucumbers obtained from the same two Minneapolis supermarkets. Use of an adequate isolation procedure and sufficient time to examine large numbers of samples have made it clear that a small percentage of raw vegetables marketed in the United States are likely to harbor Listeria spp., including L. monocytogenes, with the incidence of this pathogen being highest in root crops. Hence, the inability to detect listeriae in raw vegetables examined in the aforementioned surveys was probably because insufficient numbers of samples were examined. Although the presence of low levels of L. monocytogenes in retail raw vegetables appears to contribute only minimally to the overall risk of foodborne listeriosis in the United States, it is prudent that all produce be carefully handled and washed thoroughly, particularly when it is to be consumed by pregnant women, the elderly, and other individuals at greater than normal risk of developing listeriosis. Others in the United States have reportedly found L. monocytogenes infrequently in fresh vegetables. Lin et al. [74] determined the occurrence of L. monocytogenes and other foodborne pathogens in vegetable salads served in 31 food service establishments in Florida. Of the 63 vegetable salad samples tested, L. monocytogenes was recovered from only one (1.6%) salad consisting of iceberg lettuce, red cabbage, carrots, cucumbers, and tomatoes. Interestingly, this salad and others yielding potentially pathogenic bacteria other than Listeria were obtained from only 5 of the 31 establishments. Several of these facilities had apparently sold contaminated salads on more than one occasion, with contamination most likely a result of product mishandling by workers.

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More recently, Thunberg et al. [96] surveyed retail samples of produce in the Washington, DC area for several foodborne pathogens, including Listeria species. About half of the food types and 20% of the samples analyzed yielded Listeria spp. Products yielding listeriae included celery, field cress, lettuce, mung bean sprouts, potatoes, soybean sprouts, watercress, and yams. However, only field cress (2 of 11 samples) and potatoes (4 of 8 samples) yielded L. monocytogenes. Interestingly, the observation that a relatively high percentage (50%) of potato samples contained L. monocytogenes appears similar to the findings of Heisick et al. [59], suggesting that root crops may be particularly vulnerable to L. monocytogenes contamination. The importance of proper sanitation and handling in minimizing L. monocytogenes contamination of salads and vegetables was mentioned by Harvey and Gilmour [58]. They speculated that systematic contamination of vegetable salads by L. monocytogenes was more likely a result of improper handling by food service workers rather than from natural contamination of the raw product.

CANADA Although several instances of L. monocytogenes in Canadian vegetables have been documented (Table 16.1), only one formal Canadian publication on the subject of L. monocytogenes in produce is recorded in the scientific literature. Farber et al. [47] failed to recover any Listeria spp. from lettuce (50 samples), celery (30 samples), or tomatoes (20 samples) purchased in Ottawa during 1988. However, L. innocua was detected in 1 of 10 radish samples, which again suggests that the incidence of listeriae may be somewhat higher in root crops than in other vegetables.

WESTERN EUROPE When the first edition of this book appeared in 1991, knowledge concerning the incidence of listeriae in raw vegetables marketed in Western Europe was confined to a few scattered reports. Since then much more information has been published. An increase in the number of listeriosis cases in England, along with the possibility that some of these cases may have been food related, prompted several surveys to determine the incidence of listeriae in various foods including dairy, meat, poultry and seafood products, raw vegetables, and prepackaged salads. Working at Cambridge, Sizmur and Walker [93] examined 10 different varieties of prepackaged salads obtained from two leading area supermarkets. Overall, L. monocytogenes serotype 1/2 was isolated from 4 of 60 (6.7%) samples, with L. monocytogenes serotype 4b also being present in one of these positive samples. Prepackaged salads from which the pathogen was recovered consisted of two varieties that contained either (a) cabbage, celery, sultanas, onions, and carrots or (b) lettuce, cucumbers, radishes, fennel, watercress, and leeks. Both salad varieties contained cabbage, cucumbers, and/or radishes—three of four raw vegetables from which L. monocytogenes (predominantly serotype la) was isolated in the United States. Although no Listeria spp. were recovered from plain bean sprout salads or those that contained nuts, possibly because of a low pH, L. innocua was detected in 13 of 60 (21.7%) samples representing five different varieties of mixed vegetables and fruit salad. In addition to these findings, English investigators [52] isolated L. monocytogenes from 12.2% of vegetables sampled; the organism was only recovered from lettuce, Chinese cabbage, and green onions. On further characterization, all vegetable isolates were of serotypes other than 1 and 4. In contrast, more than 90% of isolates from turkey and beef were serotype 1, with these strains and those from seafood having greater hemolytic activity than isolates obtained from vegetables. Consequently, these authors hypothesized that characteristics of L. monocytogenes might be related to the food of origin. Several surveys of retail foods have been conducted by Danish regulatory authorities to determine approximate levels of L. monocytogenes in various food categories sold in that country [79].

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Danish regulatory policy specifies tolerances for L. monocytogenes, depending on the ability of the food to support growth of the organism and whether the food receives a terminal heat treatment or is otherwise preserved. Results of the surveys conducted during 1997 and 1998 indicated that vegetables (including sprouts) were among those foods frequently contaminated by higher populations of L. monocytogenes. Between 64.9 (1998) and 87.4% (1997) of sprouts and sliced vegetables contained less than 10 L. monocytogenes cells/g (acceptable), and 12.1 (1997) and 34.5% (1998) contained L. monocytogenes at levels between 10 and 100 cells per g (not satisfactory). Less than 1% of these products contained populations of L. monocytogenes in excess of 100 cells/g, the level that Danish regulatory policy considers unacceptable. A large study on the microbiological quality of produce and its risk to public health was conducted in Norway by Johannessen et al. in 2000 and 2001 [65]. The authors analyzed 890 samples of fresh produce for presence of L. monocytogenes and several other foodborne pathogens. The produce analyzed was varied, including lettuce, precut salads, herbs, parsley, dill, mushrooms, and strawberries. Despite the wide variety of items analyzed, the authors were able to isolate L. monocytogenes from only one sample of champignons (mushrooms) and two samples of Chinese cabbage and strawberries. While infrequent detection of L. monocytogenes suggests that this bacterium is not widespread, good hygienic practices are still warranted. Robertson et al. [88] examined alfalfa and mung bean sprouts in Norway for the presence of various pathogens. The authors were unable to detect L. monocytogenes on either type of sprout, or in irrigation water used during the sprouting process. Soriano et al. [94] surveyed and characterized Listeria spp. from raw and ready-to-eat restaurant foods in the Barcelona area. Overall Listeria species were recovered from up to 30% of lettuce and 10% of spinach samples, with L. monocytogenes present in only 1 of 10 samples each of raw and ready-to-eat lettuce. Bendig and Strangeways [17] proposed that a 74-year-old postoperative patient in a London hospital may have acquired listerial septicemia and meningitis from consuming contaminated lettuce. Although different serotypes of L. monocytogenes (l/2a and l/2c) were isolated from the patient and 1 of 11 (9.1%) samples of washed English round lettuce prepared in the hospital’s kitchen, with the pathogen absent from 44 other food samples examined, the authors concluded that consumption of washed raw vegetables may pose a potential health threat to hospital patients, many of whom are debilitated and immunocompromised. Consumption of homemade uncooked salted mushrooms containing 106 L. monocytogenes serotype 4b CFU/g was positively linked to a nonfatal case of listerial septicemia in an 80-yearold apparently healthy Finnish man [66]. During this investigation, L. monocytogenes was not detected in any vegetables. However, they isolated L. innocua from two samples each of imported and locally grown vegetables. Working in The Netherlands, van Netten et al. [99] also isolated L. monocytogenes from 2 of 20 raw mushroom samples obtained from area markets. Although their goal was not to determine the prevelance of L. monocytogenes in vegetables per se, Aguado et al. [1] reported that this pathogen was uncommon in frozen Spanish vegetables and vegetable processing machinery. Their original intent was to characterize L. monocytogenes in these locations. However, the number of frozen vegetable samples yielding L. monocytogenes was insufficient for the authors to accomplish their research as planned. Nevertheless, they were able to establish a low incidence of L. monocytogenes in frozen vegetables with 1.2% of 906 samples positive over 23 months. The bacterium was mainly found in green beans and tomato products, although occasional samples of cauliflower, peas, and artichokes also yielded L. monocytogenes. Most strains belonged to serotype 1/2a. The authors concluded that although the incidence of L. monocytogenes in frozen vegetables and on processing equipment was low, concurrent isolation of other Listeria species indicated the need for measures to further prevent establishment of L. monocytogenes.

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AFRICA Most surveys on presence of listeriae in foods have been conducted in Europe and North America. However, interest in L. monocytogenes contamination of foods has also developed in other regions not typically associated with listeriosis, including Africa. Mosupye and von Holy [76] attempted to recover L. monocytogenes from foods sold by street vendors in Johannesburg. Despite the often questionable conditions under which the street-vended foods were prepared and sold, the authors were unable to detect L. monocytogenes in any of the foods analyzed, including salad consisting of tomato and onion mixtures. The authors concluded that adequate cooking and short holding times apparently compensated for poor hygienic and handling practices.

ASIA

AND THE

MIDDLE EAST

Other countries not typically associated with listeriosis include those within Asia and the Middle East. In contrast to Africa, however, several studies have been published documenting the frequency with which fruits and vegetables are contaminated with L. monocytogenes. One of the earliest surveys of occurrence of L. monocytogenes in foods sold in Tokyo was published by Ryu et al. in 1992 [89]. Their survey included a variety of plant products, including fresh vegetables, potato salad, and pickled vegetables. L. monocytogenes was frequently isolated from meat (34%) and fish products (6.1%); however, no listeriae were recovered from vegetable products or from ready-to-eat vegetable foods such as fermented soybeans, cooked bamboo shoots, or coleslaw. In 1996, Kaneko et al. [69] conducted a similar survey of ready-to-eat foods, including fresh produce, in retail establishments and food production facilities in the Tokyo area. Only 7 of 134 samples of produce or produce-containing products contained detectable listeriae, and none contained L. monocytogenes. In contrast to the relatively low frequency of L. monocytogenes contamination of produce reported in Japan, Arumugaswamy et al. [12] surveyed various fresh and ready-to-eat foods in Malaysia and found a high incidence of L. monocytogenes contamination. About 22% of leafy vegetables analyzed contained the bacterium, with similar proportions of bean cakes and peanut sauces also reported as being positive. Although these values are higher than those reported for other countries, they were low when compared with other Malaysian vegetable products tested. Eighty percent of ready-to-eat cucumber slices and 80% of bean sprouts tested positive for L. monocytogenes. The authors suggested that a high percentage of positive samples may have resulted because many of the samples came from street vendors or small processors that often employed nonstandard sanitation practices. As was mentioned by Mosupye and von Holy [76] for South African street markets, Arumugaswamy et al. [12] likewise stressed the need for health agencies to put greater priority on foods marketed by street vendors. In stark contrast to the relatively low frequency of contamination in most other surveys, Pingulkar et al. [86] found that virtually all vegetables analyzed from a local market in India contained Listeria spp. with 73% of ready-to-eat salads from several local restaurants also positive for listeriae. Although most Listeria isolates from produce were species other than L. monocytogenes, 11% of tomatoes, 50% of coriander leaves and spinach, and 25% of cabbage samples contained L. monocytogenes. Salamah [90] conducted an extensive survey for presence of L. monocytogenes in various fresh market vegetables sold in Riyadh, Saudi Arabia. In general, the different types of produce analyzed contained L. monocytogenes, at incidence rates varying from 1.3 to 16.3%. Similar to Heisick et al. [59], Salamah found that root crops were more frequently contaminated with L. monocytogenes than vegetables grown above the ground. Conversely, Gohil et al. [53] examined 183 imported and locally grown vegetable samples in the United Arab Emirates for presence of L. monocytogenes. Unlike other surveys, they were unable

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to detect any L. monocytogenes in vegetables. However, they isolated L. innocua from two samples each of imported and locally grown produce. Although consumption of coleslaw prepared from contaminated cabbage was linked to a large Canadian outbreak of listeriosis in 1981 [91], in retrospect, it appears that this outbreak could have been avoided if the coleslaw manufacturer had realized that the cabbage farmer had fertilized the cabbage with sheep manure from a flock that was previously diagnosed as having listeriosis. Years later, van Renterghem et al. [100] suggested that L. monocytogenes dies quickly in fecal matter, and therefore animal manure may not be as important in the spread of L. monocytogenes as once thought. However, they were able to demonstrate that L. monocytogenes could be transferred from contaminated soil to vegetables. In these experiments, carrots and radishes were planted in soil that had been inoculated with L. monocytogenes (105 CFU/g soil). They found that three of six radishes but none of the carrots grown in the inoculated soil contained L. monocytogenes.

BEHAVIOR OF L. MONOCYTOGENES IN VEGETABLES Despite the 1981 listeriosis outbreak in Canada being directly linked to consumption of contaminated coleslaw [92] and an earlier cluster of listeriosis cases in Massachusetts that appears to have been epidemiologically linked to raw celery, lettuce, and tomatoes [73], until recently scientists were generally unconcerned about behavior of L. monocytogenes in raw produce. This renewed interest also prompted a series of investigations to determine behavior of L. monocytogenes on raw vegetables. Results from these studies will now be reviewed along with some data concerning thermal inactivation of L. monocytogenes in cabbage and the effect of modified atmospheric storage, chlorine, and lysozyme on growth and survival of this pathogen in various raw vegetables.

GROWTH

AND

SURVIVAL

Coleslaw was the first vegetable product that was directly linked to an actual outbreak of listeriosis in humans. Consequently, it is not surprising that growth and survival of L. monocytogenes in cabbage was initially investigated. In the first such study, Beuchat et al. [27] determined behavior of L. monocytogenes strains Scott A (clinical isolate) and LCDC 81-861 (Canadian cabbage isolate) on inoculated samples of shredded raw and autoclaved (121°C/20 min) cabbage as well as in autoclaved (121°C/15 min) salted and unsalted cabbage juice during extended storage at 5 and 30°C. Both test strains exhibited similar patterns of behavior on sterile cabbage, with populations decreasing from approximately 107 to 104–105 CFU/g during 42 days of refrigerated storage. This apparent inability of L. monocytogenes to grow on heat-sterilized cabbage at 5°C suggests that heating either decreases the availability of essential nutrients or leads to development of toxic and/or inhibitory constituents in cabbage. In sharp contrast, L. monocytogenes competed well with the normal aerobic flora and lactic acid bacteria of raw cabbage, with Listeria populations increasing approximately 4 orders of magnitude on raw cabbage during the first 25 days of refrigerated storage (Figure 16.2). Thereafter, numbers of listeriae remained unchanged on raw cabbage stored up to 64 days. Similar results were observed in subsequent studies by Hao et al. [55] and Lovett et al. [75]. Thus, L. monocytogenes can grow under conditions normally encountered during shipping and distribution of cabbage. Although both Listeria strains failed to grow in autoclaved cabbage juice containing >5% NaCl during 2 weeks of storage at 30°C, L. monocytogenes increased to 106 CFU/g in the aforementioned homemade salted mushrooms (~7.5% NaCl) during 5 months of cold storage [66]. Hence, behavior of listeriae in raw vegetables appears to be greatly affected by incubation temperature as well as concentration of salt and various growth constituents [84]. Subsequently, Conner et al. [38] more closely examined the influence of temperature, NaCl, and pH on growth of L. monocytogenes in autoclaved (121°C/15 min) clarified and unclarified cabbage

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10 9

Bacteria log10 CFU/mL

8 7 6 5 4 3 2 1 0

0

16

32

48

64

Days

n ), and total aerobic microorganisms FIGURE 16.2 Growth of monocytogenes (•), lactic acid bacteria (n (▲) on raw cabbage incubated at 5°C. (Adapted from Bhagwat, A.A., R.A. Saftner, and J.A. Abbott. 2004. Evaluation of wash treatments for survival of foodborne pathogens and maintenance of quality characteristics of fresh-cut apples. Food Microbiol. 21: 319–326.)

juice. As in the previous study, salt-free unclarified cabbage juice was an excellent growth medium for L. monocytogenes, with initial populations of ~104 CFU/mL increasing to ~109 CFU/mL after 8 days of incubation at 30°C. Beyond 8 days, populations in cabbage juice containing low levels of salt decreased rapidly, and viable cells were no longer detected after 20 days of incubation at 30°C. Growth rates of both Listeria strains at 30°C decreased markedly in cabbage juice containing low levels of salt, with inactivation of strains Scott A and LCDC 81-861 occurring in the presence of >1.5 and 2.5% NaCl, respectively. As expected, behavior of both strains was strongly influenced by acid production, with the pH of samples in which growth had occurred decreasing from 5.6 to less than 4.3 after 8 days of incubation at 30°C. Although populations of both Listeria strains failed to increase in salted and unsalted cabbage juice when the experiment was repeated at 5°C, populations in cabbage juice containing 3.5–5.0% NaCl remained relatively stable, generally decreasing only 10- to 100-fold during 70 days of refrigerated storage. Results from another study [37] suggest that viability of Listeria in similar samples of salted and unsalted cabbage juice can be reduced by adding extracts from several Chinese medicinal plants. Concern about behavior of L. monocytogenes in fresh produce has extended beyond cabbage and now includes an ever-increasing variety of fresh salad vegetables. In 1988, Steinbruegge et al. [95] first reported results of a study that examined the ability of L. monocytogenes to survive and grow on inoculated (103–105 CFU/g) samples of washed retail head lettuce during storage in sealed and unsealed plastic bags at 5, 12, and 25ºC. Although behavior of L. monocytogenes on lettuce was somewhat variable, the pathogen generally grew under conditions simulating proper refrigeration, normal handling, and ambient serving temperatures, with the pathogen increasing 1–4 orders of magnitude following 2 weeks of storage. Similar results were observed when inoculated samples of fresh lettuce juice were held at 5°C for 2 weeks. Salamah [90] reported similar increases in lettuce juices held at 26°C and also observed up to a 2-log increase at 4°C.

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Several more recent reports have appeared regarding the fate of L. monocytogenes in various types of salad greens. Butterhead lettuce (Lactuca sativa L.) supported growth of L. monocytogenes better than did endive (Cichorium endivia L.), but the bacterium was unable to grow on lamb’s lettuce (Valerianella olitoria L.). In contrast, populations of total aerobic microorganisms were unaffected by salad type. Carlin and Nguyen-the [33] offered no hypothesis as to why lamb’s lettuce failed to support growth of L. monocytogenes. Carlin et al. [34] followed up on their previous work by more closely examining several factors that affected growth of L. monocytogenes on endive, with emphasis on storage temperature, age, and quality of the endive leaves, role of epiphytic microflora, and strains and initial concentration of L. monocytogenes present. Overall, the growth rate of L. monocytogenes was essentially the same as that of the natural aerobic microflora at 10 and 20°C but slower than the native microflora at 30 and 6°C. Furthermore, the bacterium grew faster when initially present at lower (10–1000 CFU/g) rather than at higher (105 CFU/g) populations. In accordance with earlier results of Beuchat and Brackett [24], Carlin et al. [34] detected no differences in growth among the various strains tested. Carlin and co-workers [35] subsequently investigated in detail the role of indigenous microflora on growth of L. monocytogenes in unsanitized endive leaves and on leaves treated with 10% hydrogen peroxide to reduce or eliminate the indigenous microflora. These investigators also challenged L. monocytogenes with individual strains of pseudomonads and Enterobacteriaceae isolated from endive. The authors observed that reducing the native microflora by disinfection resulted in higher populations of L. monocytogenes on endive leaves. Moreover, they also observed that high populations (106–107 CFU/g) of some strains of indigenous microorganisms reduced growth of L. monocytogenes on endive. A complex mixture of various microorganisms isolated from endive completely inhibited growth of L. monocytogenes in a medium composed of endive leave exudate. Processing treatments can influence indigenous microflora and have subsequent impact on growth of L. monocytogenes. Li et al. [73] investigated the impact of heating lettuce on the development of L. monocytogenes during subsequent storage. They demonstrated that although the heat treatment was effective in reducing browning and discoloration associated with cut lettuce, it also resulted in development of higher L. monocytogenes populations than in unheated lettuce. The authors hypothesized that the enhanced growth of L. monocytogenes in heated lettuce resulted from a reduction in competitive microflora. Alternatively, they offered the notion that the effects from heating may have also contributed to more favorable growth conditions for the bacterium. Regardless of the specific reason for enhanced growth, this study clearly demonstrated that treatments designed to maintain sensory quality of products can have a detrimental effect on microbiological safety. Although botanically a fruit, tomatoes are typically used as vegetables and are among the most popular ingredients in fresh salads. Beuchat and Brackett [25] demonstrated that tomatoes are not a good substrate for growth of L. monocytogenes, probably because of their acidity. Although some growth of Listeria was evident on whole tomatoes after 10–21 days of storage at 10 and particularly 21°C, the pathogen was inactivated in chopped tomatoes (~pH 4.1) held at these temperatures. Additionally, when commercial tomato products were inoculated to contain ~106 L. monocytogenes CFU/g, populations remained reasonably stable in tomato sauce and tomato juice during 14 days of storage at 21 and particularly 5°C. However, the pathogen survived only 4 and 8 days in samples of ketchup held at 21 and 5°C, respectively, with Listeria inactivation being attributed to higher levels of acetic acid in ketchup as compared to the other tomato products. In contrast, Pingulkar et al. [86] observed that L. monocytogenes populations decreased in tomato slurry regardless of storage temperature, with inactivation increasing with temperatures. Information regarding the fate of L. monocytogenes in or on other types of salad vegetables also is limited; however, results from several studies indicate that with the exception of raw carrots [23,31], fennel, red cabbage, and Savoy cabbage [31], and beets [30], this pathogen will grow and/or survive on most other types of fresh vegetables including asparagus [17], broccoli [17], cauliflower

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[17], corn [63], green beans [63], lettuce [24,63], certain types of cabbage [38,31], celery [30], potato juice [90], and radishes [75] during the normal refrigerated shelf life of the product. Gianfranceschi and Aureli [51] noted that survival of L. monocytogenes in spinach was essentially unaffected by freezing at −50°C and extended storage at −18°C. In addition, Sizmur and Walker [93] reported that L. monocytogenes populations in several naturally contaminated vegetable salads purchased from two supermarkets in England increased approximately twofold after 4 days of refrigerated storage. Given the apparent ability of L. monocytogenes to survive and/or grow on most raw salad vegetables and the possible presence of this pathogen on many types of raw produce, health officials need to consider raw vegetables as a potential source of listerial infections. The observation that L. monocytogenes can thrive in various fresh vegetables also led to questions regarding its growth and survival in products prepared from these vegetables. In 1995, Lee et al. [72] published a study on growth and survival of L. monocytogenes in kimchi, a traditional Korean fermented vegetable product. This product can be prepared from various ingredients, but the most common type contains Chinese cabbage and various flavoring agents such as red pepper, garlic, ginger, NaCl, and pickled seafood. These ingredients are mixed and subjected to a natural lactic acid fermentation, with the product ultimately reaching a mildly acidic pH [12,13]. The authors found that populations of L. monocytogenes Scott A increased during the first 2 days of kimchi fermentation, but then decreased. Although eventually inactivated by kimchi ingredients and low pH, the pathogen still persisted after 10 days of fermentation. However, the authors concluded that kimchi could be safely produced by using ingredients of good microbiological quality.

MODIFIED ATMOSPHERE STORAGE The term modified atmosphere usually refers to systems in which the atmosphere in which a product is stored is intentionally changed to a desired gas composition. The widespread practice of packaging and storing fresh produce in a modified atmosphere has led to dramatic increases in types of produce available to consumers and in their shelf life so that most fresh vegetables (and fruits) are now available throughout the year. However, given the occasional presence of L. monocytogenes on raw produce, the ability of this pathogen to multiply relatively rapidly under microaerobic conditions at refrigeration temperatures and the present popularity of modified-atmosphere packaging, legitimate questions have been raised about the safety of refrigerated produce during long-term storage with modified atmosphere [106]. In response to these concerns, Berrang et al. [18] investigated behavior of L. monocytogenes on inoculated (103–105 CFU/g) samples of fresh asparagus, broccoli, and cauliflower during extended refrigerated storage in glass jars containing (a) 15% O2:6% CO2:79% N2, (b) 11% O2:10% CO2:79% N2, (c) 18% O2:3% CO2:79% N2, or (d) air. L. monocytogenes behaved similarly on each vegetable when the product was stored in a modified atmosphere or air. All three vegetables supported growth of L. monocytogenes at 15°C, with the pathogen attaining populations of 106 to nearly 109/g when these products were first deemed to be unfit for human consumption 6–10 days after the start of incubation. Although storage in a modified atmosphere at 15°C did not appreciably affect growth of listeriae on any of the three vegetables examined, the ability of such storage conditions to increase the shelf life of these products by 2–4 days beyond that of products packaged in air led to higher Listeria populations when these vegetables were first declared inedible by subjective evaluation. In contrast, only asparagus supported growth of L. monocytogenes at 4°C, with initial numbers being approximately 10- to 100-fold higher at the end of the product’s 21day shelf life. However, numbers of listeriae remained relatively constant on broccoli and cauliflower during 21 days at 4°C regardless of the storage atmosphere. Because these findings and those of the previous study indicate that L. monocytogenes is basically unaffected by controlledatmosphere storage, the extended shelf life gained with such storage conditions provides additional time for growth of this pathogen which, in turn, increases the public health hazard associated with

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consumption of raw vegetables. Similarly, Garcia-Gimeno et al. [50] and Kallander et al. [67] observed that use of modified atmosphere with salads neither enhanced nor repressed growth of L. monocytogenes but that the extended shelf life afforded by modified-atmosphere packaging increased the risk of foodborne illness. Subsequently, Beuchat and Brackett observed that L. monocytogenes behaved similarly when inoculated samples of iceberg lettuce [23] and tomatoes [25] were stored at 5 or 10°C (lettuce) or 10 and 21°C (tomatoes) in 3% O2:97% N2 or air. As with cabbage and asparagus, the pathogen grew on lettuce, reaching populations of 108–109 CFU/g after 10 days of storage at 10°C, with only slight growth observed in identical samples held at 5°C. However, the bacterium only achieved populations of about 105–106 CFU/g in tomatoes regardless of storage atmosphere. Vacuum packaging is another means by which produce can be held under a modified atmosphere. This practice has the effect of reducing O2 and thereby slowing respiration and senescence. Aytac and Gorris [14] investigated the effect of a moderate vacuum on growth of L. monocytogenes in chicory endive and mung bean sprouts; the organism responded differently depending on the product in question. Growth of L. monocytogenes at 5.6°C was enhanced by 400 mB of vacuum in the endive but was repressed in sprouts. The authors pointed out the need for additional barriers when such techniques are used to extend the shelf life of fresh vegetables. The atmosphere also can be changed as a result of metabolic processes of fresh fruits and vegetables. In this instance, gas-permeability characteristics of the packaging material can often drastically affect the atmosphere within the package and, consequently, the microflora in the food. Omary et al. [80] investigated the influence of packaging material on growth of L. monocytogenes in shredded cabbage packaged in films having oxygen transmission ratios (OTR) of 5.6, 1500, 4000, and 6000 cc O2/m2 per 24 h. They found that the type of packaging material used had a significant effect on growth of L. innocua (as a substitute for L. monocytogenes) in cabbage. Populations of L. innocua decreased in all samples after 14 days of storage regardless of packaging film used. However, populations of L. innocua then increased 3.5 logs CFU/g in cabbage packaged in all but the film with the highest OTR, in which populations only increased by about 2 logs CFU/g. In the latter sample, CO2 concentrations had equilibrated at near-ambient concentrations, whereas concentrations reached from 30 to 90% when films of lower OTR were used. Although most publications to date indicate that only low populations of L. monocytogenes infrequently contaminate fresh produce, the chance of this pathogen multiplying in products with extended shelf life is significant. Therefore, it appears prudent for handlers of raw produce that is intended to be held in a modified atmosphere to institute sanitation and quality control programs that will decrease the incidence of listeriae in incoming raw vegetables. In addition, holding produce at the coldest tolerable temperature will also reduce the risk of L. monocytogenes attaining high populations. It also may be necessary to shorten the marketable period for such products even though the food may appear to be acceptable.

INACTIVATION Among the most active areas of food microbiology research in the past decade has been identification of practical means of eliminating or greatly reducing populations of foodborne pathogens. Considerable research has been done to characterize the effectiveness of heat, chlorine, and lysozyme, and these will be discussed in the following text. In addition, information concerning the effect of other methods such as ozone, chlorine dioxide, high hydrostatic pressure, and irradiation has generated interest. Heat In response to the 1981 listeriosis outbreak in Canada involving consumption of contaminated coleslaw, Beuchat et al. [27] investigated thermal inactivation of L. monocytogenes in cabbage juice.

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Flasks of sterile, clarified cabbage juice adjusted to pH 4.0, 4.6, and 5.6 were inoculated to contain approximately 4 × 106 L. monocytogenes CFU/mL, placed in a shaking water bath at 50, 52, 54, 56, or 58°C, and sampled for listeriae at 10-min intervals for up to 60 min. As expected, thermal inactivation rates for Listeria in cabbage juice at 50, 52, 54, and 56°C were faster at lower pH values, with D-values of 25, 14, 6.7, and 3.6 min at pH 4.6 as compared to values of 60, 34, 8.4, and 6.8 min at pH 5.6, respectively. No viable cells were detected in cabbage juice held at 58°C for 10 min. Although inactivation rates were unaffected by addition of 1 or 2% NaCl to cabbage juice, sublethally injured cells were more sensitive to NaCl on a nonselective plating medium (Tryptic Soy Agar) than were uninjured cells, indicating increased sensitivity to NaCl. As shown by data in Figure 16.2, L. monocytogenes can multiply in raw cabbage during extended refrigerated storage; however, results from the study just discussed suggest that normal pasteurization treatments given to cabbage juice and sauerkraut are probably sufficient to eliminate any viable listeriae that may be present. Hence, unlike unfermented raw vegetables, the risk of contracting listeriosis from sauerkraut and other pasteurized fermented vegetable products appears to be minimal. Chemical Sanitizers Brackett [29] investigated the possibility of using hypochlorite solutions to inactivate L. monocytogenes on the surface of Brussels sprouts. In this study, fresh retail Brussels sprouts were inoculated to contain ~106 L. monocytogenes CFU/g, immersed in a hypochlorite solution containing 200 mg of chlorine/L, removed, air-dried for 30 min, and examined for numbers of surviving listeriae. The procedure just described decreased populations of L. monocytogenes on Brussels sprouts approximately 100-fold. However, because dipping inoculated Brussels sprouts in sterile chlorine demandfree water reduced the number of viable listeriae approximately 10-fold, the author concluded that many cells were simply washed from the surface rather than inactivated by chlorine. In a followup study with fresh retail lettuce, Beuchat and Brackett [24] also found that L. monocytogenes was still present at levels of 105–106 and 107–108 CFU/g after extended storage at 5 and 10°C, respectively, regardless of whether or not the lettuce was pretreated with a sodium hypochlorite solution to reduce the population of naturally occurring microflora. Zhang and Farber [108] evaluated several sanitizers and sanitizer combinations, including sodium hypochlorite, chlorine dioxide, trisodium phosphate, and lactic and acetic acids, for inactivation of L. monocytogenes on lettuce and cabbage. Their results generally were similar to those of Brackett [29] in that disinfectants were ineffective at reducing populations of L. monocytogenes. None of the treatments tested provided more than a 1.7-log reduction and most were under a 1-log reduction. Trisodium phosphate had no effect on listeriae, and surfactants actually reduced efficacy of sanitizers. Hypochlorite can be used very effectively to inactivate L. monocytogenes in water supplies and on the surface of previously cleaned equipment. However, current evidence indicates that chlorine dips are relatively ineffective for eliminating L. monocytogenes from contaminated raw vegetables. Similar to chlorine, ozone has also been used as an effective disinfectant for water. Consequently, it should not be surprising that it would also be considered a candidate for a practical means by which to disinfect fresh produce. Fisher et al. [49] investigated this possibility with respect to L. monocytogenes inactivation and found that ozone could effectively reduce populations of L. monocytogenes in distilled water and phosphate-buffered saline solution, and that the efficacy increased as temperatures decreased. In addition, efficacy differed among strains of L. monocytogenes. The authors reported that treating cabbage with 1% ozone for 5 min virtually eliminated all listeriae present. However, the authors did not indicate what effect this treatment had on sensory properties of the cabbage. Delaquis et al. [40] attempted to use gaseous acetic acid to inactivate several foodborne pathogens, including L. monocytogenes, on mung bean seeds. Unfortunately, treatment of mung bean

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seeds with 242 µl of acetic acid per L of air for 12 h at 45ºC yielded only sporadic reduction of L. monocytogenes, but apparently did not adversely impact germination rates. In contrast, treatment with 0.1 or 0.5% of the quaternary ammonium sanitizer cetylpyridinium chloride reportedly reduced populations of L. monocytogenes on fresh-cut broccoli, cauliflower, and radishes by as much as 4-log CFU/g [102]. After demonstrating that egg white lysozyme, a GRAS (generally recognized as safe) food additive, inhibited growth of L. monocytogenes in laboratory media and phosphate buffer [63], Hughey et al. [64] investigated the possibility of using this enzyme during refrigerated storage to inactivate L. monocytogenes on various retail vegetables, including fresh lettuce, cabbage, sweet corn, green beans, and carrots as well as previously frozen corn and green beans. In this study, 1.8-kg portions of coarsely shredded or fresh-cut and thawed frozen vegetables were treated to contain 100 mg of lysozyme/kg of vegetables and/or 5 mM of ethylenediamine tetraacetic acid (EDTA), inoculated to contain ~103–104 L. monocytogenes CFU/g, mixed by hand, and examined for numbers of listeriae during extended storage at 5°C. Overall, lysozyme was fairly effective in decreasing populations of L. monocytogenes on the surface of fresh vegetables, particularly when used in conjunction with EDTA, which presumably facilitated cell lysis by increasing contact between lysozyme and peptidoglycan in the cell wall. Listericidal effects from the combined use of lysozyme and EDTA were most pronounced on lettuce, with the pathogen no longer being detected after 12 days of refrigerated storage. Although lysozyme alone was listeriostatic, use of EDTA alone failed to prevent growth of L. monocytogenes, with the pathogen eventually attaining levels only slightly lower than those observed in untreated lettuce. Listeria behaved similarly on fresh green beans and sweet corn with two exceptions: (a) growth occurred on lysozyme-treated sweet corn and (b) combined use of lysozyme and EDTA never completely eliminated the pathogen from either product. Unlike fresh lettuce, green beans, and sweet corn, numbers of listeriae on EDTA- and lysozyme-treated raw cabbage increased during the first 20 days of refrigerated storage and then decreased 4–5 orders of magnitude during 28 days of incubation at 5°C. Although use of lysozyme combined with EDTA again was most listericidal, 41 days of refrigerated storage were required to rid this lettuce of listeriae. Unlike other fresh vegetables, the pathogen was eliminated within 9 days from untreated raw carrots as well as from those that were treated with lysozyme alone or in combination with EDTA. Hence, these findings support the notion that carrots probably contain one or more naturally occurring listericidal substances [24]. In contrast to fresh vegetables, numbers of listeriae remained relatively constant on previously inoculated frozen green beans and corn that were treated with lysozyme alone or in combination with EDTA. This apparent failure of lysozyme to inactivate listeriae on frozen vegetables may be related to loss of certain lysis-enhancing substances during processing of vegetables. These findings, together with the current use of lysozyme to prevent growth of gas-producing spore-forming bacteria in certain European cheeses, suggest that commercial use of lysozyme in combination with other previously discussed measures should help inhibit Listeria and other foodborne pathogens on fresh vegetables. Plant Components Various plant components have long been known to possess antimicrobial properties with the essential oils of herbs and spices having been studied most extensively. Kim et al. [70] demonstrated the antilisterial activity of various essential oil components in vitro and suggested that these components might also be incorporated into foods as barriers to microbial growth. Hao and Brackett [57] and Hao et al. [56] tested this suggestion by determining the efficacy of plant extracts in inhibiting growth of L. monocytogenes in beef and chicken, respectively. They found that eugenol and pimento extracts significantly inhibited growth of the bacterium on cooked chicken breasts during refrigerated storage [57]. However, they also noted that the type of food in which extracts were used was important. Unlike chicken, none of the spice extracts tested effectively inhibited

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growth of L. monocytogenes in refrigerated, cooked beef [56]. Moreover, some extracts contributed strong odors and flavors that would need consumer approval if actually used for commercial products. As mentioned previously, carrots reportedly possess some inhibitory antilisterial factors. Beuchat and Brackett [23] were the first to document this when they attempted to artificially inoculate carrots by dipping in suspensions of L. monocytogenes. They noted that populations of the bacterium decreased on both raw whole and shredded carrots but not cooked carrots. Moreover, populations of L. monocytogenes decreased in the inoculating suspensions after dipping of shredded carrots. Based on these results, they suggested that the antilisterial components of carrots are heat sensitive and released when carrot tissue is damaged. The authors suggested that phytoalexin 6methoxymellein might be one potential compound responsible for the antilisterial action. The results of Beuchat and Brackett [23] were later confirmed by Nguyen-the and Lund [77], who also noted that maceration of carrots in a high-speed blender or in liquid nitrogen also destroyed the antilisterial activity. The obvious potential for using carrot juice or its antilisterial components as a natural antimicrobial agent in foods prompted these two groups of researchers to investigate the mechanism of antilisterial activity. Nguyen-the and Lund [78] found that the antilisterial effect of carrots was suppressed by anaerobiosis, thiol compounds, bovine serum, and the free-radical scavengers histidine and diazabenzocyclooctane. However, the activity was not affected by sodium ascorbate, propyl gallate, catalase, superoxide dismutase, or chelating agents but was enhanced by Tween 20. Despite these results, these authors were unable to determine a specific compound or compounds responsible for the antilisterial activity. Beuchat et al. [26] also attempted to characterize the antilisterial components of carrots. They found that the lethal and inhibitory effects were greatest in the pH range of 5.0–6.4 and that the optimum concentration of carrot juice needed to inhibit L. monocytogenes growth was about 10%. In addition, they observed that NaCl at concentrations of up to 5% protected listeriae from the antimicrobial action of carrot juice, especially in 10% juice incubated at 5 or 12°C. Despite these observations, they were unable to identify the compounds responsible for antilisterial activity or predict the effectiveness of carrot juice as an antilisterial ingredient in food. However, Babic et al. [14] suggested that dodecanoic acid and methyl esters of dodecanoic and pentadecanoic acids identified in purified active extracts of carrots may be responsible for the observed antimicrobial activity. Looking at the potential use of carrot juice as an antilisterial treatment for foods, Beuchat and Doyle [21] determined the influence of dipping shredded lettuce in 20 or 50% carrot juice or adding up to 10% carrot juice to Brie cheese and frankfurter homogenates. Overall, both concentrations of carrot juice significantly repressed growth of L. monocytogenes in shredded lettuce stored at 5 and 12°C but not at 20°C. It is also noteworthy that the carrot juice was rather specific in its activity in that it had no discernible effect on growth of other aerobic microorganisms. In contrast to lettuce, addition of carrot juice to Brie cheese was less effective and was completely ineffective in frankfurter homogenates. Hence, it appears that carrot juice may only be of value as an antilisterial agent in some foods. One of the most well-recognized classes of antimicrobial agents arising from plant products are various sulfur compounds, particularly those associated with certain Brassica species. Kyung and Fleming [71] investigated the relative antilisterial effect of some selected sulfur compounds derived from cabbage. Of the compounds tested, they found isothiocyanate and methyl methane thiosulfonate to have the smallest minimal inhibitory concentration. However, the authors did not attempt to assess the effectiveness of these compounds to inhibit growth of L. monocytogenes in fresh produce. Moreover, the authors did point out that despite the presence of such compounds, other researchers have reported that L. monocytogenes grew well in cabbage samples. Kyung and Fleming suggested that this observation may have been due to different concentrations of these compounds in cabbage or a decrease in the levels of these components in cabbage as a result of heating.

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TABLE 16.2 Survival of Listeria spp. on Coriander Leaves Treated with Gamma Radiation and/or Chlorine and Stored at 8–10°C Storage Time Treatment Control 1 kGy Chlorine-treated 1 kGy + Chlorine

Day 0

Day 10

Day 20

+

+

+

+

+ + +

Source: Adapted from Lovett, J., D.W. Francis, and J.G. Bradshaw. 1988. Outgrowth of Listeria monocytogenes in foods. In A.J. Miller, J.L. Smith, and G.A. Somkuti. Foodborne Listeriosis. Elsevier, New York, pp. 183–187.

Processing Techniques New or newly applied food processing techniques have also offered promise for reducing or eliminating populations of L. monocytogenes in some plant-based foods. In particular, highpressure treatments can significantly reduce populations of L. monocytogenes in some semifluid or viscous products. For example, Raghubeer et al. [87] were able to reduce populations of L. monocytogenes in fresh salsa from 107 CFU/g to <0.3 CFU/g by applying 545 MPa pressure for at least 0.5 min. Irradiation also has been proposed as a means to eliminate pathogens, including L. monocytogenes from various foods. Kamat et al. [68] found that treatment of coriander leaves with 1 kGy or greater of gamma irradiation eliminated detectable levels of L. monocytogenes, whereas viable listeriae were recovered from leaves treated with 250 mg/L of sodium hypoclorite for 10 min (Table 16.2). However, in neither case was the population of L. monocytogenes in untreated leaves provided.

INCIDENCE OF LISTERIA IN FRUITS In contrast to raw vegetables, less information is available concerning the incidence of Listeria species on raw fruit (Table 16.3). The first documentation was a preliminary report [11] in which CDC officials failed to recover listeriae from seven fruit samples (e.g., cherries, pears, peaches, avocados, and tomatoes) while investigating two clusters of presumed foodborne listeriosis in Los Angeles County, California, and Philadelphia, Pennsylvania. Later, Schlech [91] mentioned that blueberries, strawberries, and nectarines were implicated in outbreaks of listeriosis. One of the few culture-confirmed cases of listeriosis resulting from consumption of a fruit product occurred in Italy. In this case, DNA fingerprinting was used to link consumption of pickled olives with a sporadic neonatal case of listeriosis [36]. Although scientific evidence is lacking, two observations, namely, infrequent association between consumption of fruit and listeriosis and the fact that most fruits grow well above the ground and are therefore not subject to frequent contact with Listeria-contaminated soil or feces, lead one to speculate that the incidence of listeriae on fruit may well be as low or lower than that observed for raw vegetables. Despite these assumptions, however, the recent Listeria monocytogenes risk assessment (see Chapter 18) [7] ranked fruits as carrying a slightly higher risk than vegetables, although still ranking it as a low predicted relative risk and contributing less than a median of 1 case per year. Although the exact reasons for the higher risk are somewhat unclear, one important factor was the higher percentage of

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TABLE 16.3 Fruit and Fruit Products from Which L. monocytogenes Has Been Recovered Country

Food

United States Czech Republic Germany

Apples, blueberries, cantaloupe, dried fruit, fruit salad, fruit products, gelatin dessert with fruit, melons, pears, pineapples, watermelon Dried fruit Fresh fruit, fruit product

Source: Adapted from Anonymous. 2003. Quantitative Assessment of Relative Risk to Public Health from Foodborne Listeria monocytogenes among Selected Categories of Ready-to-Eat Foods. http: //www.foodsafety.gov/~dms/lmr 2-toc.html, p. 465.

samples (11.8 vs. 3.6% for vegetables) with detectable contamination. In addition, the ability of certain fruits, such as melons, to support rapid growth of L. monocytogenes together with the absence of data required for more precise calculation of risk lead to a calculated high risk for fruits. FDA officials prompted an Oregon firm to issue a Class I recall for over 500,000 flavored frozen ice and juice bars that were apparently contaminated with L. monocytogenes during the latter stages of manufacture [8], which suggests postpasteurization contamination. Because raw milk also was routinely processed into frozen dairy products at this same facility, L. monocytogenes was most likely introduced into the factory environment through the raw milk supply rather than fruit juice. If this is true, then it follows that the incidence of listeriae in highly acidic fruit juice is likely to be extremely low. This view is further supported by results from a survey in which Parish and Higgins [81] failed to detect any Listeria spp. in 100 retail samples of reconstituted single-strength orange juice (pH 3.63–3.84) that were pasteurized at 30 geographically distinct dairy and nondairy facilities located across the United States and Canada.

BEHAVIOR AND INACTIVATION OF L. MONOCYTOGENES IN FRUIT AND FRUIT JUICES Although there has been a significant increase in information on the growth and survival of L. monocytogenes in vegetables, the same cannot be said for fruits. Because we currently lack sufficient information on the incidence of listeriae in raw fruits, it is not surprising that our knowledge of Listeria behavior in these products remains limited. In 1989, Parish and Higgins [82] published data from the first of two studies in which Listeria failed to grow in refrigerated orange serum adjusted to pH <4.6 and was completely eliminated from these samples after 18 to 70 days of storage, with lowest pH values proving most detrimental to survival of Listeria. However, modest growth of listeriae was observed in orange serum samples at pH 5, with the pathogen still being present at levels of 102–103 CFU/mL in the two least acidic samples after 90 days of refrigerated storage. Incubation at 30°C led to Listeria increases in serum that was adjusted to pH values of 3.6–5.0 with hydrochloric acid, inoculated to contain ~106 L. monocytogenes CFU/mL, and examined for numbers of listeriae during prolonged incubation at 4 and 30°C. As was true for cabbage juice [38], behavior of listeriae in orange serum also was markedly influenced by incubation temperature and pH. Overall, L. monocytogenes populations increased approximately 10- and 100-fold in orange serum samples adjusted to pH values of 4.8 and 5.0, respectively. As was true for unclarified cabbage juice [39], overall viability of listeriae again was greatly reduced by raising the incubation temperature, with the pathogen

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generally being eliminated from orange serum samples at pH 3.6–4.0 and 4.2–5.0 after 5 and 8 days of incubation at 30°C, respectively. The same authors [81] subsequently used several enrichment procedures to determine viability of listeriae in single-strength reconstituted samples of commercial frozen concentrated orange juice that were inoculated to contain ~l–10 L. monocytogenes CFU/mL. Although numbers of inherent microorganisms (primarily lactic acid bacteria and yeast) increased from ~102 to 108 CFU/mL after 4 weeks of incubation at 4°C, L. monocytogenes was eliminated from reconstituted frozen orange juice (average pH of 4.06) after 42 days of refrigerated storage. These findings appear to be consistent with those from the previous study involving orange serum. Acidic pH minimizes the growth and survival of L. monocytogenes on many citrus and pome fruits. However, melons and tropical fruits are often less acidic. Consequently, one would expect greater likelihood for survival and growth of the bacterium on such fruits. Behrsing et al. [16] investigated survival of L. innocua on skins of several fruits, with the assumption that L. monocytogenes would behave similarly. Indeed, they found that L. innocua survived well on bananas, passionfruit, and honeydew melon held at 8ºC for 7 days, but actually grew on the rind of cantaloupe under the same conditions. Although Ukuku and Fett [97] did not observe growth of L. monocytogenes on cantaloupe rinds, they were able to demonstrate that the cells present on the rind could be transferred to the flesh during cutting. Hence, this stresses the importance of decontaminating the exterior of such fruits, and particularly cantaloupe, before cutting. Although survival and growth of L. monocytogenes on inedible skins and rinds increases the potential for eventual consumption, the behavior of L. monocytogenes on fruit flesh is of more immediate concern with regard to listeriosis. L. monocytogenes grew well in the pulp of papaya (pH 4.87), watermelon (pH 5.50), and melon (Cucumis melo L. var. “Valenciano amarelo,” pH 5.87), achieving concentrations in excess of 108 CFU/g in the latter [82]. These results show that cut fruits could serve as a vector for L. monocytogenes and that proper sanitation and temperature control are important. As with vegetables, various microbial reduction strategies, primarily chlorine-based sanitizers, have been evaluated for efficacy against L. monocytogenes on fruits. However, thus far generally only minor reductions in numbers of listeriae have been achieved. One example for which greater reduction was reported was when apple slices were treated with a solution containing isoascorbic acid, calcium ascorbate, calcium propionate, and N-acetylcysteine [28]. Depending on pH of the solution, the authors were able to achieve L. monocytogenes reduction of <1 to >5 logs. Venkitanarayanan et al. [101] evaluated the efficacy of a combination of 1.5% lactic acid and 1.5% hydrogen peroxide to reduce populations of several foodborne pathogens on the surface of apples, oranges, and tomatoes. Exposing the fruits to this solution for 15 min at 40°C effected a >5-log reduction in populations of L. monocytogenes (Table 16.4). Moreover, the authors observed no adverse effects on sensory characteristics of the products. High-pressure processing and irradiation have also been evaluated as antilisterial treatments for fruits. Alpas and Bozoglu [2] evaluated the efficacy of high-pressure treatment for destruction of L. monocytogenes in apple, apricot, cherry, and orange juices. They were able to attain an approximately 4- and 7-log reduction in populations of L. monocytogenes when the juices were treated at a hydrostatic pressure of 250 and 350 MPa, respectively (Table 16.5). Later, Dogan and Erkmen [41] reported that the D-values for L. monocytogenes in peach juice exposed to 200, 400, and 600 MPa of hydrostatic pressure were 1.52, 3.39, and 6.17 min, respectively. The process was even more effective for orange juice, with D-values of 0.87, 1.80, and 2.87 min for the same pressure treatments, respectively. Gamma irradiation was less effective in eliminating L. monocytogenes from frozen avocado pulp [98] than from coriander leaves [68]. In the former case, irradiation was successful in reducing populations of L. monocytogenes by only 1 to 4 logs.

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TABLE 16.4 Listeria monocytogenes Populations on Apple, Orange, and Tomato Surfaces (after Treatment with 1.5% Hydrogen Peroxide at 40°C for 15 min) Product Treatment Baselinea Treated productb Treated solutionc Control productd Control solutione

Apple

Orange

Tomato

6.04 ± 0.92 ND ND 4.07 ± 0.24 5.20 ± 0.31

7.01 ± 0.09 0.97 ± 0.03 ND 4.92 ± 0.17 6.08 ± 0.48

7.20 ± 0.25 ND ND 6.11 ± 0.65 7.02 ± 0.30

a

Count (log10 CFU per product) of inoculated pathogen dried on fruit. Count (log10 CFU per treated product) of inoculated pathogen after treatment with 1.5% hydrogen peroxide at 40ºC for 15 min. cCount (log CFU/mL) of inoculated pathogen in a solution of 1.5% lactic acid plus 1.5% hydrogen peroxide 10 in which pathogen-inoculated apples were immersed at 40ºC for 15 min. dCount (log CFU per product) of inoculated pathogen after immersion in sterile deionized water at 40ºC 10 for 15 min. eCount (log CFU/mL) of inoculated pathogen in deionized water in which pathogen-inoculated products 10 were immersed at 40ºC for 15 min. b

Source: Adapted from Brackett, R.E. 1987. Antimicrobial effect of chlorine on Listeria monocytogenes. J. Food Prot. 50: 999–1003.

TABLE 16.5 Effect of High-Pressure Processing on Viability Loss of L. monocytogenes CA in Fruit Juices Log10 CFU mLa Pressurized

Product Apple juice Apricot juice Orange juice Cherry juice

Control

250 MPa, 30°C, 5 min

350 MPa, 30°C, 5 min

350 MPa, 40°C, 5 min

8.20 8.20 8.70 8.34

4.30 4.15 4.45 4.00

1.40 1.26 1.44 0.90

NDb ND ND ND

Values represent mean (n = 8) log10 CFU mL−1 of unpressurized control samples. b ND, no CFU detected in 1 mL of cell suspension from each of the samples tested. a

Source: Adapted from Alpas, H. and F. Bozoglu. 2003. Efficiency of high pressure treatment for destruction of Listeria monocytogenes in fruit juices. FEMS Immunol. Med. Microbiol. 25: 269–273.

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BEHAVIOR OF L. MONOCYTOGENES IN OTHER PRODUCTS OF PLANT ORIGIN Increased consumer demand for precooked, long-shelf-life, ready-to-eat foods containing a minimum of preservatives is making development of microbiologically safe products increasingly challenging. Not too many years ago, it was thought that no foodborne pathogen could multiply in properly refrigerated food. However, this belief has proven to be invalid following the emergence of L. monocytogenes and Yersinia enterocolitica, in particular, as causes of foodborne illness. Thus, public health officials have become concerned about the safety of many cookchilled and ready-to-eat foods of animal origin, including fermented and unfermented dairy products, luncheon meats, sausage, and precooked chicken as well as peel-and-eat shrimp. These concerns have since spread to products of plant origin, including soy milk, precooked delicatessen products such as ravioli (prepared in part from flour), and food colorants derived from red beets. Increased use of soy milk by individuals who cannot tolerate cow’s milk prompted Ferguson and Shelef [48] to inoculate commercially available pasteurized and sterile soy milk to contain 102–104 L. monocytogenes CFU/mL and incubate the products at 5 and 22°C. The pathogen attained maximum populations similar to those previously observed in cow’s milk, reaching levels of 7 × 108–3 × 109 and 8 × 108 CFU/mL of soy milk following 3 and 30 days of incubation at 5 and 22°C, respectively. Not surprisingly, generation times for L. monocytogenes in soy milk, 1.55 h at 22°C and 37.68 h at 4°C, are similar to those previously reported for the same strain of L. monocytogenes in cow’s milk. No one has yet reported the incidence of Listeria spp. on soybeans; however, because listeriae are relatively common in soil, it is possible for these organisms to find their way into soy milk processing factories and also into the finished product as a postpasteurization contaminant. If this is true, then rapid growth of L. monocytogenes in soy milk at refrigeration temperatures suggests that this product should not be overlooked as a possible vehicle of human listerial infection. Concern about behavior of Listeria in delicatessen products marketed in England and the United States prompted Beuchat and Brackett [22] to investigate viability and thermal inactivation of L. monocytogenes in commercially prepared meat, cheese, and egg ravioli purchased from Atlantaarea delicatessens. The growth portion of this study involved quantification of L. monocytogenes in inoculated (~104 and 106 CFU/g) samples of ravioli during 14 days of incubation at 5°C. For thermal inactivation tests, the three types of ravioli were inoculated to contain 3 × 105 L. monocytogenes CFU/g, stored for 0 or 9 days at 5°C, and then boiled for up to 7 min using home cooking procedures. Overall, numbers of viable listeriae decreased <10-fold during the 9-day refrigerated shelf life of the three types of ravioli. Results of thermal inactivation studies indicated that normal cooking procedures (7 min of boiling) were adequate to destroy L. monocytogenes populations of ~105 CFU/g in all three types of ravioli regardless of whether or not ravioli was refrigerated for 0 or 9 days before cooking. Although this study provides valuable information concerning behavior of Listeria in ravioli, there appears to be an urgent need for more work of this type to address the microbiological safety of precooked and ready-to-eat delicatessen products such as sandwiches, filled rolls, pizza, garlic bread, desserts, confectionery products, and chocolate, because work in England [52] and elsewhere has shown that all these products can harbor L. monocytogenes. Increased use of plant-based food colorants prompted El-Gazzar and Marth [45] to investigate behavior of Listeria in a commercial aqueous extract from the red beet root (Beta vulgaris) to which vitamin C, citric acid, and sodium propionate (1.5%) were added as preservatives. As in their previous work with milk coagulants [43,44] and annatto colorants [42], samples of beet extract were inoculated to contain 103–107 L. monocytogenes strain CA, V7, or Scott A CFU/mL and examined for numbers of survivors during prolonged storage at 7°C. Not surprisingly, the combined effect of a relatively low pH of 4.3–4.8 and sodium propionate prevented growth of listeriae in all samples of beet colorant. However, although 42–56 days of incubation at 7°C was sufficient to rid

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these extracts of 103–104 strain CA CFU/mL, this strain was still detected in 56-day-old samples that contained larger initial populations. In contrast, strains V7 and Scott A were far more resistant to the listericidal action of beet extract, with both strains being recovered at levels of 101–104 CFU/mL, depending on the initial inoculum, following 56 days of storage. Hence, unlike highly alkaline annatto extracts in which L. monocytogenes was inactivated almost instantaneously, prolonged survival of listeriae in beet colorants makes it imperative that these extracts be processed and handled carefully to prevent contamination with this pathogen. The sporadic nature of listeriosis suggests that L. monocytogenes can be an infrequent problem in unusual foods of plant origin as well as fruits and vegetables. Although this viewpoint is supported by one sporadic case of listeriosis in Canada that was linked to alfalfa tablets [39,46], the fact that L. monocytogenes can be present in virtually any ecological niche suggests that it is possible for occasional cases of listeriosis to be associated with unusual as well as common plant-based foods.

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72. Lee, S.-H., M.K. Kim, and J.F. Frank. 1995. Growth of Listeria monocytogenes Scott A during kimchi fermentation and in the presence of kimchi ingredients. J. Food Prot. 58: 1215–1218. 73. Li, Y., R.E. Brackett, J. Chen, and L.R. Beuchat. 2002. Mild heat treatment of lettuce enhances growth of Listeria monocytogenes during subsequent storage at 5°C or 15°C. J. Appl. Microbiol. 92: 269–275. 74. Lin, C.-M., S.Y. Femando, and C.-I. Wei. 1996. Occurrence of Listeria monocytogenes, Salmonella spp., E. coli and E. coli 0157: H7 in vegetable salads. Food Control 7(3): 135–140. 75. Lovett, J., D.W. Francis, and J.G. Bradshaw. 1988. Outgrowth of Listeria monocytogenes in foods. In A.J. Miller, J.L. Smith, and G.A. Somkuti. Foodborne Listeriosis. Elsevier, New York, pp. 183–187. 76. Mosupye, F.M. and A. von Holy. 2000. Microbiological hazard identification and exposure assessment of street food vending in Johannesburg, South Africa. Int. J. Food Microbiol. 61: 137–145. 77. Nguyen-the, C. and B.M. Lund. 1991. The lethal effect of carrot on Listeria species. J. Appl. Bacteriol. 70: 479–488. 78. Nguyen-the, C. and B.M. Lund. 1992. An investigation of the antibacterial effect of carrot on Listeria monocytogenes. J. Appl. Bacteriol. 73: 23–30. 79. Nørrung, B., J. Kirk, K. Andersen, and J. Schlundt. 1999. Incidence and control of Listeria monocytogenes in foods in Denmark. Int. J. Food Microbiol. 53: 195–203. 80. Omary, M.B., R.F. Testin, S.F. Barefoot, and J.W. Rushing. 1993. Packaging effects on growth of Listeria innocua in shredded cabbage. J. Food Sci. 58: 623–626. 81. Parish, M.E. and D.P. Higgins. 1989. Extinction of Listeria monocytogenes in single-strength orange juice: Comparison of methods for detection in mixed populations. J. Food Saf. 9: 267–277. 82. Parish, M.E. and D.P. Higgins. 1989. Survival of Listeria monocytogenes in low pH model broth systems. J. Food Prot. 52: 144–147. 83. Penteado, A.L. and M.F.F. Leitão. 2004. Growth of Listeria monocytogenes in melon, watermelon, and papaya pulps. Int. J. Food Microbiol. 92: 89–94. 84. Petran, R. and E. Zottola. 1989. A study of factors affecting growth and recovery of Listeria monocytogenes Scott A. J. Food Sci. 54: 458–460. 85. Petran, R.L., E.A. Zottola, and R.B. Gravani. 1988. Incidence of Listeria monocytogenes in market samples of fresh and frozen vegetables. J. Food Sci. 53: 1238–1240. 86. Pingulkar, K., A. Kamat, and D. Bongirwar. 2001. Microbiological quality of fresh leafy vegetables, salad components and ready-to-eat salads: an evidence of inhibition of Listeria monocytogenes in tomatoes. Int. J. Food Sci. Nutr. 52: 15–23. 87. Raghubeer, E.V., C.P. Dunne, D.F. Farkas, and E.Y. Ting. 2000. Evaluation of batch and semicontinuous application of high hydrostatic pressure on foodborne pathogens in salsa. J. Food Prot. 63: 1713–1718. 88. Robertson, L.J., G.S. Johannessen, B.K. Gjerde, and S. Loncarevic. 2002. Microbiological analysis of seed sprouts in Norway. Int. J. Food Microbiol. 75: 119–126. 89. Ryu, C.-H., S. Igimi, S. Inoue, and S. Kumagai. 1992. The incidence of Listeria monocytogenes in retail foods in Japan. Int. J. Food Microbiol. 16: 157–160. 90. Salamah, A.A. 1993. Isolation of Yersinia enterocolitica and Listeria monocytogenes from fresh vegetables in Saudi Arabia and their growth behavior in some vegetable juices. J. Univ. Kuwait (Sci.) 20: 283–290. 91. Schlech, W.F. 1996. Overview of listeriosis. Food Control 7: 183–186. 92. Schlech, W.F., P.M. Lavigne, R.A. Bortolussi, A.C. Allen, E.V. Haldane, A.J. Wort, A.W. Hightower, S.E. Johnson, S.H. King, E.S. Nichols, and C.V. Broome. 1983. Epidemic listeriosis: evidence for transmission by food. N. Engl. J. Med. 308: 203–206. 93. Sizmur, K.I. and C.W. Walker. 1988. Listeria in prepackaged salads. Lancet I: 1167. 94. Soriano, J.M., H. Rico, J.C. Moltó, and J. Mañes. 2001. Listeria species in raw and ready-to-eat foods from restaurants. J. Food Prot. 64: 551–553. 95. Steinbruegge, E.G., R.B. Maxcy, and M.B. Liewen. 1988. Fate of Listeria monocytogenes on ready to serve lettuce. J. Food Prot. 51: 596–599. 96. Thunberg, R.L., T.T. Tran, R. W. Bennett, R.N. Matthews, and N. Belay. 2002. Microbial evaluation of selected fresh produce obtained at retail markets. J. Food Prot. 65: 677–682. 97. Ukuku, D.O. and W. Fett. 2002. Behavior of Listeria monocytogenes inoculated on cantaloupe surfaces and efficacy of washing treatments to reduce transfer from rind to fresh-cut pieces. J. Food Prot. 65: 924–930.

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98. Valdivia, M.Á., M.E. Bustos, J. Ruiz, and L.F. Ruiz. 2002. The effect of irradiation in the quality of the avocado frozen pulp. Rad. Phys. Chem. 63: 379–382 99. Van Netten, P., I. Perales, A. van de Moosdijk, G.D.W. Curtis, and D.A.A. Mossel. 1989. Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp. Int. J. Food Microbiol. 8: 299–316. 100. Van Renterghem, B., F. Huysman, R. Rygole, and W. Verstraete. 1991. Detection and prevalence of Listeria monocytogenes in the agricultural ecosystem. J. Appl. Bacteriol. 71: 211–217. 101. Venkitanarayanan, K.S., C.-M. Lin, H. Bailey, and M.P. Doyle. 2002. Inactivation of Escherichia coli O157: H7, Salmonella enteritidis, and Listeria monocytogenes on apples, oranges, and tomatoes by lactic acid and hydrogen peroxide. J. Food Prot. 65: 100–105. 102. Wang, H., Y. Li., and M.F. Slavik. 2001. Efficacy of cetylpyridinium chloride in immersion treatment for reducing populations of pathogenic bacteria on fresh-cut vegetables. J. Food Prot. 64: 2071–2074. 103. Weis, J. 1975. The incidence of Listeria monocytogenes on plants and in soil. In M. Woodbine, Ed., Problems of Listeriosis. Surrey, U.K.: Leicester University Press, pp. 61–65. 104. Weis, J. and H.P.R. Seeliger. 1975. Incidence of Listeria monocytogenes in nature. Appl. Microbiol. 30: 29–32. 105. Welshimer, W.J. 1968. Isolation of Listeria monocytogenes from vegetation. J. Appl. Bacteriol. 95: 300–303. 106. Willcox, F., P. Tobback, and M. Hendrickx. 1994. Microbial safety assurance of minimally processed vegetables by implementation of the hazard analysis critical control point (HACCP) system. Acta Aliment. 23: 221–238. 107. Wise, C. 2004. Personal communication. 108. Zhang, S. and J.M. Farber. 1996. The effect of various disinfectants against Listeria monocytogenes on fresh-cut vegetables. Food Microbiol. 13: 311–321.

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and Control 17 Incidence of Listeria in Food Processing Facilities Jeffrey L. Kornacki and Joshua B. Gurtler CONTENTS Introduction ....................................................................................................................................683 Development of Microbial Growth Niches and Biofilms: General Principles.............................685 Maintenance and Repair Practices .......................................................................................693 Factory and Equipment Design............................................................................................697 Traditional Approaches to Listeria Control...................................................................................704 Control of Food Contamination through Effective Cleaning and Sanitation......................707 Cleaning Dry Areas ..................................................................................................707 Cleaning Wet Processing Areas ...............................................................................707 Sanitization ...............................................................................................................709 Other Approaches to Sanitization ............................................................................709 Other Factors to Consider for Control of Listeria in Food Processing Environments ................710 Traffic Patterns......................................................................................................................710 Monitoring the Finished Products, Ingredients, and Processing Environment...................711 Hazard Analysis Critical Control Point (HACCP) Concept .........................................................711 Sampling Plans for L. monocytogenes in Foods ...........................................................................713 Establishment of Barriers between Raw and Finished Product and Production Areas ...........................................................................................................715 HACCP Plan Development............................................................................................................716 The HACCP Team................................................................................................................716 Principle 1: Conduct a Hazard Analysis..................................................................716 Principle 2: Determine CCPs ...................................................................................717 Principle 3: Establish Critical Limits.......................................................................717 Principle 4: Establish Monitoring Procedures .........................................................717 Principle 5: Establish Corrective Actions ................................................................718 Principle 6: Establish Verification Procedures.........................................................718 Principle 7: Establish Record-Keeping and Documentation Procedures ................718 Incidence of Listeria spp. in Various Types of Food Processing Facilities in the United States........................................................................................................................719 Dairy Processing Facilities...................................................................................................720 Meat Processing Facilities....................................................................................................724 Poultry Processing Facilities ................................................................................................730 Egg Processing Facilities .....................................................................................................732

681

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Seafood Processing Facilities...............................................................................................732 Vegetable and Fruit Processing Facilities ............................................................................738 Incidence of Listeria spp. in Western European and Australian Food Processing Facilities.......739 Western Europe ....................................................................................................................739 Dairy Production Facilities.......................................................................................739 Meat, Poultry, and Seafood Production Facilities ...................................................741 Chocolate Production Facilities ...............................................................................741 England and the United Kingdom .......................................................................................742 English Poultry Processing Facilities.......................................................................742 English Chocolate Production Facilities ..................................................................742 United Kingdom: Miscellaneous Production Facilities ...........................................742 France ...................................................................................................................................743 Cheese Production Facilities ....................................................................................743 Poultry and Pork Production Facilities ....................................................................743 Smoked-Salmon Processing Facilities .....................................................................743 Finland ..................................................................................................................................744 Meat Processing Facilities........................................................................................744 Seafood Processing Facilities...................................................................................744 Italy .......................................................................................................................................744 Meat Processing Facilities........................................................................................744 Spain .....................................................................................................................................745 Vegetable Processing Facility...................................................................................745 The Netherlands....................................................................................................................745 Miscellaneous Production Facilities.........................................................................745 Sweden..................................................................................................................................746 Dairy Production Facilities.......................................................................................746 Switzerland ...........................................................................................................................747 Australia................................................................................................................................747 Incidence of Listeria in Household Kitchens................................................................................748 Industry-Specific Equipment, Processing Methods, and Products ...............................................749 Dairy Industry.......................................................................................................................750 Farm Environment ....................................................................................................750 Clarifiers and Separators ..........................................................................................750 Pasteurization............................................................................................................750 Pipeline and Cross-Connections ..............................................................................752 Filling and Packaging...............................................................................................752 Reclaimed and Reworked Product ...........................................................................753 Frozen Dairy Products..............................................................................................753 Fermented Dairy Products........................................................................................754 Meat Industry........................................................................................................................754 Roast Beef, Corned Beef, and Other Rebagged Products.......................................755 Frankfurters and Other Link Products .....................................................................755 Luncheon Meats .......................................................................................................756 Poultry Industry ....................................................................................................................756 Egg Industry .........................................................................................................................757 Fish and Seafood Industry ...................................................................................................757 Fruit and Vegetable Industry ................................................................................................758 References ......................................................................................................................................759

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INTRODUCTION Overwhelming evidence indicates that contamination of commercially processed foods with Listeria monocytogenes and other Listeria spp. occurs in the postprocessing environment rather than because these organisms survived heat treatments that normally render the product safe. This view is strongly supported by widespread distribution of the organism and lack of scientific evidence indicating minimum required heat treatments given to dairy, meat, poultry, seafood, and other products are inadequate to inactivate listeriae that might be reasonably expected to occur in such products before heat processing. To date, no recall of commercially prepared, Listeria-contaminated products has been unequivocally linked to the inadequacy of minimum required heat treatments, despite the microbe’s greater heat resistance as compared to many vegetative organisms. However, the clearest indication that L. monocytogenes and other Listeria spp. enter commercially processed foods as postprocessing contaminants arises because apparently healthy, nonthermally injured cells have been routinely recovered from many thermally processed dairy, meat, poultry, and seafood products and these organisms have been found in the working environments of virtually all processing facilities that have produced foods involved in Listeria-related recalls. In fact, frequent isolation of Listeria spp. from meat processing environments has resulted in a viewpoint that complete elimination of these organisms from ready-to-eat meat and poultry processing environments may not be possible with current technology [150]. Refrigerated food factories, in particular, provide conditions which allow for survival and growth of L. monocytogenes. The organism can adhere to food-contact surfaces and form a biofilm or coating which impedes effectiveness of sanitation procedures [56,69]. The refrigerated, moist environment, coupled with organic soil deposition, allows L. monocytogenes to survive and grow. Greater survival of L. monocytogenes occurred in the presence of pork serum on stainless steel, acetal-polymer, fiber-reinforced plastic, and mortar as compared to unsoiled surfaces (Figure 17.1 to Figure 17.4). L. monocytogenes is also a frequent contaminant of raw materials used in processing plants, so there is constant reintroduction of the organism into the plant environment [55]. To control this pathogen, every potential avenue of entry and cross-contamination must be controlled. This daunting task requires careful thought, continual observation, frequent and routine factory environmental sampling, and documented corrective actions.

Listeria monocytogenes survival on stainless steel: Soil effect Log CFU/coupon

6 5

A

B

A A

A A

4

A

3

soil

A

no soil

B

2

B

B

1

B

0 0

3

6

9

12

15

Days after attachment

FIGURE 17.1 Survival of Listeria monocytogenes on stainless steel: soil effect. (Adapted from Yan, Z., J.L. Kornacki, C.M. Lin, and M. Doyle. 2004. Fate of Aersolized Listeria monocytogenes in a Closed Bioaersol Chamber. International Association for Food Protection. Annual Meeting. Abstract P058.)

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Log CFU/coupon

Listeria monocytogenes survival on acetal resin: Soil effect 7 6 5 4 3 2 1 0

A

B

A

A

A B

A

A soil

B

B

no soil

B B

0

3

6

9

12

15

Days after attachment

Log CFU/coupon

FIGURE 17.2 Survival of Listeria monocytogenes on acetal-polymer: soil effect. (Adapted from Yan, Z., J.L. Kornacki, C.M. Lin, and M. Doyle. 2004. Fate of Aersolized Listeria monocytogenes in a Closed Bioaersol Chamber. International Association for Food Protection. Annual Meeting. Abstract P058.)

Listeria monocytogenes survival on FRP: Soil effect A A A A A B A A

7 6 5 4 3 2 1 0

0

3

soil

B

B

B

6 9 Days after attachment

B

12

no soil

15

FIGURE 17.3 Survival of Listeria monocytogenes on fiber-reinforced plastic: soil effect. (Adapted from Yan, Z., J.L. Kornacki, C.M. Lin, and M. Doyle. 2004. Fate of Aersolized Listeria monocytogenes in a Closed Bioaersol Chamber. International Association for Food Protection. Annual Meeting. Abstract P058.) Survival of Listeria monocytogenes on mortar: Soil effect

Log CFU/coupon

6 5

B

A

4

A

B

AA

A

soil

A

3

B

2

A

1

no soil

A

B

A

B

B

B

24

48

72

AA

AA

96

120

0 0

3

6

9

16

Time after attachment (h)

FIGURE 17.4 Survival of Listeria monocytogenes on mortar: soil effect. (Adapted from Yan, Z., J.L. Kornacki, C.M. Lin, and M. Doyle. 2004. Fate of Aersolized Listeria monocytogenes in a Closed Bioaerosol Chamber. International Association for Food Protection. Annual Meeting. Abstract P058.)

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This chapter has been specifically designed for plant managers, sanitation workers, quality-control and quality-assurance personnel, regulatory field representatives, and others who desire or have a need to understand how, where, and why Listeria establishes itself in the factory. This chapter will progress from general principles and move into specific food processing industries and products. General principles for understanding the development of microbial growth niches and biofilms in factory environments and guidelines for monitoring and reducing the presence of listeriae and other microbial contaminants in working areas that are common to many food processing facilities will be described and illustrated. No two factories are exactly alike in terms of design, equipment, maintenance, product flow, sanitation practices and procedures, distribution patterns, and managerial policies. Consequently, only general principles of cleaning and sanitation will be described for control of this organism. There will be a brief discussion of how Good Manufacturing Practices (GMPs), prerequisite programs, and Hazard Analysis Critical Control Point (HACCP) programs can all be used to sharply decrease the microbial content in any food, thereby reducing the possibility of producing a product contaminated with L. monocytogenes or any other foodborne pathogen. Specific problem areas within selected processing plants, such as pasteurizers, fillers, sausage peelers, etc., associated with the manufacture of particular products will also be identified.

DEVELOPMENT OF MICROBIAL GROWTH NICHES AND BIOFILMS: GENERAL PRINCIPLES The potential for Listeria contamination of food in the postprocessing environment theoretically exists whenever such food is not biocidally treated in the end-use container. Factory conditions that promote the growth of Listeria dramatically increase the risk of postprocessing product contamination. Many factors affect the growth of microorganisms in food processing environments, including moisture, nutrients, pH, oxidation-reduction potential, temperature, presence or absence of inhibitors, interactions between microorganisms in a population, and time. Moisture is the most critical among these as it is essential for microbial growth [63] and is often the most readily controlled of these factors. The transfer of microbes present in a nonsterile factory environment into niches that are inaccessible for cleaning and sanitation can occur via air, water, tools, workers, traffic, and other means. Attachment of Listeria cells to environmental surfaces with cell-membrane bound structures (proteins, polysaccharides, glycoproteins, etc.) occurs, given enough contact time between the cells and the surface. Furthermore, if appropriate conditions exist (sufficient nutrients, water, and time) biofilms can also develop. These biofilms further entrap Listeria in a mucilaginous matrix that can shield these organisms from cleaners and sanitizers [49,52]. Attached cells have also been associated with increased heat resistance [52,69]. The water activity in niches also impacts the type of microflora that develops therein [63]. Disruption of these niches can result in direct or indirect contamination of the product stream [63]. The probability of product contamination is affected by several variables including, but not limited to (1) proximity of microbial growth niches to the product stream, (2) number of niches, (3) spatial relationship of niches to the product stream, (4) microbial populations in niches, (5) extent of niche disruption, and (6) exposure of the product stream to the environment [63]. The chemical and physical nature of these microenvironments and the degree to which they can bind to surfaces and become entrapped in food residue is likely to play an important role in the growth and/or survival of Listeria in the factory environment. Factory structures, including equipment, as well as maintenance, repair, and practices that entrap moisture often result in microbial growth niche development [63]. Operating conditions that may release, entrap, or cause accumulations of moist residues (thus providing an environment for a potential microbial growth niche and increasing the potential for cross-contamination) in the factory environment observed by the first author have included accumulations of dust or powder (e.g., in

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FIGURE 17.5 Disassembled equipment and tool on moist floor after cleaning.

corners, on ledges, and angle iron supports, etc.), water drain lines that are not tapped to floor drains, accumulation of fatty residues on equipment surfaces, high humidity resulting in condensation, floors that are not sloped to drains, cleaning water hoses dragged from one room to another, failure to segregate forklifts used in wet raw areas from those used in finished product areas, reuse of soiled and wet cleaning “green pads,” failure to clean and sanitize tools used to repair equipment, failure to clean and sanitize equipment after repair, soiled aprons, soiled gloves, product or ingredient storage containers, packaging films stored on end in standing water, movement of soiled pallets into finished product areas, etc. (Figures 17.5 to 17.15).

FIGURE 17.6 Failure to execute captive-shoe policy.

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FIGURE 17.7 Hoses on floor. Left: Not hung to dry after use; right: moved from room to room during use.

FIGURE 17.8 Pipe cleaner (rotations promote aerosolization).

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FIGURE 17.9 Use of floor scrubbers (aerosol creation potential).

Controversy exists over the importance of aerosol-borne contamination to direct measurable product contamination. Bioaerosols could be described as solid or liquid microscopic particles suspended in air which carry microorganisms. Yan et al. [160] performed a number of trials in which various levels of L. monocytogenes were released into a 315-L bioaerosol chamber. They were unable to recover a five-strain cocktail of L. monocytogenes from settling plates exposed to 200 CFU/L for 15 min. A settling rate of about 1 log10 CFU per h was determined after release and mixing of 5 × 106 CFU/L into the chamber. However, the preceding 20-min mixing period resulted in about a 3 log10 CFU reduction of this organism at both 38 and 75% relative humidity. There was no significant difference in survival of L. monocytogenes in the air at either relative humidity

FIGURE 17.10 Inappropriate storage of rusted implements, cleaning implements, miscellaneous fittings, pipes, gloves, etc.

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FIGURE 17.11 Accumulation of moist product residue under processing equipment.

FIGURE 17.12 Open back-motor fan covers (bioaerosol potential if not covered during wet cleaning and sanitation procedures).

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FIGURE 17.13 Moist leather tool belts (cross-contamination potential).

(Figure 17.16). In one trial, 5.5 log10 CFU of L. monocytogenes was released and agar strips were used to trap airborne microbes, using a centrifugal sampler. The agar was removed from the sampler, diluted, and plated onto selective (modified Oxford medium, MOX), nonselective (trypticase soy yeast extract agar, TSAYE), and TSAYE overlaid on MOX. No recovery was found on MOX; however, TSAYE and MOX overlaid with TSAYE recovered 5.8 and 5.4 log10 CFUs, respectively. The data suggest a profound level of cell injury associated with use of the centrifugal sampler. However, differences in recovery were not nearly as profound when sedimentation plates of the same medium were used. In this instance, only 0.5 to 2 log10 CFU of cell injury occurred as compared to recovery on a nonselective medium. These findings suggest that the mixing of air such as occurs in the centrifugal sampler may have resulted in greater cell injury. This is most likely the result of the impact of desiccation on the cells. Hence, it is likely that factories with high air flow may also have greater injury of liquid aerosol-borne Listeria than those with quiescent air flow. Yan et al. [160] also exposed 100 cm2 of pre-heat-treated (71°C for 5 min) ham samples to various levels of L. monocytogenes at both 38 and 75% relative humidity (Table 17.1). Plates were opened for 15-min periods over 3 to 4 h. Levels greater than 100 CFU/L were required to contaminate ham to detectable levels when the entire ham sample was enriched and tested. The ham product used had been treated with sodium lactate and sodium diacetate by the producer. These data suggest that fluid aerosols may not be a significant source of direct measurable L. monocytogenes contamination of food treated with Listeria-inhibiting antimicrobials. De Roin et al. [50] presented evidence to suggest that frankfurters exposed to dust contaminated with L. monocytogenes could result in measurable detection. Tompkin [151] reported that in 14 years of investigational work, the air in a room has never been found to be a chronic source of contamination

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FIGURE 17.14 Wheeled bin raw-receiving area of a meat factory (cross-contamination and aerosolization risk from wheels when moved between raw and finished areas over wet areas).

FIGURE 17.15 High-pressure hoses (aersolization) and use of bristled broom to move fluids (aersolization, cross-contamination, and microbial growth niche potential).

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4.0

Log CFU/plate

3.5 3.0

38%

2.5

75%

2.0 1.5 1.0 0.5 0.0 0.0

0.5

1.0

1.5

2.0

2.5

3.5

3.0

Time after releasing (h)

FIGURE 17.16 Comparison of settling rates of a five-strain cocktail of Listeria monocytogenes at 38 and 75% relative humidity (time 0.0 is after mixing of the air).

of product contact surfaces. Nevertheless, injured Listeria could contaminate products from the air. However, moist areas in a factory environment in close proximity to product or product contact surfaces could provide a means to revive injured Listeria from the air, resulting in a microbial growth niche thus resulting in a greater risk of measurable product contamination. Contaminated air from compressed air in close proximity to the product (e.g., growth in a filter) has been implicated (and traced to a niche near the point of use), and exhaust from a small pump near the floor was suspected as the source [151]. The first author has observed moisture accumulation in low areas of compressed air lines, likely originating from condensate formation, which also resulted in microbial growth. This shows the need for use and routine replacement of point-of-use filters for compressed air lines in food processing environments. Thus, the air supply within the factory must be considered as a potential source of Listeria and other microbial contaminants. Hence, all heating, ventilation, and air conditioning (HVAC) ducts and accompanying air filters should be kept in good repair and cleaned regularly to eliminate excessive dust and dirt. Compressed air lines and filters should also be inspected regularly and be free of moisture, oil, and debris.

TABLE 17.1 Recovery from Exposed Ham of L. monocytogenes Aerosolized into a Bioaerosol Chamber at 38% Relative Humidity Exposure Time (min)

Trial

Listeria Released (CFU/L)

Con

5

30

60

120

180

240

1 2 3 4

44 2.2 × 103 1 × 105 6.6 × 105

0/3a 0/3 0/3 0/3

0/3 0/3 2/3 3/3

0/3 0/3 3/3 3/3

0/3 0/3 3/3 3/3

0/3 0/3 3/3 3/3

0/3 0/3 3/3 3/3

0/3 1/3 3/3 3/3

a

Positive samples/samples assayed.

Source: From Yan, Z., J.L. Kornacki, C.M. Lin, and M. Doyle. 2004. Fate of Aersolized Listeria monocytogenes in a Closed Bioaersol Chamber. International Association for Food Protection. Annual Meeting. Abstract P058.

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FIGURE 17.17 Failure to inspect and remove wet residue from air boxes.

MAINTENANCE

AND

REPAIR PRACTICES

Maintenance and repair practices (or failures) that may result in release, entrapment, or accumulation of moisture (if present) in the factory environment observed by the first author have included torn insulation, duct tape repairs of leaking fluid lines, torn hoses allowing entrapment of moisture between the torn outer and unbroken inner skin, broken or cracked floors or floor tiles, failure to repair stress cracks in food contact surfaces, clogged air handling unit drains resulting in fluid backup, rusted valves, roof leaks, bolt penetrations into hollow structures, torn gaskets, torn, abraded or leaking product pump gaskets, clogged drains, drains under negative pressure, inadequately vented drains, torn rubber seals around doors or ports, poorly maintained filters for point-of-use compressed air lines, etc. (Figures 17.17 to 17.25).

FIGURE 17.18 Exposed and water-soaked insulation over and adjacent to finished-product conveyor belt to freezer.

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FIGURE 17.19 Rusted electrical boxes (from failure to maintain door gasket—top photo).

FIGURE 17.20 Ill-fitting and protruding gasket in finished-product tank bottom (see Figure 17.21 for outside view).

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FIGURE 17.21 Unchanged gasket at bottom of finished-product tank (with entrapped residues—see Figure 17.20 for inside view).

FIGURE 17.22 Torn gasket.

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FIGURE 17.23 Leaking and bowed ceiling with water marks suggesting entrapped fluid.

FIGURE 17.24 Failure to effectively maintain drainage from roof.

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FIGURE 17.25 Unsealed penetrations in bowed ceiling tiles permitting leakage of fluids into factory.

FACTORY

AND

EQUIPMENT DESIGN

In addition to the building itself, all equipment within the factory should be designed to minimize cross-contamination between the factory environment and product and should also be constructed of stainless steel or other easily cleaned and sanitized nonabsorbent, nontoxic materials, such as certain types of bonded rubber and plastic. All piping in food processing facilities should be freedraining and designed to eliminate entrapment of food and cleaning and sanitizing solutions used in clean-in-place (CIP) systems. It is also important that equipment such as product conveyors is positioned high enough above the floor to minimize cross-contamination from floors and drains. Equipment designs that may result in release, entrapment, or accumulation of moisture (if present) as observed by the first author include moistened cloth conveyor belts, hollow portions of conveyorbelt rollers, sandwiched or hollow portions of slicer assemblies (e.g., shuttles, grippers, housings), moisture or steam exhaust vents resulting in condensate reflux, product accumulation, excessive surface area for equipment support pads, rotating pipe brushes, filler assemblies, brine chill solutions, open bearings, condensate in compressed air lines, motor housings, ice makers, crevices inside spiral freezers, smokehouses that have a common entrance and exit for raw and finished product, respectively, etc. (see also Figure 17.24, Figure 17.25, Figure 17.28, Figures 17.34 to 17.36, Figure 17.39, Reference 29, and Reference 133). An extensive discussion of sanitary equipment design replete with drawings and photographs can be found in Engineering for Food Safety and Sanitation: A Guide to the Sanitary Design of Food Plants and Food Plant Equipment [86]. Factory designs that may result in release, entrapment, or accumulation of moisture (if present) observed by the first author include penetrated double wall construction, flat roofs with improperly sealed penetrations, hollow areas resulting from newer construction added to older construction, negative pressure inside finished-product packaging rooms, unwise location of rest room facilities such that individuals must traverse from highly contaminated regions to finished product areas, inadequate ventilation, etc. (see Figures 17.37 to 17.39). Design features that are widely considered to be essential for all types of food processing facilities include: (1) a raw product receiving area that is completely isolated from processing and packaging areas of the factory; (2) tight-fitting exterior windows and doors that will prevent animals and insects from entering processing and packaging areas; (3) easily cleaned and sanitized walls, floors, and ceilings that are constructed of tile, metal, or appropriately sealed concrete and not

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FIGURE 17.26 Failure to maintain a gasket on an overhead light resulting in moisture entrapment and mold growth.

FIGURE 17.27 Common drain line from multiple liquid-product vessels.

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FIGURE 17.28 Angle conveyor with sandwiched surfaces and numerous bolts (entraps product residue and difficult to disassemble for cleaning and sanitization).

FIGURE 17.29 Penetrations in hollow support structures (can collect moisture and grow microbes).

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FIGURE 17.30 Entrapment of fluid between hose clamps (microbial growth and cross-contamination potential).

FIGURE 17.31 Steam vent positioned near and toward floor (introduces moisture and aerosols into factory environment).

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FIGURE 17.32 Bottom openings on an electrical control box over a product filler.

porous materials such as wood; (4) floors designed to drain rapidly and prevent pooling of water; (5) floor drains located away from packaging equipment, especially if processed foods are exposed to factory air; (6) proper screens, debris baskets, and traps on floor drains; (7) a quality control or quality assurance laboratory that is well isolated from other areas of the factory; and (8) proper means of waste disposal outside the factory to discourage congregation of insects, rodents, birds, and other animals that may harbor Listeria and other pathogenic microorganisms.

FIGURE 17.33 Drainage line untapped to drain resulting in standing water (potential to create microbial growth niches in resultant wet areas).

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FIGURE 17.34 Unsealed sewerage cover under positive pressure in production area (likely aerosolization of contaminants into production area).

In addition to these concerns, the HVAC system also must be properly designed to minimize airborne contamination [129]. Features considered to be essential for such a system include (1) intake air vents on the roof of the buildings that are located upwind from prevailing air currents but away from dumpsters, raw product receiving areas, and vents that discharge factory air; (2) installation of screens and filters inside incoming air vents to remove particulate matter and condensate; (3) easily cleanable HVAC systems; and (4) proper location of dehumidifiers and air conditioning systems so

FIGURE 17.35 Ball valves (difficult to CIP clean, entrapment of fluid and food residue within valve assembly—between ball and housing—can entrap microbes and allow growth of microorganisms).

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FIGURE 17.36 Air handling/refrigeration units (some may entrap moisture in catch pans resulting in microbial growth and contaminant release via aerosols or dripping depending upon maintenance, design, and relative humidity—the catch pans on these were appropriately tapped to drains).

FIGURE 17.37 Large metal support pads bolted to floor (readily entrap wet residue and very difficult to clean).

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FIGURE 17.38 Drain opening above cleaning implements.

that these units drain away from processing and packaging areas. All HVAC systems should be designed to produce a higher positive air pressure in processing and packaging rather than in raw receiving areas. This design readily prevents movement of airborne contaminants from raw product areas to the cleanest areas of the factory where foods are processed and packaged. The first author has isolated Listeria from cooling coils and catch pans of some HVAC systems. Tube and fin cooling coils provide a likely environment for the growth of Listeria spp. and were shown to harbor bacteria to 5 log10 to 7 log10 CFU per m2 in a well-maintained HVAC system [83]. It has been reported that air movement of 4 m/sec across a quiescent aqueous surface may be adequate to begin aerosolization of the fluid [112]. Catch-pan drain traps must be maintained because clogged traps may result in collection of moisture and growth of microbes. Hugenholtz and Fuerst [83] found that draining fluid in a condensate drip pan had counts of up to 7 log10 CFU/mL. Some HVAC systems are designed with the final filter located before, and others with the final filter located after cooling coils. In the first author’s opinion, final filters should be located after cooling coils and catch pans to prevent aerosolization of contaminants into the factory environment. Care should also be taken to periodically inspect and change filters as wet filters may become a source of microbial growth [136], which may be exhausted into the processing environment.

TRADITIONAL APPROACHES TO LISTERIA CONTROL The traditional approaches to controlling microbiological hazards associated with food products involve the simultaneous use of employee education and training programs, frequent inspection and monitoring of facilities and operations, and extensive microbiological testing of raw ingredients and unfinished and finished products.

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Food safety is everyone’s responsibility. Hence, employee education and training programs should be directed toward a thorough understanding of food hygiene, factory cleaning and sanitation requirements, and various causes of microbial contamination, including growth and survival patterns of potential contaminants such as listeriae. Trained employees should also be able to select and apply control methods that will provide consumers with safe, high-quality products. New employees should be trained and refresher courses or continuing education of all employees should occur at routine intervals. Creative approaches to motivating employees to attend such courses have been developed in some companies. Factory managers and supervisors must stress good employee hygiene and also set a good example for other workers. All individuals with obvious illnesses, infected cuts, or abrasions need to be excluded from working in processing areas or from doing other tasks that may lead to contamination of food, food contact surfaces, packaging materials or equipment. Furthermore, the use of tobacco and chewing gum, as well as the consumption of food should be banned in processing areas along with the wearing of hairpins, rings, earrings, watches, and other jewelry. Employees should always wash their hands thoroughly before starting work, on returning to work, and after touching floors, walls, light switches, any other unclean surface, and garbage. To further promote their use, hand-washing facilities should be properly designed and conveniently located near workstations. All factory workers need to be provided with hair and beard nets, as well as clean clothes, suitable footwear, and disposable gloves. Special attention is also needed to ensure that street clothes do not enter processing areas and that factory clothing, including footwear, remains inside the factory. All factory clothing should be changed daily, or more often if soiled, with the responsibility of laundering being left to the employer. These recommendations may, in some instances, be difficult for food processors to follow and enforce; however, this task will be made much easier if management can instill in workers the conviction that each employee is personally responsible for both the quality and safety of the foods that are produced and ultimately consumed by the public. The second means of controlling microbiological hazards, routine and frequent inspection and monitoring (e.g., by taking factory environmental samples for microbiological evaluation) of facilities, equipment, and operations, is necessary to ensure that GMPs (i.e., hygienic procedures that minimize the potential for production of unsafe or low-quality products) are being followed. GMPs to produce specific foods have been outlined in both advisory and regulatory documents such as GMP guidelines and the various codes of hygienic practice developed by the Codex Alimentarius Committee on Food Hygiene. Environmental samples are typically taken and analyzed for the presence of Listeria (when that is the organism of concern). However, pathogen testing in factory environments is usually limited to non-food-contact surfaces (e.g., walls, drains, floors, forklift tires, etc.). It is advisable to monitor product contact surfaces for the presence of indicator organisms (e.g., aerobic plate count, coliforms, Listeria-like or esculin-hydrolyzing bacteria) to determine the efficacy of sanitation. Some factories use ATP bioluminescence assays, available from several manufacturers [65], on environmental samples to determine the efficacy of cleaning as this can be done in real time. Microbiological test results are typically not available until days later and, therefore, often after the surface has been soiled again from subsequent production. Such samples are tested after cleaning and sanitation and preoperation. These have routinely been used to validate a sanitation break in production. Acceptable preoperational contact surface data, in conjunction with extensive finished-product testing and other investigational data, could be helpful in limiting the scope of contamination investigations to selected lots by providing evidence that the manufactured product was previously not contaminated. Routine sampling of postsanitation/preoperation food contact surfaces can save the factory much trouble by focusing recall-related efforts on selected product lots as opposed to all product lots. A variety of techniques have been developed for taking microbiological samples in the factory environment. Traditional techniques include swab, contact plate, and sponge-based approaches [61]. Each approach offers advantages and disadvantages, depending upon the nature of the specific sample area (Table 17.2).

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TABLE 17.2 Performance Capabilities of Environmental Sampling Techniques Sample Irregular Surfaces

Qualitative Assays

Traditional swabs

Yes

Yes

No

Sponges

Yes

Yes

Yes

Contact plates

Yes

Noa

No

Tongue blades

Yes

Yes

Yesb

Sampling Technique

a

Sample Heavily Soiled Surfaces

Possible with nontraditional approaches. Biofilm removal possible.

b

Environmental samples are most easily collected using swabs or sponges. Only polyurethane or expanding cellulose sponges should be used for this purpose, because other types, including retail cellulose sponges, contain inhibitory agents that not only prevent recovery of L. monocytogenes and Staphylococcus aureus but also interfere with recovery of Brochothrix thermosphacta, Aeromonas hydrophila, Pseudomonas putrefaciens, and P. fluorescens, as well as Escherichia coli, Serratia marcescens, and Enterobacter cloacae [63,104]. It is important to stress that laboratory personnel should never attempt to isolate pathogenic microorganisms from such samples unless the laboratory is completely separate from the factory. Analysis of environmental and, if necessary, food samples for microbial pathogens is best left to outside commercial testing laboratories that are ISO 17025 compliant or otherwise certified. However, it is strongly recommended that coliform and standard aerobic plate counts be obtained for samples from the factory environment and the food during all stages of production to monitor the extent of postprocessing contamination and thus to quickly identify any problems associated with inadequate cleaning and sanitizing. Coliform organisms are commonly regarded as being indicators of postprocessing or postsanitation contamination and the possible presence of pathogens; however, the presence or absence of coliforms in food or environmental samples does not guarantee the presence or absence of foodborne pathogens. In fact, little, if any, correlation has often been observed between the presence of coliforms and Listeria in finished products. Therefore, routine testing of environmental samples for Listeria spp. and other foodborne pathogens by outside laboratories remains a critical component of any sanitation verification program. It is a common misconception that only one assay can be done per sponge, swab, or other similar environmental surface sample. This has resulted in some individuals sampling the same surface more than once and testing each of these samples, ostensibly from the same surface, for a different organism or assay of interest. However, this need not and should not be done. Each time a sample is taken off a surface, that surface is altered. Some microbes are removed although others may remain. Hence, the microbes recovered from the same surface should not be expected to yield the same results. Appropriate aliquots of diluent from a single sample can be subdivided for quantitative or qualitative (enrichment) analysis of each organism or group of organisms of interest. One example of this occurred when the first author and others took 49 duplicate samples in a dry factory environment for the analysis of Enterobacter sakazakii. In this instance, each of the 49 areas was sampled twice. The first author reported recovery of the organism from 12 sites whereas the other laboratory reported recovery from 11 sites. However, only 7 sites were common. In addition, the first author reported that 7 total ribotypes of the organism were recovered from 49 samples, whereas analysis of the duplicate set resulted in detection of 14 ribotypes and only four ribotype patterns in common for a total of 17 distinct ribotypes of the organism recovered [96,99]. The same phenomenon

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is likely to be true for L. monocytogenes, as multiple and varied ribotypes were found in factory environmental samples depending upon the enrichment medium used [132]. Many believe that it is important to sample a defined surface area (e.g., 4 in.2); however, it is the first author’s conviction that this approach, which may have some value in routine product contact surface testing, is detrimental to investigational sampling of the factory environment, particularly relating to Listeria or other pathogen surveys. Numerous areas are unnecessarily precluded from meaningful pathogen sampling because of rigid adherence to sampling a defined surface area. Important, but precluded, areas may include product-pump-bearing seals, small penetrations in walls, and equipment that may entrap moisture, irregular surfaces, etc. (Figures 17.5 to 17.15; 17.17 to 17.38).

CONTROL OF FOOD CONTAMINATION CLEANING AND SANITATION

THROUGH

EFFECTIVE

Any discussion related to control of L. monocytogenes in food processing facilities would not be complete without mention of appropriate approaches to cleaning and sanitation. However, a detailed discussion of cleaning and sanitation is beyond the scope of this chapter, and many excellent resources exist [79,84,107,109]. However, some general principles will be described. Cleaning can be defined as the physical removal of visible dirt, impurities, and other extraneous matter commonly referred to as soil. Such soil may entrap and provide nutrients to microbes, including Listeria, although profound differences exist in cleaning and sanitization of wet processes (such as meat, poultry, and fluid dairy factories) as opposed to dry facilities (e.g., chocolate, dry blend factories, dry areas of dry milk processing facilities, etc.) or dry areas. Cleaning Dry Areas Dry approaches to cleaning (e.g., appropriate vacuum removal of residues, brushing, scraping, and damp mopping) should be used in dry facilities processing dry products. The use of unfiltered portable dry vacuum devices is discouraged as the exhaust may simply redistribute the residue to be removed. However, some factories have excellent central vacuum systems. The general absence of moisture in these environments suppresses the growth of microbes, preventing the development of microbial growth niches. Application of water to such environments is strongly discouraged, as this may result in creation of microbial growth niches and greater potential for product contamination. Cleaning Wet Processing Areas The first step in any cleaning procedure should be physical removal of visible residues. This can be accomplished by appropriate brushing, sweeping, and application of hot water (in wet processes). Wet cleaning is done through proper use of solutions of soaps, detergents, surfactants, and abrasive agents. In contrast, sanitizing causes inactivation of most microorganisms left on cleaned surfaces by exposing them to heat or chemical agents such as chlorine, iodine (iodophor), acid anionic, or quaternary ammonium compounds. Hence, the routine use of good cleaning and sanitizing practices is of utmost importance in controlling microbiological safety and quality of finished products. In establishments such as those that produce fluid milk and ice cream, adherence to good cleaning and sanitation practices that involve both equipment and the factory environment may be the only means of preserving product quality beyond initial pasteurization of ingredients. Each food processing facility needs to institute and enforce an effective cleaning and sanitizing program that will ensure manufacture of safe products. Management personnel need to develop standard operating procedures for every job in the factory as part of this program along with master schedules with the frequency of cleaning and sanitizing procedures, so that the workers will recognize their individual responsibilities and maintain accurate records regarding routine sanitation practices. Management personnel also need to instill the great importance of good cleaning and

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sanitizing practices in their employees through the use of continuing education programs that deal with current issues such as Listeria. Such cleaning and sanitizing responsibilities should never be assigned to new untrained employees. Floors, drains, walls, ceilings, and each piece of equipment in the factory should be cleaned and sanitized on a regular basis, with the frequency of cleaning and sanitizing being dependent on the extent to which the particular item becomes contaminated during normal operation and whether or not a product is likely to come in contact with the item during processing and packaging. The extent to which a particular area becomes contaminated can be determined by careful observation and appropriate sampling and testing of that area. All food contact surfaces such as tables, peelers, slicers, collators, overhead shielding, conveyors, conveyor belts, chain rollers, supports, and other intricate equipment directly associated with processing, filling, and packaging operations need to be cleaned and sanitized daily, sometimes more often, particularly around filling and packaging operations. A regular cleaning and sanitizing schedule must also be adopted for non-food-contact surfaces such as floors, walls, ceilings, floor drains, pipes, blowers, HVAC ducts, coils and pans from dehumidifying and air conditioning units, light fixtures, material-handling equipment, and wet and dry vacuum canisters. Listeria spp., including L. monocytogenes, have been most frequently isolated from floor drains and floors, as will be shown, thus suggesting that these areas may function as reservoirs for listeriae in food processing facilities. All floors and drains, including drain covers and baskets, in production and refrigerated storage areas should be thoroughly cleaned and sanitized daily; however, high-pressure hoses should never be used in these areas, because such practices readily promote the spread of listeriae to nearby equipment and other areas of the factory through splashing and the production of aerosols. Managers of food processing facilities must be sure that proper equipment is available for daily cleaning and sanitizing operations. Absorbent articles such as sponges and rags should never be used in the factory environment because these items, when moistened, can support the growth of high numbers of diverse microflora. Various types of metal scrapers can be used for removing hard mineral deposits, with disposable paper towels being best suited for eliminating excess moisture and accidental spills. Brushes are readily cleaned and sanitized, unlike sponges and rags, and are therefore suitable for widespread use in the factory. However, to avoid cross-contamination, separate color-coded brushes with nonporous plastic or metal handles should be used for scrubbing (1) exterior and interior surfaces of equipment, (2) raw and finished product areas, (3) food contact and non-food-contact equipment surfaces, and (4) floor drains. Brushes, particularly those used to scrub floor drains, are best cleaned and stored in an effectively maintained sanitizing solution after use. Water temperatures sufficient to liquefy fats and solubilize proteins should be employed. There is disagreement as to the appropriate water temperatures to use to accomplish this purpose and required temperatures can vary depending upon the nature of the soil and the method of cleaning (e.g., CIP vs. manual cleaning, cleaning chemicals used, etc.). It should be recognized that stainless steel and metal surfaces in factories act as heat sinks, and water hose exit temperatures may need to be appreciably higher (e.g., often 20–30°F) than the area’s targeted surface temperature. This is especially important to recognize in those factories where the environment is refrigerated. If water must be applied, then use of low-pressure high-volume delivery is to be preferred over high pressure. Moisture from a high-pressure hose may project food residue and moisture to places inaccessible for cleaning and result in microbial growth niche development. In many instances (e.g., meat processing facilities), surfaces soiled with fatty residues will need to be warmed to 140°F to liquefy the fat and move it away from the surface. Temperatures as high as 160–170°F may need to be used to achieve a surface temperature of 140°F. Data supporting these temperatures can be found [117,138,152]; however, it is best to check with one’s supplier of cleaning agents and sanitizer chemicals and seek advice related to proper water temperatures specific to the factory environment, equipment, and soil type. Second, application of suitable cleaning agents should be used. It is important to understand the nature of the soil in the factory to best understand which cleaner to use. Relevant literature and commercial suppliers of cleaning and sanitizing chemicals can provide guidance. Bohner and

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Bradley [32] published a very useful chart in this regard. Cleaning should be in the direction of product flow and from top to bottom. Subsequent to cleaning, scrubbing may be necessary depending upon the cleaning approach. This is typically followed by rinsing. Moisture on the floor and walls should be squeegied to appropriately trapped drains. Sanitizing is the final step in eliminating L. monocytogenes, other foodborne pathogens, and the myriad spoilage organisms present in the production environment. Because the presence of organic debris, particularly if proteinaceous, readily decreases the effectiveness of sanitizing agents against most microorganisms, including listeriae [30], it is important to remember that every item must first be thoroughly cleaned before being sanitized. Sanitization In those relatively rare instances in which application of aqueous sanitizer in dry factory environments is necessary, it can sometimes be done by carefully damp-wiping surfaces with a clean cloth previously immersed in appropriate concentrations of sanitizer. Damp mopping of floors with sanitizer may also be done. Thorough drying of the factory equipment and environment is critical and should be done after application of sanitizer to both wet and dry factories. The Code of Federal Regulations [68] specifies concentrations of sanitizers that may be used in food processing facilities. Research has demonstrated that L. monocytogenes is sensitive to sanitizing agents commonly employed in the food industry. According to several authors [105,122], chlorine-based, iodine-based, acid anionic, and quaternary ammonium-type sanitizers were effective against L. monocytogenes when used at concentrations of 100 ppm, 25–45 ppm, 200 ppm, and 100–200 ppm, respectively. However, 21 CFR 178.1010 indicates that these can be used at the 200-ppm level on finished product contact surfaces without the requirement of a subsequent water rinse, with the exception of iodine-based sanitizers where the maximum level on product contact surfaces not receiving a subsequent water rinse is 25 ppm. These concentrations may have to be adjusted to compensate for in-plant use, as well as oxidation and reduction factors relating to water quality and hardness. However, recommended concentrations should not be markedly exceeded, because the use of extremely concentrated sanitizing solutions heightens the danger to employees, increases the risk of chemical contamination of food and, in some instances, causes corrosion of equipment. Because foaming chlorine-based sanitizers are corrosive, their use should be primarily confined to floors, floor drains, walls, and ceilings. These areas can, alternatively, be flooded or foamed with quaternary ammonium-type sanitizers; however, fogging exterior surfaces with quaternary ammonium-type sanitizers is frequently regarded as being ineffective and dangerous for employees. Quaternary ammonium-based sanitizers are also not recommended for use on food contact surfaces and should never be used in cheese or sausage factories, because lactic acid starter culture bacteria are rapidly inactivated by small residues of these sanitizers. In contrast, acid anionic and iodine-type sanitizers are best suited for equipment surfaces, with the former readily neutralizing excess alkalinity from cleaning compounds and preventing formation of alkaline mineral deposits. Application of sanitizer should be done in the order of the process flow and from the bottom to the top of equipment to prevent recontamination of sanitized equipment surfaces from the floor. Many factories employ frequent applications of sanitizer to the wet processing environment. However, establishment of application frequencies should be based upon appropriate in-factory research related to their efficacy. Thorough drying of cleaned and sanitized surfaces before startup is essential. Sanitizers are typically inactivated over time by organic matter and the remaining moisture may become a microbial growth niche. Other Approaches to Sanitization Sometimes pieces of equipment cannot be adequately or efficiently broken down for complete and effective cleaning and sanitization. Examples of such equipment include some slicers [29],

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sausage peelers, angle conveyors, etc. Some companies have relied upon novel applications of moist heat or steam [150]. However, observations and validation of such approaches should be undertaken to ensure that they are appropriate (i.e., do not add excessive moisture to the environment or damage the equipment) and effective. The use of steam should be confined to closed systems because of potential hazards associated with aerosol formation, liberation of more moisture in the factory environment for future microbial growth, and burn risks associated with human exposure. Custom-designed CIP systems have been installed in many food processing facilities, particularly dairies, for automated cleaning and sanitizing of pipelines, tanks, vats, heat exchangers, homogenizers, and other equipment in processing lines. Presumably adequate by design, CIP systems should also be reviewed for proper timing, flow rate, temperature, pressure, and sanitizer strength as recommended by chemical suppliers. Proper operation of the entire system should be verified from data collected on recording charts, which can be stored for future reference. It is important that equipment be designed for ease of disassembly for cleaning and sanitization. Alternative approaches to effectively clean and sanitize equipment in which sandwiched surfaces cannot be broken down for cleaning should be researched.

OTHER FACTORS TO CONSIDER FOR CONTROL OF LISTERIA IN FOOD PROCESSING ENVIRONMENTS TRAFFIC PATTERNS Employee movement within food processing facilities can also have a major impact on the microbiological quality of finished products. Therefore, traffic patterns need to be developed that restrict or preferably eliminate movement of workers between raw, processing, filling, packaging, and shipping areas. Managers need to educate employees about the spread of Listeria and other microbial contaminants from clothing, boots, and tools to all areas of the factory, and they need to situate locker rooms, changing areas, and lunch and break rooms to minimize traffic through production areas. Issuing different-colored outer garments to workers in various areas of the factory has proved helpful in monitoring employee movement. Because L. monocytogenes and other microbial pathogens are commonly associated with raw products of both plant and animal origin, employees working in raw product receiving areas (including maintenance personnel) and individuals who deliver raw products, particularly milk haulers, should be denied access to all processing areas. When necessary, employee movement between raw product and processing areas of the factory should only be allowed after completely changing outer garments, as well as scrubbing and disinfecting boots. In many factories workers are encouraged to use disinfectant-containing footbaths that are placed in all doorways leading into the factory, as well as between raw product and production areas. However, if disinfectant footbaths are used, routine monitoring of sanitizer concentration should be done; otherwise, these will become a source of moisture and a potential Listeria growth niche. Experience demonstrates that the undersides of these footbaths can entrap wet residue and become microbial growth niches as well. Hence, they should be cleaned, sanitized, and thoroughly dried before use on a daily basis. These footbaths need to be monitored throughout the day for sanitizer strength and cleanliness. These are not recommended for use in dry processing areas as they contribute moisture to such environments. Instead, sanitary shoe covering or bootchanging areas should be provided at key areas of the plant. Because a great variety of microorganisms are carried on street clothing, it may also be prudent for managers to consider limiting the number of visitors and tour groups going through the factory. Large glass observation windows provide ample opportunity for visitors to view processing areas and simultaneously prevent introduction of additional microbial contaminants.

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MONITORING THE FINISHED PRODUCTS, INGREDIENTS, AND PROCESSING ENVIRONMENT The final means of controlling microbial hazards in finished products is through rigorous microbiological testing of ingredients, as well as the unfinished and finished product. Analysis of samples for pathogens or, more commonly, indicator (aerobic plate count, coliforms, yeasts, and molds) or spoilage organisms is crucial to ascertaining that good manufacturing, handling, and distribution practices are being followed. If such analysis records are maintained in an appropriate database, then trend analysis can be a powerful tool for solving problems [57]. However, the data are only as valid as the accuracy of the assay, the sanitary manner in which the sample was taken and analyzed, and the statistical confidence associated with the sampling plan (see section titled “Sampling Plans for L. monocytogenes in Foods”).

HAZARD ANALYSIS CRITICAL CONTROL POINT (HACCP) CONCEPT Traditional approaches for controlling microbial contaminants are being used by many food companies around the world; however, cases of foodborne illnesses still occur. The need for a modified approach to food-safety assurance led to the development of the HACCP concept, which can be used to identify and control biological, chemical, and physical hazards in foods from raw material production, procurement, and handling to manufacturing, distribution, and consumption of the finished product. A detailed discussion of HACCP is beyond the scope of this chapter; however, a brief explanation will assist the reader in understanding how the concept, along with GMPs and prerequisite programs, can be used to reduce the level of L. monocytogenes in food processing facilities and subsequently in cooked, ready-to-eat (RTE) foods. The HACCP concept was developed by the Pillsbury Company with the cooperation and participation of the National Aeronautics and Space Administration (NASA), the Natick Laboratories of the U.S. Army, and the U.S. Air Force Space Laboratory Project Group [28]. Development of the HACCP system began in 1959, when Pillsbury was asked to produce a food that could be used in the space program. There needed to be assurance (as close to 100% as possible) that the food produced for the space program would not be contaminated with bacterial or viral pathogens, toxins, and chemical or physical hazards that could cause illness or injury. The HACCP concept was developed and first presented to the scientific community at the 1971 Conference for Food Protection. The HACCP concept was first used in the acidified and low-acid canned-food industry and was then adopted by a number of companies during the 1970s and early 1980s. The food industry expressed considerable interest in the application of HACCP after an important 1985 National Academy of Sciences publication strongly recommended the HACCP concept. Consequently, in 1988 the National Advisory Committee on Microbiological Criteria for Foods (NACMCF) was established and embraced the HACCP concept. In 1989, the committee developed a HACCP document as a guide for maintaining uniformity of the principles and definitions of terminology [113]. Since then, the NACMCF has made several refinements and improvements in the HACCP concept and published revisions in 1992 [114] and 1997 [115]. The 1997 document, entitled “HACCP Principles and Guidelines” [115], contains many additions and includes a section on prerequisite programs. Prerequisite programs are essential to successful development and implementation of a HACCP plan [141] and form the foundation upon which the plan is built. Many of the prerequisite programs are based on the current GMPs in the Code of Federal Regulations [67] and in the Codex Alimentarius General Principles of Food Hygiene [91] for foods intended for international trade. In addition to specific items in the GMPs, prerequisite programs can include other activities such as ingredient specifications, supplier approval programs, ingredient-to-product traceability, and consumer-complaint-management programs. A summary of prerequisite program activities is presented in Table 17.3 [141].

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TABLE 17.3 Summary of Prerequisite Program Activities Facilities Adjacent properties Building exterior Building interior Traffic flow patterns Ventilation Waste disposal facilities Sanitary hand-washing facilities Water, ice, culinary steam Lighting Raw materials controls Specifications Supplier approval Receipt and storage Temperature control Testing procedures Sanitation Master schedules Pest control program Environmental surveillance activities Chemical Training Personal safety GMPs HACCP Production equipment Sanitary design and installation Cleaning and sanitation Preventive maintenance Calibration of equipment Production controls Product zone controls Foreign material control Metal protection program Allergen control Glass control Storage and distribution Temperature control Transport vehicle cleaning and inspection Product controls Labeling Product traceability Customer and consumer complaint investigations Source: Adapted from Sperber, W.H., K.E. Stevenson, D.T. Bernard, K.E. Deibel, L.J. Moberg, L.R. Hontz, and V.N. Scott. 1998. The role of prerequisite programs in managing a HACCP system. Dairy Food Environ. Sanit. 18: 418–423.

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Prerequisite programs are not part of the formal HACCP system and are established and maintained separately. There are some circumstances where the existence of a prerequisite program does not preclude use of specific activities with a HACCP system [141]. For example, sanitation procedures are normally part of a prerequisite program; however, some manufacturers manage selected sanitation procedures as critical control points (CCPs) in their HACCP systems. This has been done frequently in the meat and dairy industries in which sanitation procedures for meat slicers, ice cream fillers, and other pieces of equipment were established as CCPs to help prevent recontamination of processed products by L. monocytogenes [141]. The existence and effectiveness of prerequisite programs should be assessed during the design and implementation of each HACCP plan. Well-developed and consistently performed prerequisite programs can simplify the HACCP plan, so it is imperative that all food processors establish, document, and maintain effective prerequisite programs to support their HACCP plans [141]. In the absence of appropriate prerequisite programs, it would be wrong to assume that a company with a HACCP plan, even one that is well designed, is immune from having serious product contamination events. The first author has observed factories with seemingly acceptable HACCP plans that have had serious food contamination problems [97]. Listeria and many other microorganisms can establish themselves in niches with potential to inoculate the product stream because of unsanitary operating practices, unsanitary maintenance and repair practices, or unsanitary equipment or factory design (see Figures 17.5 to 17.37). If such niches are in the post-CCP environment, serious contamination can occur, as evidenced by a Michigan food factory that distributed products across the country from which 80 people developed listeriosis, and 21 died in 22 states [38,39,110].

SAMPLING PLANS FOR L. MONOCYTOGENES IN FOODS Prevention of microbiological hazards is clearly of considerable importance when one considers the serious health problems that may develop in certain individuals who consume Listeria-contaminated foods. Consequently, the International Commission on Microbiological Specifications for Foods (ICMSF) [87] initially proposed that L. monocytogenes be placed in the same category with agents that pose severe health hazards to the general population, such as Brucella (strains abortus, suis, melitensis), Burkholderia cocovenans, the neurotoxin produced by Clostridium botulinum, C. butyricum, and C. baratti, C. perfringens type C, enterohemorrhagic E. coli O157:H7 and O111:NM, Mycobacterium bovis, Salmonella typhi (serotypes paratyphi A, B, and C), Shigella dysenteriae I, Vibrio cholera 01 and 0139, aflatoxins, vCJD/BSE, the parasite Taenia solium, and hepatitis A virus [10,87]. In February 1988, the ICMSF considered application of its sampling plans to assess acceptability of foods with respect to L. monocytogenes. (The reader must be cautioned from the start that no microbiological sampling plan other than one that involves total destructive sampling of all products manufactured can ever provide complete consumer protection.) According to terminology developed previously by the ICMSF, sampling plans for L. monocytogenes in RTE foods would follow the recommendations made for cases 13, 14, and 15 [87,88]. These cases include those considered a “severe hazard for (a) the general population or (b) restricted populations, causing life-threatening or substantial chronic sequelae or illness of long duration [88].” Listeria monocytogenes is now considered to fit subcategory 2 in relation to immunocompromised individuals [88] along with Campylobacter jejuni serovar O:19 and serotypes associated with Guillain-Barre syndrome, enteropathogenic E. coli (EPEC and ETEC strains) in food intended for infants, Clostridium botulinum types A and B (infants), Enterobacter sakazakii (infants), C. perfringens type C (enteritis necrotans) for protein-deficient persons, Vibrio vulnificus and hepatitis A virus for patients with liver disease, and the parasite Cryptosporidium parvum with immunocompromised persons.

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Case 13 applies when conditions under which the product is normally handled and consumed after sampling reduce the degree of hazard associated with the product, whereas cases 14 and 15 refer to situations in which hazard levels remain constant or increase, respectively. Using a statistically based two-class attribute sampling plan, n (i.e., number of sample units to be examined from a particular lot) would equal 15, 30, or 60 for cases 13, 14, and 15, respectively, and c (i.e., the maximum allowable number of sample units containing L. monocytogenes) would equal 0 for all three cases. However, it is possible to reduce the number of actual tests done through use of validated sample compositing schemes [47]. The FDA [80] has a compositing scheme that involves taking 10 × 50 g (or mL) subsamples. These are then divided into two sets of 5 × 50 g samples, which are combined and stomached in 250 mL of buffered Listeria broth each. Aliquots of 50 g (or mL) from each of the two stomached preparations are added to two separate 200-mL BLEB enrichments and the assay continued. The assay, when performed in this manner, is effectively on two 25-g (mL) portions of the sample. An attribute sampling plan was also proposed in the United States by a working group of the NACMCF [20]. According to this plan, which was developed for RTE shrimp and crabmeat, n (i.e., number of samples for foods produced in facilities employing HACCP and GMP systems) would equal 10, whereas c (i.e., mandatory standard for L. monocytogenes that should not be exceeded) would equal “negative.” Thus, with the exception of n, this plan is similar to the two-class attribute plan proposed by the ICMSF. Before recommending any Listeria-sampling plan, there must be good epidemiological or risk assessment data indicating that the product or product group to be sampled has been implicated in foodborne listeriosis. In addition, there must be good reason to believe that introduction of a sampling program will substantially reduce the risk of contracting listeriosis by consumption of such products. The number of samples required to detect a contaminated lot increases as the incidence of food contamination decreases. A food production lot contaminated at 10% incidence would require 30 × 25 g samples to be taken to detect with 95% confidence whether or not the lot was contaminated (e.g., at least 1 positive in the 30 samples assayed). However, this assumes a random distribution of contaminants and a 100% accurate test [23]. A lot contaminated at a 1% level would require 299 such samples (Table 17.4). This is a time-consuming, costly, and ineffectual approach to food safety, if used alone. This is also an approach of diminishing returns as factory processes move toward safety. This illustrates the importance of a comprehensive approach to food safety, including GMPs, monitoring of the factory environment with documented corrective actions, and HACCP.

TABLE 17.4 Test Number Needed to Detect One or More Positives per Lot Percent Positives % Positive 100 10 1 0.1 0.01

Number of Analytical Units to Be Tested (n) 90% Confidence

95% Confidence

99% Confidence

3 23 230 2,303 23,026

4 30 299 2,996 29,963

4 46 461 4,605 46,052

Source: Adapted from APHA. 2001. Sampling plans, sample collection, shipment, and preparation for analysis, in F.P. Downes and K. Ito (eds.), Compendium of Methods for the Microbiological Examinations of Foods, 4th ed. American Public Health Association, Washington, DC, chap. 2.

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The International Dairy Foods Association (IDFA) made a series of recommendations concerning sampling plans for listeriae in milk, soft cheeses, and vegetables based on information collected in 1988 [10]. L. monocytogenes is commonly found in raw milk; however, minimum required pasteurization (71.7°C/15 sec) should eliminate this hazard. Therefore, the IDFA recommended that manufacturers institute monitoring programs to prevent postpasteurization contamination rather than routine sampling plans of pasteurized end products as the most appropriate means of protecting the consumer. Many varieties of Listeria-contaminated cheese have also been identified since 1985, with contamination most frequently being reported in surface-ripened cheeses. Because L. monocytogenes can grow rapidly in Brie and Camembert cheese during the late stages of ripening, a two-class attribute-sampling program should be considered if such cheeses are destined for consumption by pregnant women, immunocompromised adults, or the elderly. However, because no sampling program can ensure that such products are completely free of L. monocytogenes, public health interests are far better served by application of HACCP principles during cheese manufacture and ripening. The ICMSF recognized that raw vegetables may also become contaminated with L. monocytogenes, but routine testing of raw vegetables is unlikely to markedly reduce the risk of contracting listeriosis. Hence, consumers of raw vegetables are urged to wash all such products vigorously before consumption. End-product sampling programs are clearly not the answer to protecting consumers from listeriosis or other types of foodborne illness. However, microbiological sampling is recommended by many regulatory agencies and the World Health Organization as part of the HACCP approach to prevent opportunities for contamination, survival, and growth of L. monocytogenes as well as other microbial pathogens in raw materials, factory environments, and food products during manufacture, storage, distribution, sale, and use. The statistical principles for taking appropriate microbiological samples can be found in a variety of sources [23,88,139].

ESTABLISHMENT OF BARRIERS AND PRODUCTION AREAS

BETWEEN

RAW

AND

FINISHED PRODUCT

Food processors must strive to prevent the spread of microbial contaminants from heavily contaminated raw product receiving areas to processing and packaging areas because air, water, waste products, and anything else that comes in contact with the finished product must be considered as a potential source of contamination. In addition to construction of physical barriers between such areas in food processing plants, all incoming cases, pallets, containers, forklifts, and cleaning materials such as brushes and other equipment must be assumed to harbor listeriae along with other microbial contaminants and, therefore, should never be allowed to enter processing and packaging areas. Ideally, separate equipment, including tools employed by maintenance personnel, should be available for use in raw and finished product areas. If this is not possible, then all equipment should be cleaned and sanitized before entering processing and packaging areas. As previously stressed, all areas within food processing facilities should be kept dry and as free as possible from processing waste to minimize microbial growth. Also, floor drainage problems that lead to pooling of water must be eliminated, as well as cracks and holes in floor tiles and grouting in which water and food particles can accumulate. Because L. monocytogenes has been recovered from condensate in many factories, it is imperative to keep all processing and packaging equipment and walls, floors, and ceilings as condensate-free as possible. In the event that dripping condensate cannot be prevented by manipulation of temperature and humidity in processing and packaging areas, deflector shields should be installed to prevent direct contact between the exposed product and dripping condensate.

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HACCP PLAN DEVELOPMENT HACCP is a process management system that is designed for use in all segments of the food industry from production agriculture to consumption of the finished product. The HACCP approach for controlling biological hazards in food is based on seven principles [115]: 1. 2. 3. 4. 5. 6. 7.

Conduct a hazard analysis Determine the critical control points (CCPs) Establish critical limits Establish monitoring procedures Establish corrective actions Establish verification procedures Establish record-keeping and documentation procedures

THE HACCP TEAM A HACCP team should be formed before development of a HACCP plan. The HACCP plan should be a team effort involving representatives from a wide variety of factory responsibilities. This will ensure emotional buy-in necessary if the plan is to have wholehearted support of the factory. The insights gained from a wide diversity of backgrounds will also be invaluable. One individual fully versed in the principles of HACCP should be appointed as the team leader. This individual can, but need not, be an outside consultant, and should begin the process of introducing HACCP by educating team members in its basic principles. Once educated, the team is ready to begin developing the plan. Principle 1: Conduct a Hazard Analysis Hazard analysis is the key element in developing an effective HACCP plan. Oddly enough, this is often the most neglected or inadequately developed aspect of many HACCP plans. The purpose of hazard analysis is to determine which of the potential hazards associated with a food or a manufacturing process presents a reasonable risk to consumers. The hazard analysis involves two stages. The first stage, hazard identification, involves the review of ingredients used in the product, the activities conducted at each step in the process, the equipment used, the final product and its method of storage and distribution, and intended use and consumers of the product. Hazard identification focuses on developing a list of potential food hazards associated with each process step. In stage two, hazard evaluation, each potential hazard is evaluated based on severity and its likelihood of occurrence. It is important that food processors conduct a hazard analysis on all existing and any new products to be manufactured, because microbiological safety of processed foods not subjected to a microbicidal treatment (e.g., heating, irradiation) is directly related to the quality of raw materials. Furthermore, contaminated raw materials, even when given an adequate microbicidal treatment, may introduce pathogens into the food processing environment. Any hazard analysis must begin with identification of hazards associated with raw materials, with particular attention being given to raw products of animal origin (i.e., milk, meat, poultry, and seafood), all of which may harbor L. monocytogenes and other foodborne pathogens. Heat treatments, acidulation, fermentation, salting, and drying are designed to destroy or inhibit growth of pathogenic and spoilage microorganisms; nevertheless, other operations such as slicing and dicing, cooling of cooked products, and filling and packaging may allow pathogenic organisms to contaminate the final product. Therefore, all hazards associated with manufacturing procedures and postprocessing contamination, as previously discussed, must be fully understood along with consequences of processing failures and errors. A thorough review of operations, facilities, and processes should

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be done to ensure that this occurs. It is advisable for a thorough in-factory risk assessment to be made involving inspection of the premises for potential microbial, chemical, or physical hazards. Inspection or sampling with a view to understanding the microbial ecology of the ingredients, process, equipment, and overall factory environment should be done. It will be necessary to seek counsel from those trained to engage in such activities (e.g., factory qualityassurance personnel, experienced food safety scientists or outside consultants, etc.). Food processors should also be familiar with the effect of various physicochemical factors (e.g., pH, water activity, preservatives, and type of packaging with or without modified atmosphere) on the behavior of pathogenic organisms, including L. monocytogenes, in the product during processing, distribution, storage, and use by the consumer. The National Advisory Committee HACCP document [115] contains a series of questions regarding ingredients, intrinsic factors of the food during and after processing, processing procedures, microbial content of the food, factory and equipment design and use, and packaging when conducting a hazard analysis. It should be noted that any change in raw materials, product formulation, processing, packaging, distribution, or intended use of the product should prompt an immediate reassessment of hazards, as these changes have the potential to adversely affect product safety. Careful hazard analysis should lead to the ready identification of CCPs. Principle 2: Determine CCPs A CCP is a step at which control can be applied and is essential to prevent or eliminate a food safety hazard or reduce it to an acceptable level. Complete and accurate identification of CCPs is fundamental to controlling food safety hazards, and CCPs must be carefully developed and documented. Examples of CCPs may include thermal processing, chilling, and product formulation control. Principle 3: Establish Critical Limits A critical limit is a boundary of safety and is used to distinguish between safe and unsafe operating conditions at a CCP. Each CCP will have one or more control measures to ensure that the identified hazards are prevented, eliminated, or reduced to acceptable levels. Critical limits may be based on extrinsic factors such as temperature, humidity and time or intrinsic factors such as physical dimensions, moisture level, water activity (aw), pH, titratable acidity, salt concentration, available chlorine, viscosity, or preservatives. Critical limits must be scientifically based and may be obtained from regulatory standards and guidelines, scientific literature, experimental results, and experts. However, care should be taken when establishing a critical limit because exceeding a critical limit is an indication that the food produced may be unsafe for consumption. Hence validation of critical limits should be done (see subsection titled “Validation”). Principle 4: Establish Monitoring Procedures Monitoring is a planned sequence of observations or measurements to assess whether a CCP is under control and is used to produce an accurate record for future use in verification. Monitoring facilitates the tracking of an operation and is used to control the process. Monitoring is also used to determine when there is loss of control and deviation at a CCP (i.e., exceeding or not meeting a critical limit). Monitoring procedures must be effective to determine deviations and then documented corrective actions must be taken. Most monitoring procedures need to be rapid and often include visual observations and measurement of temperature, time, pH, and moisture level. Microbial tests are seldom effective for monitoring owing to their time-consuming nature and problems with ensuring detection of contaminants (e.g., because of statistics associated with sampling plans—see section titled “Sampling Plans for L. monocytogenes in Foods”).

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Principle 5: Establish Corrective Actions Corrective actions are necessary when there is a deviation from an established critical limit. Foods that may be hazardous are prevented from reaching consumers through the establishment of corrective actions. When there is a deviation from critical limits, corrective actions are needed to: • • •

Determine and correct the cause of noncompliance Determine the disposition of noncompliant product Record the corrective actions that are taken

Specific corrective actions should be developed for each CCP and included in the HACCP plan. Plans should avoid vague language related to future decisions that may or may not be made related to disposition of the product. The time to determine the specific corrective actions is during the HACCP plan development, rather than waiting until there is a deviation from a critical limit during production of a product. Corrective actions should be clearly and precisely stated. Principle 6: Establish Verification Procedures Verification determines the validity of the HACCP plan and is used in evaluating whether the facility’s HACCP system is functioning according to the HACCP plan. An effective HACCP system requires little end-product testing, because sufficient validated safeguards are built in early in the process. However, verification of the efficacy of the HACCP plan can and has been done using finished product testing. Nevertheless, firms should rely on frequent reviews of their HACCP plan, verification that the plan is being correctly followed, and review of CCP monitoring and corrective action records. Validation Another important aspect of HACCP plan development is validation that the initial plan is scientifically and technically sound, that all hazards have been properly identified, and that if the HACCP plan is properly implemented, these hazards will be effectively controlled. Subsequent validations are done and documented by the HACCP team or independent experts as needed. Validations are conducted when there is an unexplained system failure, when a significant product, process, or packaging change occurs, or when new hazards are recognized. Incorporation of CCPs into a HACCP plan implies that they have been validated. Validation is so important that it is being considered as a candidate worthy of the status of an eighth HACCP principle [140]. Validation of critical limits at CCPs has been done in a variety of ways. These include tradition (not advised), regulations, published data, and challenge studies. However, some multivariable processes are difficult to completely control in the factory or cost prohibitive to model in a laboratory study. In these instances, harmless surrogates may be used to validate (or verify) processes [98]. Surrogates avoid the risk associated with introduction of pathogens and offer the advantage of assessing the efficacy of the process under actual use conditions. Parameters for selection of appropriate surrogates were listed by USFDA-CFSAN [22]. In recent times, USDA–FSIS has stressed the importance of validation [59,60]. Principle 7: Establish Record-Keeping and Documentation Procedures Establishment of an effective record-keeping system is an integral part of a HACCP system. Records are the only reference available to trace the production history of a finished product. If questions arise concerning the safety of a product, a review of records may be the only way to prove that the product was prepared and handled in a safe manner in accordance with the company’s HACCP plan [85].

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A well-developed and implemented HACCP plan built on a strong foundation of GMPs and prerequisite programs can reduce the incidence of L. monocytogenes in food processing facilities and in cooked RTE foods. In conclusion, to reduce the incidence of L. monocytogenes in the food supply, food processors must develop and implement HACCP plans that are built on a strong foundation of GMPs and prerequisite programs which in turn will decrease the incidence of listeriosis.

INCIDENCE OF LISTERIA SPP. IN VARIOUS TYPES OF FOOD PROCESSING FACILITIES IN THE UNITED STATES Interest in the extent of Listeria contamination in various food processing facilities has grown over the past 2 decades, because L. monocytogenes was not identified as a serious foodborne pathogen until 41 cases of listeriosis in Canada, including 17 deaths, were linked to consumption of contaminated coleslaw in 1981 [134]. Despite further evidence 2 years later suggesting possible involvement of pasteurized milk in an outbreak of listeriosis in Massachusetts, public health officials in the United States and elsewhere did not yet regard the presence of L. monocytogenes in food as a major threat to public health. However, this situation changed dramatically in June 1985 when up to 300 cases of listeriosis, including 85 deaths, were eventually linked to consumption of contaminated Mexican-style cheese in southern California. This listeriosis outbreak, along with America’s major outbreak of salmonellosis, in which over 16,000 individuals in the Chicago area became ill during March and April 1985 after consuming a particular brand of pasteurized milk contaminated with Salmonella Typhimurium [103], prompted U.S. Food and Drug Administration (FDA) officials to begin testing various types of domestic and imported cheese for Listeria. FDA officials also developed the Dairy Safety Initiative Program, which included microbiological surveillance of finished dairy products and the factory environment in which they were produced along with in-depth inspection of fluid milk factories and eventually all types of dairy processing facilities located throughout the United States. The subsequent discovery of L. monocytogenes in cooked RTE meat, poultry, and seafood products by FDA and U.S. Department of Agriculture (USDA) officials led manufacturers of foods other than dairy products to become concerned about the incidence of listeriae in their products and processing facilities. Once FDA and USDA officials announced their plans to review GMPs that were presumably being used by most American firms, the food industry launched a Herculean effort to identify Listeria spp. and eliminate such problems within the food processing environment before governmental inspectors arrived. Considering the adverse publicity and potential monetary losses that could result from discovery of L. monocytogenes in the finished product and the factory environment, it is not surprising that very little information concerning the incidence of listeriae and other microbial contaminants in food (except Class I recalls) and food processing facilities has been released to the scientific community. Vast amounts of data have been generated since 1985 by local, state, and federal government inspectors, as well as private microbiological testing and consulting laboratories and the food manufacturers themselves; however, much of the information which now follows is either of a general nature describing particular niches within food processing facilities from which listeriae have been isolated or consists of limited results from academic surveys which describe the actual incidence of Listeria contamination in a relatively small number of food processing facilities. This is because many industry-generated data are kept proprietary, at least in part because of concerns about liability-related issues associated with misuse of this information by others. Food companies should be encouraged, not discouraged, to generate such proprietary information, so they have the courage to find real problem areas and correct them. Requiring companies to make results of environmental testing public creates an incentive against responsible rigorous inspectional sampling to find and correct real problem areas. Food processors should be encouraged to thoroughly monitor, understand, and control the microbial ecology within their factories.

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DAIRY PROCESSING FACILITIES Following several dairy-related outbreaks of salmonellosis and listeriosis, FDA officials, in cooperation with state governments and the dairy industry, intensified surveillance of various types of dairy processing facilities under the Dairy Safety Initiative Program which began April 1, 1986 [100]. Under this program, state officials were requested to sponsor a series of statewide meetings to discuss foodborne illness associated with Grade A and non–Grade A dairy products and to intensify their surveillance and inspection efforts in dairy processing facilities. Nationally, FDA officials vowed to (1) conduct intensified check ratings in every interstate milk shipment (IMS) milk pasteurization plant, (2) conduct similar inspections at non–Grade A (non-IMS) milk pasteurization plants, (3) initiate a microbiological surveillance program designed to detect pathogenic microorganisms in finished products, (4) intensify and upgrade training and standardization practices for federal and state milk specialists, rating officers, and sanitarians, and (5) regularly prepare national reports which summarize the status of the U.S. dairy industry. In the first of these reports [4] covering the 6-month period from April to September 1986, 9 of 357 (2.5%) milk pasteurization factories examined produced various dairy products contaminated with L. monocytogenes. A subsequent report in February 1987 indicated generally similar contamination rates with 16 of 620 (2.6%) and 3 of 620 (0.48%) dairy processing facilities manufacturing finished products containing L. monocytogenes and L. innocua, respectively [5]. Eight months later, FDA officials reported that 11 of 604 (1.8%) IMS and 18 of 412 (4.4%) non-IMS milk pasteurization factories had produced products contaminated with Listeria spp., principally L. monocytogenes [6]. Extensive follow-up efforts in milk processing plants producing Listeria-contaminated products uncovered various defects in factory design and pasteurization equipment. Nevertheless, FDA officials have maintained that listeriae entered these products as postpasteurization contaminants. This view is strongly supported by FDA’s success in isolating Listeria from numerous floor drains in processing and other areas, wooden (porous) walls, floors and ceilings, wooden pallets, external surfaces of milk cartons, and sweetwater (refrigerated water) from leaking pasteurizer plates. Although not clearly identified in FDA’s list of environmental samples that harbored Listeria, FDA officials [66] noted the following problem areas related to environmental, postpasteurization contamination of dairy products with listeriae: (1) improperly operating high-temperature short-time (HTST) and vat pasteurizers, (2) leaking or cracked storage tanks, jacketed vessels, and valves, (3) inadequate sanitizing regimens, (4) cross-connecting pipes which allow commingling of raw and pasteurized product, (5) use of contaminated rags and sponges, (6) exposure to contaminants in unfiltered air and condensate, (7) filling and packaging operations, (8) conveyor belts, (9) use of returned product and reclaiming operations, (10) walls, floors, and ceilings, particularly in walk-in refrigerators, (11) formation of aerosols, (12) traffic patterns within the factory, (13) entrances and floor mats, and (14) personal cleanliness of employees and others in the factory. In reality, L. monocytogenes and other foodborne pathogens have been detected in environmental samples from many of these problem areas, as indicated in the following surveys of dairy factories in California and Vermont. In response to these federal programs, officials of the Milk and Dairy Foods Control Branch of the California Department of Food and Agriculture published [41] results from a statewide survey in which 597 environmental samples were collected from 156 milk processing facilities during the first half of 1987 and analyzed for listeriae. Overall, Listeria spp. were identified in the working environment of 46 (29.5%) milk-processing facilities with 31 of these 46 (67.4%) Listeria-positive factories being contaminated with L. monocytogenes. Furthermore, L. monocytogenes and other Listeria spp. were most frequently observed in factories producing fluid milk products, followed by those that manufactured frozen dairy products (i.e., ice cream and novelty desserts) and cultured milk products (i.e., yogurt and cottage cheese), with lowest contamination rates being associated with production of miscellaneous products and cheese. In all likelihood, this unusually low incidence of listeriae in California cheese factories was a direct result of massive cleanup efforts that were instituted following the 1985 listeriosis outbreak in the Los Angeles area involving Mexican-style cheese.

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TABLE 17.5 Evaluation of Listeria Species Isolates Based on Area of the Processing Plant Area Designation Processing Entrance (to processing) Entrance (other than processing) Raw milk receiving/storage Coolers and freezers Dry storage Othera Total

Number of Sites

Number of Positive

% Positive

145 53 37 33 30 16 31 346

52 21 11 13 14 7 4 122

35.9 39.6 29.7 39.4 46.7 43.8 12.9 —

a

Includes areas such as common hallways, testing laboratories, and wheels of forklifts.

Source: Adapted from Pritchard, T.J., C.M. Beliveau, K.J. Flanders, and C.W. Donnelly. 1994. Increased incidence of Listeria species in dairy processing plants having adjacent farm facilities. J. Food Prot. 57: 770–775.

Pritchard et al. [127] sampled 30 dairy processing plants in Vermont. Of the 346 sites tested, 122 (35.3%) contained one or more species of Listeria. Coolers and freezers had the highest rate, with 14 of 30 sites (46.7%) being positive for Listeria (Table 17.5). Other sites that resulted in high positive rates included dry storage areas (39.6%) and raw milk receiving and storage areas (39.4%). Pritchard et al. [127] also noted that plants producing dairy ingredients, frozen milk products, or fluid milk had significantly higher incidence rates of Listeria than expected. Facilities producing cultured dairy foods or a combination of cultured dairy foods and fluid milk were found to have significantly lower incidence rates of Listeria than expected. These researchers also observed that when dairy farms were contiguous to the processing facilities, these plants were more likely to be contaminated than plants without on-site dairy farms. More recently, Pritchard et al. [128] surveyed 21 dairy processing factories, including 5 fluid milk, 12 cultured diary, 3 frozen dairy, and 1 fluid milk/cultured product processing facilities, respectively. These factories ranged from small, single-room farmstead fluid milk operations to facilities producing millions of pounds of cheese per year. Environmental and processing equipment (non–product-contact surfaces) were evaluated by the sponge sampling technique of Silliker and Gabis [137]. Three different enrichments recovered Listeria species in 21.2% (80) of samples, including 26 more samples than if a single enrichment had been used. L. innocua, L. monocytogenes, and L. seeligeri were recovered in 59 (73.8%), 35 (43.8%), and 5 (6.3%) of samples, respectively. Among the 21 factories surveyed, Listeria-positive equipment samples were obtained from 6 (28.6%) plants. However, 19 of the 21 plants (90.5%) had positive environmental sites. Listeria species were found in 17 (17.9%) of the 215 equipment samples taken. L. monocytogenes was recovered from 11 of these 17 Listeria-species-positive sites, including three holding tanks, two table tops, three conveyor/chain systems, a pasta filata wheel, a pint milk filler, environmental samples and a brine prefilter machine. Listeria species were recovered from 63 of 163 (41.1%) of which 24 were positive for L. monocytogenes. A comparison of the incidence of listeriae in different milk processing areas and sample sites (Table 17.6) supports the widespread belief that listeriae most frequently enter products after, rather than before, pasteurization, with the prevalence of these organisms in the factory environment increasing as the product passes through processing, filling, packaging, and storage areas. This apparent movement of listeriae through milk processing facilities is most readily seen in

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TABLE 17.6 Incidence of Listeria-Indifferent Milk Processing Areas and Sample Sites Facility Working Area and Sample Site Raw milk receiving room Drain Condensate Other a Processing room Drain Condensate Other a Filling/packaging room Drain Condensate Conveyor Other a Cold storage room Drain Condensate Conveyor

Number of Samples Examined

Number (%) of Positive Samples L. monocytogenes

All Listeria spp.

30 32 1

1 (3.3) 0 0

1 (3.3) 1 (4.5) 0

150 76 21

4 (2.7) 1 (1.3) 3 (14.3)

14 (9.3) 3 (3.9) 6 (28.6)

60 36 15 10

7 (11.7) 1 (2.8) 5 (33.3) 0

12 1 7 2

105 44 14

12 (11.4) 0 4 (28.6)

(20.0) (2.8) (20.0) (20.0)

17 (16.2) 1 (2.3) 9 (64.3)

a

Includes wooden blocks, pallets, case dollies, and utility tables.

Source: Adapted from Charlton, B.R., H. Kinde, and L.H. Jensen. 1990. Environmental survey for Listeria species in California milk processing plants. J. Food Prot. 43: 198–201.

results obtained from sampling conveyor belts and floor drains. However, sporadic isolation of listeriae from condensate, as well as wooden blocks, pallets, case dollies, and utility tables points to additional routes by which these organisms can be disseminated in dairy processing plants. The low incidence of listeriae in raw milk receiving rooms as compared to other areas of the factory may at first seem surprising; however, these findings most likely reflect difficulties encountered in adequately cleaning and sanitizing equipment in processing, filling, and packaging areas of factories rather than what could be interpreted as a near absence of listeriae in California raw milk. In an environmental survey of 39 frozen milk product plants in California, Walker et al. [158] collected 922 samples and found 111 (12%) positive for Listeria spp. L. monocytogenes was the only specie recovered from 5 (12.8%) plants and L. innocua was the only specie recovered from 13 (33.3%) plants. Both species were isolated from 9 (23.1%) plants. The highest recovery rates of Listeria were found in the batch flavoring, freezing, ingredient blending, and packaging and filling areas of plants surveyed. Working at the University of Vermont, Klausner and Donnelly [94] performed a large-scale survey to identify sources of Listeria (and Yersinia) contamination in fluid milk, cheese, and noncheese processing facilities. Overall, 66.7, 9.5, and 23.8% of samples collected from floors and other non-food-contact surfaces at 34 fluid milk, cheese, and non-cheese factories were positive for Listeria spp., with L. monocytogenes and L. innocua being identified in 1.4 and 16.1% of 361 samples examined, respectively. As expected, the percentage of Listeria-positive samples was higher among those from floors (12.0–27.9%) than from other non-food-contact surfaces (8.1%) (Table 17.7) and wet (85.7%) rather than dry (14.3%) areas. According to these investigators, paperfiller beds, whey drainage pans on cheese presses, and case-washing areas were particularly prone

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TABLE 17.7 Incidence of Listeria and Yersinia on Floors and Non-Food-Contact Surfaces of 34 Fluid Milk, Cheese, and Non-Cheese Factories in Vermont Type of Sample Floor areas Coolers Processing Entrance Mats/footbaths Other areas Non-food-contact surfaces Total

Number of Samples Analyzed

43 117 64 25 38 74 361

Number (%) of Positive Samples Listeria

Yersinia

12 21 11 3 10 6 63

9 14 18 1 6 4 52

(27.9) (17.9) (17.2) (12.0) (26.3) (8.1) (17.5)

(20.9) (12.0) (27.7) (4.0) (15.8) (5.4) (14.4)

Source: Adapted from Klausner, R.B. and C.W. Donnelly. 1989. Personal communication.

to contamination with Listeria and Yersinia. The fact that 20.9% of all positive samples contained both Listeria and Yersinia suggests that yersiniae might be somewhat useful as a potential indicator of Listeria contamination within the dairy processing environment. Kabuki et al. [91a] examined L. monocytogenes contamination patterns in Latin-style freshcheese processing plants in the United States. Three plants were evaluated over a 6-month period by collecting 246 environmental samples. L. monocytogenes was found in 11.0% of environmental samples and in 6.3% of cheese samples tested. L. monocytogenes was found to be most prevalent in crates, drains, and floors at 55.6, 30.0, and 20.6%, respectively. However, only one of three plants tested positive for L. monocytogenes on finished product contact surfaces. The most common subtype detected in two plants (20/36 isolates) was ribotype DUP-1044A, linked in 1998 to a multistate listeriosis outbreak. In one plant, this subtype was widespread and found in finished products. As noted, L. monocytogenes, Yersinia spp., and most other foodborne pathogens are more commonly found in wet rather than dry processing areas. However, the fact that listeriae (1) were recovered from whey drainage pans, (2) were routinely shed in whey during cheese-making experiments, (3) grew in samples of refrigerated milk and whey, and (4) survived the typical spraydrying process used to manufacture nonfat dry milk suggests that these organisms should be of concern to manufacturers of dry dairy products. In light of these concerns, Gabis et al. [70] determined the incidence of Listeria in the working environment of 18 dry milk and whey processing facilities throughout the United States. The authors supplied environmental sampling kits containing sterile cellulose sponges, fabric-tipped swabs, and other necessities to all firms participating in the survey along with instructions as to how and where to collect samples. All samples were then sent to a central laboratory and, within 48 h of collection, were analyzed for listeriae according to the FDA procedure. Overall, only 2 of 410 (0.24%) samples examined were positive for Listeria spp., with L. monocytogenes and a Listeria species other than L. monocytogenes being isolated from floor drains in a raw milk receiving area and from a composite sample from several floor drains and trenches in a powder production area, respectively (Table 17.8). Allowing factory employees to choose specific sampling sites, as well as the number of samples to be analyzed, may have somewhat biased these results; however, the incidence of listeriae and hence the risk of postprocessing contamination appears to be many times lower in dry rather than wet dairy processing facilities. Nevertheless, since manufacturers of nonfat dry milk and dry whey are not immune to the Listeria problem, they should take appropriate action to eliminate this organism from the processing environment, thereby greatly reducing the chance of producing a contaminated product.

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TABLE 17.8 Incidence of Listeria in the Working Environment of 18 Nonfat Dry Milk and Whey Processing Facilities Located throughout the United States Work Area Raw milk receiving Wet processing Cheese factory Whey factory Dryer Bagging room Heating, ventilating, and air conditioning system Miscellaneous Total

Number of Samples Analyzed

Number (%) of Positive Samples L. monocytogenes

Other Listeria spp.

62 151 22 23 38 27 53

1 (1.6) 0 0 0 0 0 0

0 0 0 1 (4.3) 0 0 0

34 410

0 1 (0.24)

0 1 (0.24)

Source: Adapted from Gabis, D.A., R.S. Flowers, D. Evanson, and R.E. Faust. 1989. A survey of 18 dry dairy product processing plant environments for Salmonella, Listeria and Yersinia. J. Food Prot. 52: 122–124.

MEAT PROCESSING FACILITIES Unlike dairy processing facilities in which raw milk is pumped into the factory, pasteurized, and then either packaged immediately or pumped to closed vats for processing into cream, butter, ice cream, cheese, or other dairy products, raw meat processing factories are actually open-air disassembly line operations in which animals are slaughtered, eviscerated, and broken down to obtain various cuts of meat, hides for leather, and other items of commercial value. Considering that domestic cattle, sheep, and pigs frequently shed L. monocytogenes asymptomatically in fecal material, it is not surprising that surveys have shown this pathogen to be not only ubiquitous but also endemic to slaughterhouses and meat-packing facilities. Rivera-Betancourt et al. (2004) reported the prevalence of L. monocytogenes, E. coli O157:H7, and Salmonella in two geographically distant commercial beef processing plants in the United States. Plant sampling took place during the 5-month period of April, May, July, August, and October at two beef processing plants, one in the northern (plant A) and one in the southern (plant B) U.S. region. Detection of Listeria spp. was reported as being accomplished by the AOAC official method 996.14 and specific species confirmed biochemically using the AOAC official method 992.19. Cattle hides and environmental samples were taken as an indicator of Listeria residing in and entering the plant. The prevalence of both Listeria spp. and L. monocytogenes on hides was higher in plant B. The prevalence of both Listeria spp. on holding-pen fence panels was significantly different between the two plants A and B, at 14.7 and 40%, respectively. L. monocytogenes prevalence during each month of sampling was 2.0% for plant A, but it varied between 3.8 and 48.6% for plant B. The authors attributed this difference to regional differences in Listeria prevalence on animal hides as the cattle came from a 150-mi radius of each plant. Environmental samples (812 and 797, respectively) other than those obtained from cattle pens were gathered from 11 environmental sites sampled at each plant. Samples were taken from the slaughter floor, fabrication floor, and locker rooms at various times during processing (Table 17.9 and Table 17.10). Significant findings included L. monocytogenes recovery from floor drains toward the end of operation in plant B and before operation on the brisket saw. L. monocytogenes was also recovered from one source

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TABLE 17.9 Prevalence of Listeriae in Samples from the Slaughter Floor Environment of Two Beef Processing Plants Sampling Site Floor drains, before operation Plant A Plant B Floor drains, late in operation Plant A Plant B Product contact surfaces, before operation Plant A Plant B Product contact surfaces, late in operation Plant A Plant B Brisket saw, before operation Plant A Plant B Brisket saw, taken during lunch break Plant A Plant B Splitting saw, taken before operation Plant A Plant B Splitting saw, taken during lunch break Plant A Plant B Trolleys, late in operation Plant A Plant B

Listeria spp. Prevalence (%)

L. monocytogenes Prevalence (%)

0/50 (0.0) 0/50 (0.0)

0/50 (0.0) 0/50 (0.0)

2/74 (2.7) 4/75 (5.3)

0/74 (0.0) 1/75 (1.3)

0/49 (0.0) 0/50 (0.0)

0/49 (0.0) 0/50 (0.0)

0/74 (0.0) 1/75 (1.3)

0/74 (0.0) 0/75 (0.0)

0/20 (0.0) 1/11 (9.1)

0/20 (0.0) 1/11 (9.1)

0/30 (0.0) 0/17 (0.0)

0/30 (0.0) 0/17 (0.0)

0/20 (0.0) 0/20 (0.0)

0/20 (0.0) 0/20 (0.0)

0/27 (0.0) 0/29 (0.0)

0/27 (0.0) 0/29 (0.0)

1/73 (1.4) 2/73 (2.7)

0/73 (0.0) 0/73 (0.0)

Source: Adapted from Rivera-Betancourt, M., S.D. Shackelford, T.M. Arthur, K.E. Westmoreland, G. Bellinger, M. Rossman, J.O. Reagan, and M. Koohmaraie. 2004. Prevalence of Escherichia coli O157: H7, Listeria monocytogenes, and Salmonella in two geographically distant commercial beef processing plants in the United States. J. Food Prot. 67: 295–302.

on the fabrication floor in plant B, the conveyor belts, where the incidence of contamination late in operation was as high as 14.2%. L. monocytogenes prevalence in locker rooms of plants A and B (145 and 150 samples, respectively) was 3.3% for plant B (recovered only from floor drains) and 0% for plant A. The authors hastened to point out that prevalence reports for all three pathogens (L. monocytogenes, E. coli O157:H7, and Salmonella) occurring concurrently in beef receiving and processing plants in the United States are scarce or nonexistent. The authors concluded that the level of L. monocytogenes recovered from animal hides and holding pens is an excellent means of determining plant carcass-contamination patterns.

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TABLE 17.10 Prevalence of Listeriae in Fabrication Floor Environmental Samples in Two Beef Processing Plants Sampling Site Product contact surfaces, prior to operation Plant A Plant B Product contact surfaces, late in operation Plant A Plant B

Listeria spp. Prevalence (%)

L. monocytogenes Prevalence (%)

0/100 (0.0) 6/99 (6.1)

0/100 (0.0) 6/99 (6.1)

0/150 (0.0) 25/148 (16.9)

0/150 (0.0) 21/148 (14.2)

Source: Adapted from Rivera-Betancourt, M., S.D. Shackelford, T.M. Arthur, K.E. Westmoreland, G. Bellinger, M. Rossman, J.O. Reagan, and M. Koohmaraie. 2004. Prevalence of Escherichia coli O157: H7, Listeria monocytogenes, and Salmonella in two geographically distant commercial beef processing plants in the United States. J. Food Prot. 67: 295–302.

Initiation of the USDA–FSIS testing program for listeriae in cooked and RTE meat products in September 1987 prompted immediate action by the meat industry. However, even before government testing began, meat processors became concerned about the incidence of listeriae in the working environment. In June 1987, results from a large-scale survey were reported in which nearly 2,300 environmental samples were collected from over 40 meat processing facilities nationwide and analyzed for listeriae [11]. Fourteen processing areas within these factories yielded evidence of being contaminated with L. monocytogenes or other Listeria spp. Overall, listeriae were recovered from 21% of the environmental samples examined. (These results also compare favorably with those of a much smaller survey [14] in which Listeria spp. were detected in 9 of 27 (33%) meat processing environmental samples.) Problem areas in which >20% of the samples were positive included drains, trenches, floors, exhaust hoods, cleaning aids (sponges, brooms, hoses, and mops), product contact surfaces (peelers, conveyors, and slicers), and wash areas. Sampling of surfaces in contact with sliced luncheon meat revealed Listeria contamination rates of 9.3, 32.3, and 23.6% before, during, and after production, respectively. Similarly, listeriae were recovered from 2.8, 14.5, and 25.5%, respectively, of food contact surfaces examined before, during, and after production of frankfurters. From September 1987 to October 1991, USDA–FSIS inspectors sampled over 15,000 processed meat products, including cooked beef, sliced hams from cans, cooked sausage, jerky, cooked poultry, salads and spreads, and imported meats [149]. The overall incidence of L. monocytogenes during this sampling period was 1.6%, with 235 products testing positive for L. monocytogenes This led to 25 recalls of product from the market during 1989–1991 [149]. During the USDA’s microbiological monitoring of over 13,000 lots of RTE meat products, from January 1, 1993 to September 30, 1997, 2.9% of the lots tested positive for L. monocytogenes (Levine, personal communication 1998). Results from a large-scale 1987 survey sponsored by the American Meat Institute [3,14] support the notion that Listeria spp. are widely distributed within the environment of many meat-processing facilities, and as in the earlier study by Flowers [11], also point to floors, drains, cleaning aids, wash areas, sausage peelers, and food contact surfaces as significant problem areas, with between 20 and 37% of such samples harboring listeriae. In addition, Listeria or Listerialike organisms have been reported in RTE meat and poultry factories in a variety of areas, including those associated with brine chillers, ceiling refrigeration units, peelers, conveyors, slicers, dicers, can

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openers, spiral freezers, and even packaging machines (Table 17.11). Identification of listeriae in condensate and compressed air and on walls and ceilings strongly indicates that these organisms are also airborne and very widespread in at least some meat processing facilities. Recently, the USDA published its interim final rule [153]. According to this rule, RTE meat processing facilities were given three alternatives with respect to Listeria testing of the plant environment. Factories that must test according to alternative 3 are those which have no postprocessing antimicrobial or microbicidal treatment (e.g., after the smokehouse or other typical treatment). Factories which must conform to alternative 3 must test the environment for Listeria or an appropriate indicator (e.g., presence of the genus Listeria, esculin-hydrolyzing bacteria, Listeria-like organisms, etc.) at a level relative to the size of the facility. Factories that have one additional postprocessing antimicrobial treatment (e.g., use of an antimicrobial substance in the product formulation that is still active after processing) or process must test according to alternative 2. Factories that fall into alternative 2 must test their environment, but at a reduced level as compared to alternative 3. Factories using two antimicrobial processes or ingredients, in addition to the antimicrobial process normally used, are considered alternative 1 factories and are not required to test the production environment. Recognizing the potential opportunity for Listeria to contaminate meat during packaging, one major manufacturer of processed meat products attempted to obtain “near–operating room conditions” in its packaging room by cleaning the area for 3 days and then fogging the entire packaging room with 200 ppm quaternary ammonium compound [9,11]. Despite these efforts, listeriae were still detected in 1 of 19 environmental samples obtained from the packaging room. After this exercise, the firm packaged processed meat products in this room over a 2-week period. Despite adherence to normal cleaning and sanitizing procedures at the end of each workday, the overall incidence of listeriae in the packaging room increased, with 3 of 20 (15%), 6 of 20 (30%), and 8 of 20 (40%) samples testing positive for Listeria spp. 3, 6, and 8 days after the room was initially cleaned and fogged, respectively. Owing to the increased concern about L. monocytogenes in meat products, there has been a concerted effort to minimize the risk of postprocess contamination during the production of processed meats. In one study, cited by Tompkin et al. [149], swab samples were collected from packaging lines and floors where exposed RTE products were transported, chilled, stored, or packaged. The incidence of Listeria at these locations, from August 1989 to January 1992, is summarized in Figure 17.39. The overall trend is toward improved control of Listeria with fewer positive samples being evident following the inception of a Listeria-control program. The results show a strong seasonal effect for the presence of Listeria in finished product environments, with fewer positive samples being detected during the winter months. In addition, Tompkins et al. [149] also reported results of a 3-year study in which about 100 packaging lines were tested for Listeria (Table 17.12). The percentage of Listeria-negative samples increased from 44 in 1989 to 64 in 1991. The percentage of lines that exceeded the company’s established criterion of 5% of samples positive for Listeria decreased from 29 in 1989 to 13 in 1991. As improvements were made, attention was given to chronically positive lines. Examples of contaminated sites on packaging lines included hollow rollers for conveyors, on and off valves and switches, rubber seals around doors, fibrous conveyor belts, and areas of equipment that were inaccessible to cleaning. Tompkin [151] listed many such places (see Table 17.11). The authors also noted that occasional lapses in cleaning and sanitizing procedures resulted in a fairly rapid loss of control. They observed that a certain sequence of events can lead to periodic contamination of packaging lines. The floor is particularly difficult to render Listeria-negative, and this situation provides a ready source of organisms to contaminate the packaging line during production or while cleaning, allowing the establishment of sites for microbial multiplication. This sequence of events can be prevented by striving for Listeria-negative floors, effectively cleaning and sanitizing packaging lines at the end of each day’s production, eliminating inaccessible sites in the equipment, and by providing adequate preventive maintenance of the equipment. The authors summarized their report with the comment, “. . . for the present, it must be concluded that existing technology cannot eliminate Listeria from the cooked product

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TABLE 17.11 Sites Yielding Listeria or Listeria-Like Organisms in Ready-to-Eat Meat/Poultry Factories Sites

Source

Product

Chill unit doors Rubber door seals Rubber coated fabric Hinges extending width of door Bump guards Uncleanable areas in metal-tometal sandwiched steel framing Brine-saturated insulation on ammonia line to brine chilling unit Hoses and spray nozzles used to spray franks for easier peeling Rubber seals on stainless steel door at exit end of tunnel Condensate from the unit

Frankfurters/similar linked products Frankfurters/similar linked products Frankfurters/similar linked products Frankfurters/similar linked products Frankfurters/similar linked products Frankfurters/similar linked products

Gears in peeler (personal observation) On/off valves for steam and water lines near peeling equipment Wet insulation (from condensate) behind a fiberglass wall Vacuum-based casing removal system Meat entrapped between two-ply Plexiglas shield guard—under conveyer Fluid entrapped in a hollow split sprocket Hollow rollers Fibrous/fabric conveyor belts Wheel bearings

Frankfurters/similar linked products Frankfurters/similar linked products

Brine chiller Continuous chamber

Ammonia cooling unit Exit Tunnel for product on hanging racks Ceiling refrigeration unit (in staging cooler for peeler) Peeler or peeler area

Hopper (post-peeler) for catching product

Inclined conveyers-peeler to packaging

Conveyers (other)

Slicers

Buildup inside safety cover over gear and drive belt and over finished product conveyer Worn hydraulic seals at base of slicer Slicer blade guides (personal observation) Slicer blade housing (R. Behling [29], personal communication) Product grippers for slicers (personal observation)

Can opener Dicer

Product residue inside dicer blade guides (personal observation) Hollow support rods

Frankfurters/similar linked products Frankfurters/similar linked products Hams Frankfurters/similar linked products

Frankfurters/similar linked products Frankfurters/similar linked products Frankfurters/similar linked products

Frankfurters/similar linked products Cooked products Cooked products Cooked products (personal observation) and breaded products Sliced meat products (lunch meats, canned ham, pepperoni) Sliced meat products (lunch meats, canned ham, pepperoni) Cooked products Cooked products Cooked products Sliced meat products (lunch meats, canned ham, pepperoni) Diced cooked meat/poultry Cooked products (continued)

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TABLE 17.11 (CONTINUED) Sites Yielding Listeria or Listeria-Like Organisms in Ready-to-Eat Meat/Poultry Factories Sites Spiral freezer

Packaging machine

Source Wet insulation at entrance over product (personal observation) Entrapped product residues in belt (personal observation) Electrical control box/wet residue over product inside spiral conveyor (personal observation) Standing water inside spiral freezer after defrosting (personal observation) Crack in stainless steel covering on top edge of packaging machine Stainless steel rods for pushing product into carton Air duct at bottom for blowing bags open prior to insertion of product

Product Cooked products Cooked products Cooked products

Cooked products

Cooked products Cooked products Cooked products

Source: Adapted from Tompkin. R.B. 2002. Control of Listeria in the food-processing environment. J. Food Prot. 65(4): 709–723.

FIGURE 17.39 Incidence of Listeria on packaging lines and in the environment (floors) from August 1989 to January 1992, inclusive. (From Tompkin, R.B., L.N. Christiansen, A.B. Shaparis, R.L. Baker, and J.M. Schroeder. 1992. Control of Listeria monocytogenes in processed meats. Food Aust. 44: 370–376.)

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TABLE 17.12 Listeria Contamination on Packing Lines from 1989 to 1991 Year

Number of Lines

1989 1990 1991

96 106 97

Percentage of Lines Positive for Listeria at 0%

≤5%

>5%a

44 59 64

27 27 23

29 14 13

a

Exceeds company guideline. Source: Adapted from Tompkin, R.B., L.N. Christiansen, A.B. Shaparis, R.L. Baker, and J.M. Schroeder. 1992. Control of Listeria monocytogenes in processed meats. Food Aust. 44: 370–376.

environment of processing plants.” However, this does not mean that this organism’s growth or prevalence in the factory is beyond reasonable control. Since Listeria spp., including L. monocytogenes, have been found in up to 50% of raw beef, pork, and lamb marketed in the United States [132], complete elimination of listeriae from meat processing environments appears highly improbable. However, the American Meat Institute has developed a series of interim guidelines [3], which, if followed, will reduce the incidence of listeriae and decrease the overall microbial load in the working environment. A detailed description of these guidelines appears later in this chapter.

POULTRY PROCESSING FACILITIES Reports have shown that up to 50% of all raw poultry sold in the United States contains various Listeria spp., including L. monocytogenes, with fecal material from infected flocks cited most frequently as the source of contamination. Researchers at the University of California–Davis investigated the prevalence of listeriae in processing samples from one chicken [73] and one turkey slaughterhouse [74] during three or four separate visits. According to these investigators, no Listeria spp. were isolated from feathers, incoming chiller water, or scalding water, the latter of which aids in feather removal (Table 17.13). Nonetheless, L. monocytogenes and L. innocua were identified in samples of overflow chiller water and feather picker drip water obtained from the chicken slaughterhouse, with both organisms being detected in recycled water used to clean gutting equipment. Incidence rates for L. monocytogenes in chicken and turkey processing facilities were generally similar, with the percentage of Listeria-positive samples increasing approximately 2- to 2.5-fold during the latter stages of processing. However, L. welshimeri and L. innocua were absent from most chicken and turkey processing samples, respectively. Only two poultry slaughterhouses were examined in this survey; however, the inability of these researchers to detect L. welshimeri in fresh chicken meat and L. innocua in fresh turkey meat routinely processed at these facilities suggests that L. welshimeri and L. innocua might be able preferentially to colonize the gastrointestinal tract of turkeys and chickens, respectively. These findings, along with the ability of these investigators to further demonstrate an increasing incidence of Listeria spp. on the gloves and hands of poultry workers from the beginning to the end of processing (Table 17.14) confirm that these contaminants move along the processing line with the raw product.

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TABLE 17.13 Incidence of Listeria spp. in One Chicken and One Turkey Slaughterhouse in California Number of Chicken/Turkey Sample Scalding water overflow Feather picker drip water Incoming chiller water Overflow chiller water Recycled water for cleaning gutters

Slaughterhouse Samples Analyzed 16/15 16/15 16/0 16/15 16/15

Number (%) of Positive Samples L. monocytogenes

L. innocua

L. welshimeri

Total

0/0 0/1 (6.7) 0/0 2 (12.5)/0 1 (6.3)/2 (13.3)

0/0 3 (18.8)/0 0/0 0/0 5 (31.3)/0

0/0 0/1 (6.7) 0/0 0/1 (6.7) 0/3 (20.0)

0/0 3 (18.8)/2 (13.3) 0/0 2 (12.5)/1 (6.7) 6 (37.5)/5 (33.3)

Source: Adapted from Genigeorgis, C.A., D. Dutulescu, and J.F. Garayzabal. 1989. Prevalence of Listeria spp. in poultry meat at the supermarket and slaughterhouse level. J. Food Prot. 52: 618–624, 630; Genigeorgis, C.A., P. Oanca, and D. Dutulescu. 1990. Prevalence of Listeria spp. in turkey meat at the supermarket and slaughterhouse level. J. Food Prot. 53: 282–288.

Considering the fecal carriage rate for listeriae in domestic birds, the current assembly-line methods for processing poultry, and the fact that Listeria spp. (including L. monocytogenes) and salmonellae have been isolated from up to about half of all raw chickens marketed in the United States, one can speculate that the poultry and meat industries face similar problems in controlling the spread of listeriae and other organisms in the work environment. If one draws a parallel between methods used to process meat and poultry, then floors, drains, cleaning aids, wash areas, and food contact surfaces emerge as likely niches for Listeria spp., including L. monocytogenes, in poultry processing facilities. Berrang et al. [29a] uncovered L. monocytogenes in drains on the raw side of a poultry processing plant. These isolates appeared to be identical on a molecular level to

TABLE 17.14 Incidence of L. monocytogenes and L. innocua on the Hands and Gloves of Poultry Meat Processors Assigned to Three Different Stations in a Slaughterhouse

Sample Postchilling handlers Leg/wing cutters Leg/wing packers

Number of Chicken/Turkey Slaughterhouse Samples Analyzed

L. monocytogenes

L. innocua

L. welshimeri

Total

20/30 11/30 44/30

2 (10.0)3 (10.0) 4 (36.4)/3 (10.0) 20 (45.5)/5 (16.7)

2 (10.0)/0 1 (9.1)/0 11 (25.0)/0

0/2 (6.7) 0/7 (23.3) 0/7 (23.3)

4 (20.0)/5 (16.7) 5 (45.5)/10 (33.3) 31 (70.5)/12 (40.0)

Number (%) of Positive Samples

Source: Adapted from Genigeorgis, C.A., D. Dutulescu, and J.F. Garayzabal. 1989. Prevalence of Listeria spp. in poultry meat at the supermarket and slaughterhouse level. J. Food Prot. 52: 618–624, 630; Genigeorgis, C.A., P. Oanca, and D. Dutulescu. 1990. Prevalence of Listeria spp. in turkey meat at the supermarket and slaughterhouse level. J. Food Prot. 53: 282–288.

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L. monocytogenes isolated from fully cooked products in the same plant, suggesting environmental cross-contamination from the raw to the finished areas of the processing facility.

EGG PROCESSING FACILITIES The discovery of L. innocua and, to a lesser extent, L. monocytogenes in 15 of 42 (36%) samples of frozen, raw, commercial liquid whole egg obtained from 6 of 11 manufacturers located throughout the United States suggests that listeriae- as well as salmonellae-laden poultry feces may contaminate the surface of eggs before breaking, and that these organisms in turn may be spread to various areas within the egg processing environment. Fortunately, the Egg Products Inspection Act of 1970 led to regulations, which now require that all egg products be pasteurized to eliminate salmonellae (and L. monocytogenes). However, as is true for fluid milk, there is ample opportunity for recontamination of liquid egg products in the factory environment (e.g., with listeriae, salmonellae, and nonpathogenic organisms) after pasteurization, which can greatly decrease the shelf life, microbial quality, and acceptability of the finished product. Prudent producers of such products should be certain that floors, drains, cleaning aids, wash areas, and food contact surfaces, as well as eggbreaking and egg-separating, pasteurization, and packaging equipment are thoroughly cleaned and sanitized on a regular basis to eliminate potential problems involving listeriae, salmonellae, and high levels of spoilage organisms. Special care should be taken to control listeriae in the postpasteurization environment and on equipment. Effective cleaning and sanitation of these environments should include the complete breakdown of positive displacement pumps, including the back plate, for effective cleaning and sanitation. Gaskets should be checked for leaks and kept on a preventative maintenance program.

SEAFOOD PROCESSING FACILITIES FDA officials began testing a wide range of domestic and imported fish and seafood products for listeriae and other organisms of public health significance after L. monocytogenes was recovered from fresh frozen crabmeat in May 1987. The results from these analyses led to numerous Class I recalls of Listeria-contaminated products, and government officials also released additional findings that were obtained during visits to various seafood processing facilities. The literature has well established the link between environmental contamination and crosscontamination of seafood, especially RTE seafood products during processing [54,64,118]. Between January and April 1988, inspectors from the Oregon Department of Agriculture analyzed 480 environmental swab samples from 17 seafood processing facilities located throughout Oregon [12,66,157]. Only 4% of all samples were positive for Listeria spp.; however, 10 of 17 (60%) factories yielded evidence of Listeria contamination in the work environment. Specific locations from which listeriae were isolated included the following: (1) a fiberglass tote in a walk-in cooler, (2) a drain in a walk-in cooler, (3) a phosphate recirculation system on a shrimp processing line, (4) an ice tote in a cold room, (5) a floor gutter near a shrimp peeler, (6) a wooden door frame in a crab freezing room, (7) tires on heavy machinery, (8) a cold saturated brine solution, (9) the framework of a fish dumpster, (10) floor and wall junctions in a cooler, and (11) seagull droppings on an office manager’s window. Additional environmental niches within processing plants that are strongly suspected of harboring listeriae include walls, floors, ceilings, condensate, pooled water, and processing wastes. Hence, this information, along with other observations that virtually all Listeria cells recovered from processed seafoods have been healthy rather than thermally or otherwise injured, suggest that the presence of listeriae in processed seafood is almost exclusively the result of recontamination after processing. L. monocytogenes and other Listeria species have been isolated from different types of raw and

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processed seafood. Several studies [51,58] have been done to detect potential sources of this pathogen in seafood processing plants so product contamination could be minimized. Eklund et al. [58] surveyed cold-smoked salmon processing plants to determine occurrence and sources of L. monocytogenes. These authors observed that cleaning and sanitizing procedures adequately eliminated L. monocytogenes from the processing line and equipment, but recontamination occurred soon after processing was resumed. They also identified the external surfaces of fresh and frozen fish as the primary source of L. monocytogenes in cold-smoked fish processing plants (Table 17.15). During filleting, rinsing, and brining operations, the bacterium is transferred to the exposed flesh and, as the product moves through the processing steps, equipment, personnel, and other surfaces which the product contacts become contaminated. These then serve as secondary sources of contamination. Destro et al. traced the transmission of L. monocytogenes in a shrimp processing plant [51], using two molecular typing methods: random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). The breakdown of 115 L. monocytogenes isolates examined follows: 25 were recovered from the plant environment (floors, walls, and pipes); 15 were from equipment and utensils, including tables, plastic boxes, knives, and trays; 9 were found in water used in shrimp processing; 7 were isolated from the hands of employees; and 59 were from the shrimp. The results from this study indicated that environmental strains all fell into composite groupings unique to the environment, whereas strains from both water and utensils shared another composite profile group. The L. monocytogenes isolates from fresh shrimp belonging to one profile group were found in different areas of the processing line. This same profile group was also present on the hands of employees from the processing and packaging areas of the plant.

TABLE 17.15 Incidence of Listeria in a Cold-Smoked Salmon Processing Plant Area in Plant

L. monocytogenes

L. innocua

Raw Product and Processing Area Thawing water for fish (from tank) Rack from bottom of thawing tank Filleting table Rinse water Skins from raw salmon Slime from raw salmon Drip from raw salmon Trimming from raw salmon

11/59 2/2 1/9 6/6 3/7 4/4 8/9 15/26

15/59 0/2 0/9 3/6 4/7 3/4 3/9 14/26

Finished Product and Processing Area Salmon sides from smokehouse Trim table Trim machine Skins from skinning machine Fillet midline trimmings Product trimmings from slicers

9/9 1/8 6/15 29/30 8/20 17/35

7/9 0/8 2/15 8/30 0/20 20/35

Source: Adapted from Eklund, M.E., F.T. Poysky, R.N. Paranjpye, L.C. Lashbrook, M.E. Peterson, and G.A. Pelroy. 1995. Incidence and sources of Listeria monocytogenes in cold-smoked fishery products and processing plants. J. Food Prot. 58: 502–508.

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This study showed that there were many different sources of L. monocytogenes in shrimp processing plants. Lappi et al. (2004) monitored two RTE crawfish processing plants over a period of 2 years to determine the efficacy of Listeria control strategies including targeted sanitation procedures and worker education 1 [102]. Environmental, as well as raw material and finished product samples, were taken weekly during the months of April to June and tested for L. monocytogenes in addition to Listeria spp. Before implementation of the control strategies, Listeria spp. prevalence stood at 5.2% in the processing environment, 29.5% in raw crawfish, and 0% in the finished product. However, 1 year after implementing the control strategies, Listeria spp. prevalence jumped to 10.8% in the environment, 57.5% in raw crawfish, and 1% in finished product. L. monocytogenes prevalence increased from 3.9 to 10.9% in the raw materials and remained static in the environment. Thimothe et al. (2004) conducted a longitudinal study using processing plants in the smokedfish industry as a model to examine the sources and dissemination of L. monocytogenes in RTE food processing plant environments [148]. During regular monthly sampling (February–December of one calendar year) contamination rates of environmental sources were assessed along with those for raw and finished seafood products. The sampling methodology was coupled with molecular subtyping to determine the extent of L. monocytogenes transmission in the plants. The researchers chose to examine two plants on the east coast and two on the west coast, each producing a variety of products including hot- or cold-smoked fish and shellfish, as well as smoked-fish salads and retorted (shelf stable) smoked products in some instances. The authors stated that although the varying prevalence of L. monocytogenes in processing plants is well established [58,64,81], there is only limited information available on L. monocytogenes transmission from raw materials and environmental sources to finished products. Twelve to fourteen environmental samples were taken on each sampling date and included drains in the raw, in-process, and finished product area, floors, cart wheels, underneath tables, knives, slicing machines, scales, skinning machines, employee hands, gloves, and aprons, as well as door knobs. Flat surface areas were swabbed in 24 × 24 in. areas, and all accessible surfaces of a given drain were swabbed. All plants were relatively new and had been remodeled in the past 4 to 10 years, except for plant 1, which was located in a building almost 100 years old. Results compiled from all plants confirmed that 71 of 553 environmental samples (12.8%) tested positive for L. monocytogenes in all four plants, with 151 of the 553 samples (27.3%) testing positive for all presumptive Listeria spp. (Table 17.16). The prevalence of L. monocytogenes and Listeria spp. differed significantly among the four plants (P < 0.0001). The environmental samples also displayed a statistically significant positive association (P = 0.0005) between L. monocytogenes and Listeria spp. prevalence. Drain samples were the most common source of L. monocytogenes and Listeria spp., although prevalence varied from plant to plant (Table 17.16). L. monocytogenes was isolated from drain samples in both the raw and finished areas of plants 1, 2, and 3, but not in plant 4 (Table 17.16 and Table 17.17). The second highest source of isolation was from non-food-contact surfaces (e.g., floors, floor mats, wheels of rolling carts, trash containers, and the undersides of tables). L. monocytogenes was found in these areas in plants 1 and 3, but not in plants 2 and 4, although plant 2 had a 38.9% prevalence of Listeria spp. in these areas. Other common sources of contamination included wheels of rolling carts, floors, and stress mats. Seven of 10 swabs from wheels of rolling carts in plant 1 were positive for L. monocytogenes and the stress mats in the finished product handling area of plant 3 were positive 5 of 11 times. Two of the four plants also had waste-material containers that tested positive for L. monocytogenes. Employee contact surfaces (e.g., gloves, aprons, door handles, and switches) tested positive for L. monocytogenes and Listeria spp. at 10 and 16%, respectively. Plant 2 had the highest prevalence (21.2%) of L. monocytogenes–positive employee samples, although six of seven samples were from a raw material employee’s apron. Bacterial prevalence on food contact surfaces was relatively low,

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TABLE 17.16 Prevalence of Listeriae in Four Smoked-Fish Processing Plants; Percentage of Positive Samples (Total Samples Taken) Product Sampled/ Sampling Site Unprocessed fish L. monocytogenes Listeria spp. (including L. monocytogenes) Finished product L. monocytogenes Listeria spp. (including L. monocytogenes) Environmental surfaces L. monocytogenes Listeria spp. (including L. monocytogenes) Food contact samples L. monocytogenes Listeria spp. (including L. monocytogenes) Employee contact samples L. monocytogenes Listeria spp. (including L. monocytogenes) Non-food-contact samples L. monocytogenes Listeria spp. (including L. monocytogenes) Floor drains L. monocytogenes Listeria spp. (including L. monocytogenes)

Plant 1

Plant 2

Plant 3

Plant 4

Plants Total

10.0 (60) 18.3 (60)

3.0 (66) 9.0 (66)

0 (66) 21.2 (66)

2.4 (42) 19.0 (42)

3.8 (234) 16.7 (234)

3.3 (60) 11.7 (60)

0 (66) 0 (66)

1.5 (65) 15.4 (65)

0 (42) 9.5 (42)

1.3 (233) 9.0 (233)

0 (126) 10.3 (126)

12.8 (553) 27.3 (553)

29.8 (131) 42.7 (131)

7.0 (143) 31.5 (143)

14.4 (153) 24.2 (153)

6.1 (33) 12.1 (33)

0 (33) 27.3 (33)

12.5 (32) 21.9 (32)

0 (27) 0 (27)

4.8 (125) 16.0 (125)

16.1 (31) 19.4 (31)

21.2 (33) 27.3 (33)

4.5 (44) 13.6 (44)

0 (27) 3.7 (27)

10.4 (135) 16.3 (135)

37.8 (37) 59.5 (37)

0 (36) 38.9 (36)

13.6 (44) 22.7 (44)

0 (45) 6.7 (45)

12.3 (162) 30.2 (162)

60.0 (30) 80.0 (30)

7.3 (41) 31.7 (41)

30.3 (33) 42.4 (33)

0 (27) 33.3 (27)

23.7 (131) 45.8 (131)

Source: Adapted from Thimothe, J., K.K. Nightingale, K. Gall, V.N. Scott, and M. Wiedmann. Tracking of Listeria monocytogenes in smoked fish processing plants. J. Food Prot. 67: 328–341.

with plants 2 and 4 testing negative for L. monocytogenes, and plant 4 also testing negative for Listeria spp. One finished product meat-and-bone separator in plant 1, used to make smoked-fish salad, was positive for L. monocytogenes. Other finished product food contact areas that were positive for L. monocytogenes included two samples from a slicing machine and two samples from a scale in plant 3. The authors did regression analysis and determined that there was a correlation between L. monocytogenes prevalence in the environment and raw materials (P = 0.027). The correlation was even stronger (P < 0.0001) between L. monocytogenes prevalence in environmental and finished product samples, although no positive correlation existed between Listeria spp. prevalence in the environmental samples vs. finished product samples. A positive statistical association was found

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TABLE 17.17 Listeria monocytogenes Prevalence Among Environmental Samples in Four Smoked-Fish Processing Plants (Positive Samples/Total Samples Taken) Plant 1 Food contact samples

Meat/bone separator (1/10); hand utensil (1/1)

Employee contact samples

Aprons, raw area (4/10); gloves, finished area (1/10)

Non-foodcontact samples

Floors (4/10); cart wheels (7/10); tubs (3/4)

Drain samples

Cold-smoking ovens (5/10); raw area (8/10); finished area (e.g., slicing) (5/10)

Plant 2

0 Aprons, raw area (6/11); door handle, finished area (1/11)

Raw area (2/11); finished area trench (1/11)

Plant 3

Plant 4

Slicer faceplate (2/11); scale (2/11)

0

Aprons, raw area (1/11); gloves, finished area (1/11)

0

Floor mats (5/11); waste totes (1/11)

0

Finished area (e.g., slicing) (5/11); raw area (e.g., racking) (2/11); raw area (e.g., skinner) (3/11)

0

Source: Adapted from Thimothe, J., K.K. Nightingale, K. Gall, V.N. Scott, and M. Wiedmann. 2004. Tracking of Listeria monocytogenes in smoked fish processing plants. J. Food Prot. 67: 328–341.

between Listeria spp. prevalence in the environment and L. monocytogenes prevalence in the environment (P = 0.0005) and in the finished product (P = 0.031). Molecular examination of the 71 L. monocytogenes isolates from environmental samples elucidated 14 distinct ribotypes. Only two of these ribotypes were found in the environment of more than one plant. Eight different ribotypes were uncovered from environmental samples of plant 1. One of these ribotypes was isolated at 8 of 10 sample-collection times representing 44% of all environmental samples recovered. Plant 2 yielded four different ribotypes from environmental samples, with one ribotype being isolated seven times and the three other ribotypes being isolated only one time each. One ribotype isolated from raw fish later the same day was isolated from the apron of a raw material employee. Four ribotypes were also isolated from environmental samples in plant 3 with one ribotype being isolated 18 times and the other three ribotypes being isolated only 1 or 2 times. The authors concluded that the variance of L. monocytogenes prevalence in environmental samples (0–30%) is consistent with previous studies that have reported L. monocytogenes prevalences ranging from 5 to 30%. Each of three plants had at least one predominant L. monocytogenes strain isolated throughout the study. The authors concluded that it is difficult, however, to differentiate between a persistent subtype in a processing plant and the persistent reintroduction of a specific subtype, since L. monocytogenes can enter the plant through numerous routes, including equipment, employees, and raw material [150]. Three persistent strains in plants 1, 2, and 3 were never uncovered in the raw product. Two ribotypes found on raw materials were not isolated from environmental samples in the same plant. Plant 1 harbored a persistent strain (determined via ribotyping) that was isolated

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throughout the plant and on the wheels of carts and conveyers on 4 of 10 sampling dates. This plant also had the most lax policies to prevent movement of raw materials, equipment, and employees between various areas of the plant. One persistent strain in plant 3 represented 82% of all L. monocytogenes–positive environmental samples. The authors concluded that their data support the hypothesis that L. monocytogenes on raw material represents a separate population from persistent plant subtypes and that L. monocytogenes on raw material does not always become established in the plant environment. They left open the possibility that some of the persistent subtypes may have been introduced into the environment via raw materials before the study or were being introduced to the plant concurrent with the study through other sources (e.g., employees, equipment). Thimothe et al. (2002) examined the prevalence of L. monocytogenes and Listeria spp. in two crawfish processing plants in southwest Louisiana. Each plant was 2 to 3 decades old and processed between 60,000 and 100,000 lb of fresh and frozen crawfish meat per year. Seasonal production is from January to July and the authors chose to take samples between April and June. A total of 130 samples were taken from plant I on five occasions, and on eight occasions a total of 207 samples were collected from plant H. Samples including both crawfish and the environment remained consistent throughout the study. Samples were taken between the middle and end of the processing day. The sample pool of 337 samples from both plants yielded 31 positive (9.2%) for Listeria spp. whereas the incidence of Listeria spp. in whole crawfish stood at 29.5% (23 of 78 positive). Four Listeria spp. (L. innocua, L. welshimeri, L. grayi, and L. monocytogenes) were identified in the crawfish samples. Although 18% of crawfish sampled were positive for L. innocua, only 3.8% tested positive for L. monocytogenes. In stark contrast, only 8 of 181 (4.4%) environmental samples tested positive for L. monocytogenes and Listeria spp. The drains tested positive for Listeria spp. in 6 of 39 instances (15.4%), and gloves and aprons tested positive in 2 of 39 instances (5.1%), with 38 food contact surfaces consistently testing negative (Table 17.18). Drains in the peeling room and boiling room tested positive one time each for L. innocua and L. monocytogenes, respectively. The L. monocytogenes isolate in the boiling room was subsequently identified as ribotype DUP-1045B, the same ribotype previously reported as a contaminant in smoked-fish processing plants [119]. A drain in the peeling room of plant H tested positive for L. innocua on three occasions. These results corroborate other reports of recovering Listeria spp. and L. monocytogenes most frequently from food processing plant drains [118,131]. All samples in the crawfish picking room were negative. L. innocua was

TABLE 17.18 Listeriae Prevalence in Two Crawfish Processing Plants; Percentage of Listeriae (Listeriae-Positive Samples/Total Samples Taken) Plant H Product samples (Total) Raw product samples Finished products samples

13.5 (13/96) 27 (13/48) 0 (0/48)

Environmental samples (Total) Food contact samples Employee contact samples Non-food-contact samples Drain samples

4 0 4.2 0 12.5

(4/111) (0/23) (1/24) (0/40) (3/24)

Plant I 16.7 (10/60) 33.3 (10.30) 0 (0/30) 6 0 6.7 0 20

(4/70) (0/15) (1/15) (0/25) (3/15)

Plants Total 14.7 (23/156) 29.5 (23/78) 0 (0/78) 4.4 0 5.1 0 15.4

(8/181) (0/38) (2/39) (0/65) (6/39)

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isolated once in each plant on employee contact surfaces in the boiling rooms. These were pooled composite swab samples that included swabs of aprons and gloves of employees in contact with raw material. All finished products also tested negative for listeriae. This study stands in stark contrast to a previous report suggesting worker or environmental cross-contamination in 17% of frozen partially cooked crawfish tail meat with 3% of whole boiled crawfish testing positive for L. monocytogenes [108].

VEGETABLE

AND

FRUIT PROCESSING FACILITIES

Consumption of coleslaw prepared from contaminated cabbage was directly linked to the first documented outbreak of foodborne listeriosis in 1981; however, the incidence of listeriae in raw vegetables and fruits and, particularly, the prevalence of these organisms in work environments of vegetable and fruit processing facilities have received relatively little attention. Nevertheless, the long-recognized association of listeriae with soil and the discovery of Listeria spp., including L. monocytogenes, on raw vegetables suggest that these organisms are almost certainly present in vegetable and fruit processing facilities. The presence of Listeria spp. in vegetable processing facilities has also been observed by the first author. Soil and production-area samples from one potato processing factory in The Netherlands have yielded L. monocytogenes, L. innocua, and L. seeligeri (see Table 17.19 and Table 17.20).

TABLE 17.19 Incidence of L. innocua in Working Environments of 15 Food Processing Facilities in The Netherlands Number of Positive Samples/Sample Analyzed (%)

Environmental sample Drains Condensed/stagnant water Floors Residues Processing equipment Miscellaneous Total

Fluid Dairy Factory

Ice Cream Factory

Italian-Style Cheese Factory

Frozen Food Factory

Potato Processing Factory

na = 5 2/4 (50.0) 2/5 (40.0)

n=1 4/4 (100.0) 4/8 (50.0)

n=5 19/42 (45.2) 7/20 (35.0)

n=3 2/3 (66.7) NA

n=1 7/13 (53.8) 7/10 (70.0)

0/2 NAb 0/10 0/13 4/34 (11.8)

8/16 (50.0) 4/12 (33.3) 7/20 (35.0) 2/8d (25.0) 29/68 (42.6)

14/44 (31.8) 16/71 (22.5) 6/68 (8.8) 12/103e (11.7) 74/348 (21.3)

2/4 (50.0) NA 1/6 (16.7) 15/78 (19.2) 20/91 (22.0)

9/13 (69.2) 5/15c (33.3) NA 4/17f (23.5) 32/68 (47.1)

Note: NA = not applicable. a

Number of factories analyzed. Not analyzed. cIncludes one sample positive for L. seeligeri. dConveyor belt (two of two positive). eRaw milk (two of two positive), untreated effluent. fPotato delivery soil (two of three positive), sand from effluent treatment (two of two positive). b

Source: Adapted from Cox, L.J., T. Kieiss, J.L. Cordier, C. Cordellana, P. Konkel, C. Pedrazzini, R. Beumer, and A. Siebenga. 1989. Listeria spp. in food processing, non-food and domestic environments. Food Microbiol. 6: 49–61.

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TABLE 17.20 Incidence of L. monocytogenes in Working Environments of 15 Food Processing Facilities in The Netherlands Number of Positive Samples/Sample Analyzed (%) Environmental Sample

Fluid Dairy Factory (na = 5)

Ice Cream Factory (n = 1)

Italian-Style Cheese Factory (n = 5)

Frozen Food Factory (n = 3)

Potato Processing Factory (n = 1)

Drains Condensed/ stagnant water Floors Residues Processing equipment Miscellaneous Total

0/4 0/5

0/4 0/8

2/20 (4.8) 0/20

1/3 (33.3) NA

0/13 0/10

0/2 NAb 0/10

1/16 (6.3) 0/12 6/20 (30.0)

2/44 (4.5) 7/71 (9.9) 2/68 (2.9)

0/4 NA 0/6

1/13 (7.7) 0/15 NA

1/13 (7.7) 1/34 (2.9)

1/8c (12.5) 8/68 (11.8)

0/103 13/348 (3.7)

2/78 (2.6) 3/91 (3.3)

1/17d (5.9) 2/67 (3.0)

Note: NA = not applicable. a

Number of factories analyzed. Not analyzed. cSponge (one of one positive). dPotato delivery soil (one of three positive). Source: Adapted from Cox, L.J., T. Kieiss, J.L. Cordier, C. Cordellana, P. Konkel, C. Pedrazzini, R. Beumer, and A. Siebenga. 1989. Listeria spp. in food processing, non-food and domestic environments. Food Microbiol. 6: 49–61. b

INCIDENCE OF LISTERIA SPP. IN WESTERN EUROPEAN AND AUSTRALIAN FOOD PROCESSING FACILITIES Information concerning the extent of Listeria contamination in European food processing facilities is limited. However, existing information indicates that European and American food companies are experiencing similar problems regarding listeriae in the manufacturing environment. Furthermore, because similar food production, processing, and packaging methods and cleaning and sanitation practices are employed in both Western Europe and North America, much of the following information regarding the incidence of Listeria contamination within Western European foodprocessing facilities is probably applicable to manufacturers of similar products in the United States and Canada. The following are selected examples of Listeria recovery in other processing facilities in other parts of the world.

WESTERN EUROPE Dairy Production Facilities In 1988, Cox [44,45] presented some preliminary data concerning prevalence of Listeria spp. within one blue and six soft cheese factories in Western Europe, as well as in one ice cream factory and eight chocolate factories. As expected, listeriae generally occupied similar environmental niches in both soft (Table 17.21) and blue cheese factories; however, Listeria contamination was far more common in ripening than production areas of the one blue cheese factory examined (Table 17.22). Ripening practices for blue cheese, including maintenance of a relatively moist environment, appear

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TABLE 17.21 Prevalence of L. monocytogenes and Nonpathogenic Listeria spp. within the Working Environment of German Factories Producing Soft Smear-Ripened Cheese

Environmental Sample Smear liquid and smearing machine Other machinery Ripening boards Condensate and cooling water Floor drains

Number of Samples Analyzed

Number (%) of Positive Samples L. monocytogenes

210 251 69 36 74

2 12 0 1 3

(0.9) (4.8) (2.8) (4.1)

Nonpathogenic Listeria spp. 33 31 2 2 29

(15.7) (12.3) (2.9) (5.6) (39.2)

Total 35 43 2 3 32

(16.7) (17.1) (2.9) (8.3) (43.2)

Source: Adapted from Terplan, G. 1988. Listeria in the dairy industry—situation and problems in the Federal Republic of Germany. Foodborne Listeriosis—Proceedings of a Symposium, Wiesbaden, West Germany, September 7, pp. 52–70.

to be the likely reason for higher rates of Listeria contamination in ripening than production areas. Some environmental niches in this blue cheese factory were not sampled; however, results for soft cheese factories point to walls, air coolers, stagnant water, and condensate as possible problem areas in blue cheese factories as well.

TABLE 17.22 Incidence of Listeria spp. in Several Western European Blue and Soft Cheese Factories Percentage of Samples Yielding Listeria spp. Environmental Sample Drains Floors Residues Equipment Walls Air coolers Stagnant water Condensate Brine Miscellaneous

Soft Cheese Factory

Blue Cheese Factory

22 20 NAc 0 33 22 14 5 NA 19

71a/80b 5/83 23/46 0/NA NA/NA NA/NA NA/NA NA/NA 0/NA NA/NA

Note: NA = not applicable. a

Production areas. Ripening areas. cNot analyzed. b

Source: Adapted from Cox, L.J. 1988. Listeria monocytogenes—a European viewpoint. General Assembly of IOCCC, Hershey, PA, April 28–30; Cox, L.J. 1988. Prevention of foodborne listeriosis—the role of the food processing industry. WHO Informal Working Group on Foodborne Listeriosis, Geneva, Switzerland, February 15–19.

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TABLE 17.23 Incidence of Listeria spp. in the Production Environment of One Western European Ice Cream Factory Environmental Sample

Percentage of Samples Yielding Listeria spp.

Listeria Populations (CFU/g or mL)

Drains Conveyors Stagnant water Floors Residues/waste products

100 75 66 63 50

≥106 102 NR 10–106 10–104

Note: NR = Not reported. Source: Adapted from Cox, L.J. 1988. Listeria monocytogenes—a European viewpoint. General Assembly of IOCCC, Hershey, PA, April 28–30; Cox, L.J. 1988. Prevention of foodborne listeriosis—the role of the food processing industry. WHO Informal Working Group on Foodborne Listeriosis, Geneva, Switzerland, February 15–19.

Samples from at least half of the drains, conveyors, stagnant water, floors, and residue and waste products from one Western European ice cream factory contained populations of Listeria spp. ranging from 10 to >106 CFU/g or mL (Table 17.23). This factory manufactured all of its ice cream from commercially produced reconstituted powdered milk (a product from which Listeria has not yet been isolated) rather than fresh milk. These findings strongly suggest that Listeria contamination in dairy processing facilities is not always linked to incoming raw milk or milk haulers. Meat, Poultry, and Seafood Production Facilities Gudbjornsdottir et al. [77] reported results of 36 surveys taken for presence of L. monocytogenes in 13 meat processing plants (two poultry plants, five seafood plants, and six meat plants) in the Faroe Islands, Finland, Iceland, Norway, and Sweden. A combined sum of 2,522 samples were taken from the environment, personnel, raw material, and raw or RTE products. Environmental and personnel samples were taken before process start-up and then 2 h after start-up. Cleaning resulted in 11.5 and 8.3% of 661 samples positive for Listeria spp. and L. monocytogenes, respectively. However, the incidence increased to 26.3% for Listeria spp. and 14.9% for L. monocytogenes 2 h after start-up. The incidence of L. monocytogenes in the plants ranged from 0 to 34.6% after cleaning. Drains and floors in 11 of 13 plants had detectable levels of L. monocytogenes. L. monocytogenes was not detected in any air samples. The incidence of L. monocytogenes in personnel samples was roughly the same after cleaning (7.6%) and during processing (6.3%). Three of five seafood plants and five of six meat plants had no L. monocytogenes detected on personnel. This is in contrast to 42.3% of personnel samples in poultry plants testing positive for Listeria spp. One positive personnel sample of note was from a quality-assurance individual who moved between various areas of the plant. Chocolate Production Facilities A 1988 report by Cox [44] indicated that 8 of 32 (25%) and 10 of 59 (17%) samples obtained from damp, wet, and dry areas of eight Western European chocolate factories were positive for Listeria spp. Growth of listeriae in chocolate is very unlikely given its low water activity; however,

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contamination of the finished product during packaging is clearly possible. The relatively low risk of producing Listeria-contaminated chocolate can be further reduced by development of adequate cleaning and sanitation programs and by maintaining production and packaging areas as dry as possible.

ENGLAND

AND THE

UNITED KINGDOM

English Poultry Processing Facilities In one Western European survey, Hudson and Mead [82] determined the incidence of Listeria spp. at 10 different sites within one large English poultry processing facility. According to these authors, scald water, feathers, and chill water, as well as swab samples from defeathering machines and conveyors leading to the chiller were free of listeriae; however, L. monocytogenes was routinely isolated from automatic carcass openers and was also present in samples from evisceration-line drains, neck-skin trimmers, and conveyors on which carcasses travel to the packing area (Table 17.24). Only one to three samples from each site were analyzed in three successive visits; however, the areas from which L. monocytogenes was recovered in this poultry processing facility were generally similar to those observed by Genigeorgis et al. [73,74] for chicken and turkey slaughterhouses in California (see Table 17.13). English Chocolate Production Facilities Listeria spp., including L. monocytogenes, have also been detected in commercially produced chocolate that was marketed in England [75]. United Kingdom: Miscellaneous Production Facilities Evans et al. (2004) examined refrigeration systems of chilled rooms in 15 food processing plants in the United Kingdom [62]. The plants processed a variety of foods including vegetarian meals, ready-prepared meals, pies, cooked meats, ready-prepared Chinese meals, raw and cooked poultry, raw meat, dairy products, mechanically recovered meat, salads, and ready-prepared pasta meals. The study examined 336 locations on chilled-room walls, evaporators, and drip trays for Listeria spp. The investigators determined that the chilled rooms were rarely cleaned and temperatures in chilled rooms ranged from −1 to +16.9°C. A total of 891 sites examined for aerobic plate count

TABLE 17.24 Incidence of Listeria spp. in the Working Environment of One Poultry Processing Facility in England Type of Sample Transport crates Automatic carcass opener Evisceration line drain Neck skin trimmer Conveyor to packing area

Number of Samples Analyzed 9 3 3 3 3

Number (%) of Positive Samples L. monocytogenes

L. innocua

0 3 (100) 2 (66.7) 2 (66.7) 1 (33.3)

1 (11.1) 0 0 0 0

Source: Adapted from Hudson, W.R. and G.C. Mead. 1989. Listeria contamination at a poultry processing plant. Lett. Appl. Microbiol. 9: 211–214.

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resulted in 25% of samples with bacterial counts greater than 105 CFU/cm2. However, all 336 samples were negative for Listeria spp.

FRANCE Cheese Production Facilities Large quantities of French Brie cheese were contaminated with L. monocytogenes in 1986, as noted in a previous chapter. Therefore, emphasis was first placed on determining prevalence of listeriae in cheese factories. Results of one small-scale environmental survey of French cheese factories [27] identified L. monocytogenes in one floor sample, and L. innocua was recovered from boards, wheels, and equipment (7 of 22 samples), brushes (1 of 6 samples), and filtered air (1 of 19 samples). A French cheese factory was sampled for Listeria contamination from 1988 to 1990; of the 344 samples collected and analyzed for Listeria, 61 isolates (44 L. monocytogenes and 17 L. innocua) were recovered from four varieties of cheese, cheese brines, processing equipment, and the plant environment [90]. All L. monocytogenes–positive samples were from the ripening and rind-washing stages and not before, suggesting that the cheese contamination occurred at these points in the manufacturing process [90]. Terplan [146] surveyed German factories producing soft smear-ripened cheese and isolated nonpathogenic Listeria spp. from smear liquid, various pieces of machinery (especially smearing machines), and floor drains, with L. monocytogenes being detected far less frequently than other listeriae (see Table 17.21). Hence, opportunity exists for contamination of both mold and bacterial surface-ripened cheese during the later stages of manufacture and storage. Poultry and Pork Production Facilities Chasseignaux et al. [42] determined the incidence of L. monocytogenes in the processing environments and finished products from a poultry (plant A) and pork production plant (plant B) in France over a period of 1 year and 4 months, respectively. A total of 232 samples from plant A and 116 samples from plant B were taken from the environment and equipment. Finished products in plants A and B, respectively, tested positive 40 and 35.7% of the time, whereas environmental and equipment samples were only positive 18.3 and 16.4% of the time. In another study, Chasseignaux et al. [43] reported three pork and two poultry processing plants in France for L. monocytogenes. The pathogen was found in 23.7% of 497 samples (263 samples taken during production and 234 taken after cleanup and sanitation). From a total of 100 samples taken during production, 38% were positive for L. monocytogenes. The authors reported this level as being higher than the 26% reported by Lawrence et al. (1994) in poultry production facilities and lower than the 55% in a pork processing plant reported by Salvat et al. [133].

SMOKED-SALMON PROCESSING FACILITIES Dauphin et al. (2001) reported the incidence and PFGE characterization of L. monocytogenes in three cold-smoked salmon processing plants in France [48]. Samples were taken from the products in all three plants but only from the processing environment in two of three plants. A total of 44 environmental samples taken from plant 1 came from food contact areas, transport boxes, equipment, floors, and worker’s hands. A combined total of 59 of 141 samples (42%) tested positive for L. monocytogenes from all three plants. However, 64 and 84% of the salmon and environmental samples, respectively, tested positive for the bacterium in plant 1. Swabs from workers’ hands in plant 2 tested positive for L. monocytogenes on 2 of 2 occasions. Following PFGE typing, one pulsotype was shown to predominate and persist in plant 1. The results indicated that the finished product was most likely contaminated from persistent environmental strains rather than from the raw product.

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FINLAND Meat Processing Facilities Lunden et al. (2004) assessed the incidence of L. monocytogenes in three RTE pork and beef processing plants and one poultry processing plant in Finland [106]. Overall, 596 L. monocytogenes isolates were recovered from the processing plants over several years. Samples were taken from walls, floors, drains, equipment, products, and raw materials. Plant A yielded L. monocytogenes isolates (18 total) from the environment (2), equipment (9), and product (7), whereas plant B yielded isolates (92 total) from the environment (24), equipment (49), product (18), and raw material (1). In plant C L. monocytogenes isolates (307 total) were recovered from the processing environment (43), equipment (199), product (63), and raw material (2). Plant D (the poultry processing facility) yielded L. monocytogenes isolates (179 total) from the environment (38), equipment (104), and product (37). Contaminated environmental surfaces included slicing machines, spiral freezers, packing machines, conveyers, dicing machines, peeling machines, weigh scales, packing machines, and various components of said equipment (e.g., control panel, motor, lubricant, chamber, funnel, etc.). These processing machines were persistently contaminated in all four plants. Multiple isolates recovered from finished products had identical PFGE patterns to those from the processing machines. The 596 isolates uncovered were subdivided into 47 groupings based on PFGE typing. Persistent L. monocytogenes strains were classified as those that were found five or more times over a period of ≥3 months. Nonpersistent strains were those that were found fewer than five times, or only within a limited time frame (<3 months). Overall, 19 of the 47 PFGE groups were determined to be persistent, with the remaining 28 being nonpersistent. Seafood Processing Facilities Autio et al. [25] were unable to prove the spread of L. monocytogenes through heavily contaminated air in a food processing plant in Finland. Furthermore, neither environmental nor smoked-salmon products tested positive for L. monocytogenes for 5 months after comprehensive disinfection of a processing plant with hot water, hot air, and hot steam.

ITALY Meat Processing Facilities Peccio et al. (2003) examined the occurrence and characteristics of L. monocytogenes in two meat producing plants in northeast Italy [123]. Over a 20-month period, 72 and 68 samples, respectively, were taken from a cattle slaughterhouse and a swine meat processing plant. All of the 46 environmental samples taken from the bovine slaughterhouse were L. monocytogenes negative except for three samples (6.5%), all from knives. However, in the same plant, 4 out of 26 raw product samples (15.4%) were positive for L. monocytogenes. Other environmental samples that were all negative for L. monocytogenes included swabs taken from a refrigerated room, tables, saws, and floor drains, as well as other unspecified areas. Results from the pork processing plant were similar, with 2 of 51 environmental samples (3.9%) positive for L. monocytogenes, including one kneader and one mincer sample. However, the pathogen was recovered from 5 of 7 (29.4%) samples of raw pork products. L. monocytogenes was not recovered from tables, a meat stuffer, or other environmental samples. All seven L. monocytogenes–positive samples from the beef processing facility were identical when they were characterized via PFGE. However, in the pork processing plant, six different PFGE profiles were found among the seven isolates.

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SPAIN Vegetable Processing Facility Aguado et al. [2] reported the incidence of L. monocytogenes in a vegetable processing facility during a 23-month study in Spain. The 906 sampler taken included environmental and RTE vegetable samples. Listeriae incidence in frozen vegetables was 1.2, 8.5, 0.2, and 0.1% for L. monocytogenes, L. innocua, L. welshimeri, and L. seeligeri, respectively. Of 166 environmental samples taken, 11 of 166 (6.6%) were positive for L. innocua and 2 of 166 (1.2%) were positive for L. monocytogenes. These isolates were taken from various points along the processing chain (e.g., washing tunnels, conveyer belts, floors, and machinery).

THE NETHERLANDS Miscellaneous Production Facilities In one large European survey during the latter half of 1986, Cox et al. [46], investigated the incidence of Listeria spp. in the processing environment of 17 establishments in The Netherlands that produced fluid dairy products, ice cream, Italian-style cheese, frozen food, potato products, and dry culinary foods. A total of 608 samples were collected from drains, floors, condensed and stagnant water, residues, processing equipment, and other areas and were analyzed for listeriae using the original USDA or FDA method with or without modification. All presumptive Listeria isolates were then speciated according to results from conventional biochemical tests. Despite use of GMPs in these factories, Listeria spp. were recovered from all types of food processing facilities examined, with the exception of two that produced dry culinary products. Overall, 181 of 608 (29.8%) samples yielded Listeria spp. with L. innocua, L. monocytogenes, and L. seeligeri being identified in 87.3, 14.9, and 0.5% of all positive samples, respectively. Only five samples contained both L. monocytogenes and L. innocua; however, the actual number of such samples is probably somewhat greater, because a limited number of presumptive Listeria isolates from each sample were chosen for confirmation. L. innocua was most prevalent in establishments that produced processed potato products followed by those that produced ice cream, frozen food, Italian-style cheese, and fluid dairy products, with the organism generally being isolated from drains, floors, and condensed and stagnant water (Table 17.19). In contrast, L. monocytogenes was detected in 11.8% of all environmental samples obtained from one ice cream factory, but was found in 2.9, 3.0, 3.3, and 3.7% of similar samples from establishments that manufactured fluid dairy products, potato products, frozen food, and Italianstyle cheese, respectively (Table 17.20). Only one ice cream factory was examined in this survey; however, the results are as expected when one recalls that Cox [44,45] previously found that listeriae were widespread in another Western European ice cream factory and also were present in very large numbers, particularly in floor drains (see Table 17.23). Given such populations of listeriae in ice cream factories and the current extruding, molding, and freezing methods used to produce ice cream, particularly ice cream novelties, one can easily postulate many routes whereby listeriae may recontaminate the finished product, as has been reported in the United States. Results concerning the incidence of Listeria spp. as well as L. innocua and L. monocytogenes in various work environments of 15 food processing facilities are summarized in Table 17.25. Overall, these findings are comparable to what has been previously noted for similar food processing facilities in the United States; for example, Listeria spp. and L. innocua were most frequently recovered from drains, followed by condensed and stagnant water, floors, residues, and processing equipment. This same trend is readily apparent for all five types of food processing facilities listed in Table 17.19 with a few minor exceptions, which probably resulted from the number of samples analyzed. Thus a logical pattern emerges in which L. innocua moves from floor drains to pools of condensed and stagnant water, which then come into direct contact with floors and residues.

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TABLE 17.25 Overall Incidence of Listeria spp. in Working Environments of 15 Food Processing Facilities in The Netherlands Environmental Sample

No. of Samples Analyzed

Drains Condensed/stagnant water Floors Residues Processing equipment Miscellaneous Total a b

66 43 79 97 104 219 608

No. (%) of Positive Samplesa Listeria spp. 36 20 36 32a 20 37 181b

(54.5) (46.6) (45.6) (33.0) (19.2) (16.9) (29.8)

L. innocua 34 20 33 24 14 33 158

(51.5) (46.5) (41.8) (24.7) (13.5) (15.1) (26.0)

L. monocytogenes 3 (4.5) 0 4 (5.1) 7 (7.2) 8 (7.7) 5 (2.3) 27 (4.4)

One sample yielded L. seeligeri. Five samples yielded both L. monocytogenes and L. innocua.

Source: Adapted from Cox, L.J., T. Kieiss, J.L. Cordier, C. Cordellana, P. Konkel, C. Pedrazzini, R. Beumer, and A. Siebenga. 1989. Listeria spp. in food processing, non-food and domestic environments. Food Microbiol. 6: 49–61.

Once present in open areas of the work environment, employees can spread L. innocua by various means, to processing equipment that comes into direct contact with the product. L. monocytogenes, unlike L. innocua, was far less prevalent in all types of food processing facilities and was distributed fairly evenly within the factory environment, with incidence rates ranging between 2.3 and 7.7%. L. innocua is by definition nonpathogenic; however, as L. innocua and L. monocytogenes (and possibly other Listeria spp.) occupy similar environmental niches, it follows that detection of listeriae anywhere within the manufacturing environment should prompt immediate corrective action.

SWEDEN Dairy Production Facilities Waak et al. [156] reported the incidence of Listeria spp. in raw whole milk from farm bulk tanks, as well as from raw milk stored in a Swedish dairy. Raw whole milk samples (25 mL) were taken from 153 farms between the months of December and March and then again from August to September, minus 12 farms that were excluded from the study because of cessation of production. Raw whole milk samples (295 total) at the dairy processing plant were taken from three storage silos with respective capacities of 40,000, 40,000, and 100,000 L each. These samples were taken in the months of November to April and July. Equipment at the dairy plant was also sampled for the prevalence of Listeria spp. Two plate coolers, eight different-sized gaskets from the plate cooler and two silo pumps, and rinse water from silo sampling cocks were microbiologically examined. Weekly sampling of milk products and dairy plant drains resulted in 440 surface samples from blue mold cheese, 70 whey samples, 149 swab samples from pasteurized-product-area exposed drains, and 116 cheese-washing liquid samples. Only 1% (3 samples) of the farm milk tank samples tested positive for L. monocytogenes, with Waak et al. [156] reporting that these results are similar to findings from other countries [53]. The three samples contained L. monocytogenes at levels of <10, 60, and <10 CFU/mL, respectively. Listeria prevalence in dairy plant silos was significantly higher than on the farm because of the high probability of cross-contamination from previously positive bulk milk loads. The fact that one silo had nearly twice the prevalence of L. monocytogenes as the remaining two

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silos was attributed to either persistent contamination within the silo or a greater number of L. monocytogenes–positive shipments going into that particular silo. The incidence was also higher from January to March than in other months of the year as is now well documented. Farm milk was more frequently contaminated with L. innocua (3.4%) than L. monocytogenes (2.3%); however, just the opposite was true in sampling dairy plant silos, which had levels of 19.6% and 8.5%, respectively. The authors concluded that because silo milk was stored longer than farm milk, the lactoperoxidase system of raw milk may have been more effective against L. innocua than L. monocytogenes [125]. However, these results are in contrast to those of Canillac and Mourney [37] who found no L. monocytogenes in raw milk whereas 25% of the samples were L. innocua positive, and Harvey and Gilmour [78], who reported L. monocytogenes in 33.3% of raw milk samples from dairy silos and Listeria spp. in 54% of samples. All processed milk product and environmental samples (e.g., rinse water, surface swabs, gaskets, and floor drains) tested negative for Listeria spp. The authors concluded that the lack of Listeria detection might be the result of a rigorous in-house sanitation program, as well as the fact that an alternative non-enrichment-based Listeria method was used for processed milk products and environmental tests in contrast to traditional enrichment protocol for milk. These findings differ from previous studies that have reportedly recovered Listeria spp. in large numbers from dairy-processingplant floors, drains, presses, conveyer belts, cheese-ripening areas, and brushing machines [40,78,95,111].

SWITZERLAND Swiss officials tracing the source of contamination in the 1987 listeriosis outbreak involving consumption of Vacherin Mont d’Or soft-ripened cheese recovered the epidemic strain of L. monocytogenes from smear brine, curing brine, wastewater sinks, wooden cheese hoops, and wooden boards used in 10 different cheese factories that manufactured Listeria-contaminated cheese [36]. Additionally, nearly half of the 12 cellars used to ripen cheese contained listeriae, with the pathogen being detected on 6.8% of the wooden shelves and 19.8% of the brushes used in the ripening cellars. Although not noted in the report, one would suspect that L. monocytogenes was also present in commonly recognized environmental niches such as drains, floors, stagnant water, and various food contact surfaces within the cheese factories and ripening cellars. Thus, brushing cheese with saltwater and ripening hooped cheese on wooden shelves appear to be two important means for dissemination of listeriae within cheese factories.

AUSTRALIA Information concerning the prevalence of listeriae in food processing facilities located in other parts of the world is currently limited to a few Australian studies. Following the isolation of L. monocytogenes from ricotta cheese in 1987, the Victorian Dairy Industry Authority and the Department of Agriculture and Rural Affairs conducted a joint survey to determine the extent of Listeria contamination in the working environments of 52 Melbourne-area factories producing pasteurized milk and different types of cheese [155]. Overall, various Listeria spp. were detected in 141 of 763 (18.5%) environmental samples from 21 of 52 (40.4%) factory environments, with L. monocytogenes, L. seeligeri, and L. ivanovii being identified in 132 (93.6%), 8 (5.7%), and 1 (0.7%) of these Listeria-positive samples, respectively. More important, L. monocytogenes was present in all but one of the Listeria-positive factories. As expected from other surveys conducted in the United States and Western Europe, factory sites most frequently contaminated with listeriae once again included drains and floors in coolers, surfaces of manufacturing and packaging equipment, and conveyors. Even though strict cleaning and sanitizing programs were implemented at many of these facilities, Listeria spp. were very difficult to eliminate from the working environment, with these organisms being continuously isolated from one factory over a period of 5 months.

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TABLE 17.26 Three-Year Study in 12 Australian Dairy Processing Plants to Determine Environmental Diversity and Identify Major Environmental Niches of L. monocytogenes Factory Type Cheese Milk Ice cream Mixed produce Total

Number of Factories

Number of Samples

7 2 1 2 12

319 87 53 106 565

Listeria spp. (%) 34 51 9 22 116

(11) (59) (17) (22) (21)

L. monocytogenes (%) 26 20 3 19 68

(8) (23) (6) (18) (12)

L. innocua (%) 9 42 7 8 66

(3) (48) (13) (8) (12)

L. grayi (%)

Mixed (%)

2 (1) — — — 2 (0.4)

3 11 1 5 20

(1) (13) (20) (5) (4)

Source: Adapted from Australian Dairy Authorities’ Standards Committee. 1994. Australian Manual for Control of Listeria in the Dairy Industry, pp. 1–35.

Sutherland and Porritt [145] conducted a 3-year study in 12 Australian dairy processing facilities to assess the environmental diversity and identify the major environmental niches for L. monocytogenes. A total of 565 environmental samples were collected and tested. The overall incidence of Listeria-positive samples was 21% (Table 17.26), with approximately half of these samples (12%) positive for L. monocytogenes. Cheese, ice cream, and mixed-product plants all had similar incidences of L. monocytogenes and Listeria spp. The incidence of L. monocytogenes in mixed-product factories (18%) was comparable to the higher levels found in milk factories. Sutherland and Porritt [145] also highlighted the following four major routes by which L. monocytogenes enters a dairy processing facility: 1. Ingredients: especially raw milk 2. Inward goods: including milk crates and crate washers, vehicles (trucks, road and rail tankers), and wooden pallets 3. Environment: including air and internal air quality 4. Personnel: especially outside contractors and visitors Once L. monocytogenes is inside the processing plant, these authors [145] found numerous areas in which this organism can survive, grow, and potentially contaminate products. Conveyor systems, drains, and floors were the most common isolation sites. Other areas of concern were traffic flow, cooking units, and internal air quality. Complete elimination of listeriae from dairy processing facilities may, in some instances, be nearly impossible; however, the likelihood of producing Listeria-contaminated products can be greatly reduced by following GMPs, which include implementation of rigorous cleaning and sanitizing programs for equipment used at critical points during manufacture and packaging of these foods.

INCIDENCE OF LISTERIA IN HOUSEHOLD KITCHENS Thus far, this chapter has dealt exclusively with Listeria contamination in commercial food processing facilities; however, because of the relatively high incidence of Listeria spp. (including L. monocytogenes), salmonellae, and other foodborne pathogens in fresh beef, pork, lamb, and poultry available to the general public at butcher shops and supermarkets, safe home preparation of these foods must be reemphasized. In 1989, Cox et al. [46] isolated nine strains of listeriae from 7 of 35 (20%) household kitchens surveyed in The Netherlands. Overall, L. monocytogenes was recovered from four dishcloths and one refrigerator, with two dishcloths and two dustbins from two

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other households yielding L. innocua and L. welshimeri, respectively. Considering results from commercial food processing facilities, one might expect to recover Listeria spp. from household kitchen areas such as drains, U-tubes, and drain boards. If this is true, then garbage disposal systems could conceivably lead to problems resulting from production of aerosols. Further work is needed to clarify the public health significance of listeriae in the kitchen environment; however, you may recall from the previous chapter that L. monocytogenes was found in many refrigerated foods belonging to an Oklahoma woman who contracted listeriosis after consuming contaminated turkey frankfurters that were eventually recalled nationwide. Centers for Disease Control and Prevention (CDC) officials also isolated L. monocytogenes from 15 of 25 (60%) refrigerators that were used by apparent victims of foodborne listeriosis [21]. Hence, consumers should regularly clean and sanitize kitchen areas, sinks, and refrigerators. Such efforts can help prevent potential problems involving listeriosis and other forms of foodborne illness in the home.

INDUSTRY-SPECIFIC EQUIPMENT, PROCESSING METHODS, AND PRODUCTS The discovery of Listeria spp., including L. monocytogenes, in various fermented and unfermented dairy products, raw and RTE meats, poultry products, seafoods, and vegetables has prompted food manufacturers to renew their concern for factory hygiene and product safety. Failsafe procedures for production of Listeria-free foods largely do not yet exist; however, specific guidelines have been developed for controlling listeriae and other microbial contaminants within American dairy [4,16,35,76,100,129,144,], meat [1,3,136], poultry [18,136], and seafood [66,72,157] processing facilities, with Denmark [19], England [89,101,126], France [17], and Australia [24] also addressing the elimination of listeriae from fluid milk and cheese operations during all facets of production, distribution, and retail sale. In response to the discovery of L. monocytogenes in RTE foods and delicatessen products, European public health officials have expressed particular concern about contamination of these products during retail slicing and storage. They have also warned grocery store managers to give particular attention to storage temperatures for refrigerated foods in display cases and the potential sale of products beyond their normal code dates. Most of these guidelines stress the need to (1) decrease the possibility that raw products will contain listeriae, (2) minimize environmental contamination in food processing facilities, and (3) use processing methods that will eliminate listeriae from food. Following these proposed guidelines, which will be discussed in detail shortly, will decrease the possibility of producing foods contaminated with L. monocytogenes and other foodborne pathogens. In addition, diligent attention to cleaning and sanitation and overall GMPs will lead to lower microbial populations in processed foods, which will, in turn, increase the shelf life of the finished product. Any approach to controlling the spread of listeriae and other microorganisms in food processing facilities is complicated by the enormous variety of foods being processed today along with the variable quality of incoming raw products, design of the factory, sanitary design of the processing and packaging equipment, and processing methods. However, this subject can be simplified by first focusing on problem areas such as factory design, general factory environment, heating and air conditioning systems, traffic patterns, and personnel cleanliness that are common to all food processing facilities described earlier in this chapter. Once Listeria control measures for these problem areas are understood, attention can be given to specific processing steps which are unique to the dairy, meat, poultry, seafood, and vegetable industries. It is now appropriate to briefly examine some of the industry-specific equipment and processing methods, many of which have been cited as critical control points for the production of Listeria-free dairy, meat, poultry, seafood, and vegetable products. This information will be useful

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to enhance the effectiveness of preexisting cleaning and sanitation programs; however, the reader is reminded that food processing facilities, even though they manufacture similar products, are all unique in terms of factory design, raw product quality, product flow, handling, and processing methods. Therefore, no universally acceptable cleaning and sanitation program can be developed for the safe production of a given product.

DAIRY INDUSTRY Farm Environment Listeriae are widespread in the environment. Therefore, any quality control program should first contain a plan to minimize contamination of raw milk with Listeria and other microorganisms on the dairy farm. Along with good animal husbandry practices, including the use of only high-quality feed and silage, farm workers should also give attention to cleanliness of the milk house and milking equipment. Most important, teats and udders of all cows should be properly sanitized and dried before milking equipment is attached. Bulk tanks in which raw milk is stored also need to be properly maintained and inspected regularly. Clarifiers and Separators All raw milk should be filtered and subsequently clarified and separated by centrifugation to remove extraneous matter and somatic cells (i.e., leukocytes) before pasteurization. As L. monocytogenes is sometimes found in leukocytes, clarifiers and separators should be well isolated from the pasteurizer and all finished product areas of the factory. Sealed containers should be used to dispose of all clarifier and separator waste, both of which may contain high levels of listeriae. Special care should also be taken to clean and sanitize separators, clarifiers, and surrounding areas. Pasteurization Proper pasteurization using a vat or high-temperature short-time (HTST) pasteurizer is the only commercially practical means by which all non-spore-forming pathogens, including L. monocytogenes, can be inactivated in raw milk. Thus, it is imperative that all pasteurization equipment be designed, installed, maintained, and operated properly. Continuous-flow HTST pasteurization is used to process virtually all fluid milk and ice cream mix; however, vat (or batch) pasteurization is employed by many smaller firms, particularly those involved in cheese making, when the volume of incoming raw milk is too small to justify the use of a continuous-flow HTST system. If vat pasteurization is used, raw milk must be heated to a minimum of 62.8°C (145°F) and then maintained at that temperature for at least 30 min. In theory, vat pasteurization is a relatively simple process, with raw milk being pumped into a steam- or hotwater-jacketed vat and held for the prescribed time. However, FDA inspections conducted as part of the Dairy Initiative Program mentioned earlier have uncovered numerous problems with vat pasteurizers, including improper equipment design, the absence of proper outlet valves and airspace thermometers, and improperly operated air-space heaters. The latter problem is particularly critical, because the air-space temperature above the product in the vat must be at least 2.8°C (5°F) higher than that of the product at all times to ensure proper pasteurization. Operators of such pasteurizers should be made accountable for proper performance, as well as proper cleaning and sanitizing of the equipment. In addition, recording charts showing time and temperature relationships along with other data for each vat of product pasteurized should be kept for at least 3 months. As mentioned earlier, continuous-flow HTST pasteurization at 71.7°C (161°F) for a minimum of 15 sec is the principal method for processing raw milk. An in-depth discussion of the many intricate problems associated with HTST-pasteurization equipment is beyond the scope of this book; however, a basic knowledge of HTST pasteurization is essential to appreciate the seriousness of

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some of the recently identified problems that have been linked to faulty maintenance and operation of the equipment. Interested readers may consult the “HTST Pasteurizer Operation Manual” [8] for more detailed information on HTST pasteurization. All HTST pasteurizers consist of five basic components, as shown in Figure 17.40: (1) plate heat exchanger—a series of thin, gasketed stainless-steel plates divided into three sections (heater, regenerator, and cooler) for heating incoming raw milk and cooling outgoing pasteurized milk; (2) constant level tank—provides a constant level of raw milk to the HTST system; (3) timing pump—a positive displacement pump that establishes the holding time and temperature relationship for pasteurization; (4) holding tube—a length of pipe in which fully heated milk is held for the required holding time; and (5) flow diversion valve—a three-way valve that will allow properly pasteurized milk to enter the regenerator section of the plate heat exchanger or divert improperly pasteurized milk to the constant level tank for repasteurization. In addition to these five components, a source of steam or hot water is required to heat incoming raw milk, a safety thermal limit recorder is needed to activate the flow diversion valve in the event of improper pasteurization, and a cold milk recorder is required to record the temperature of outgoing pasteurized milk. Finally, auxiliary components that may be added to HTST units for additional processing of milk or milk products include a booster pump, homogenizer as a timing pump, stuffing pump, and flavor treatment or vacuum units. Inspections of HTST pasteurizers conducted in conjunction with the FDA Dairy Initiative Program uncovered numerous problems relating to proper installation and maintenance of these units. Problems most commonly associated with HTST pasteurization equipment have included stress cracks and pinholes in the heat-exchanger plates, leaking gaskets, improper flow diversion valves, and inadequate cleaning and sanitizing of the pasteurization unit. Positive pressure should be maintained between the product and heating medium, as well as the product and cooling medium (sweetwater), to prevent Listeria-contaminated raw milk or sweetwater from mixing with the pasteurized product in the event that some of the heat-exchanger plates contain stress cracks or pinholes. Operators should examine all pasteurization plates for defects every 6 months using the standard dye test. Sweetwater and glycol solutions should also be routinely examined for microbial contaminants, as these coolants may harbor L. monocytogenes, Yersinia enterocolitica, and Salmonella typhimurium for extended periods along with large populations of psychrotrophs [16,124]; the latter

Raw Milk Diversion Line Flow-Diversion Valve

Pasteurized Milk

Cold Past. Milk Out

Holding Tube

Cooler

Regenerator

Raw Milk In

Heater Timing Pump Homogenizer

Raw Milk Constant Level Tank

FIGURE 17.40 Schematic diagram of milk flow through an HTST pasteurizer. (Adapted from Anonymous. 1987. HTST Pasteurizer Operation Manual. Oregon Association of Milk, Food and Environmental Sanitarians, Oregon State University, Corvallis, OR.)

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are particularly detrimental to product shelf life. Operators of HTST pasteurizers must be responsible for proper operation of these units and retain accurate records and chart recordings for each lot of pasteurized product for at least 3 months. The inability of L. monocytogenes to survive the minimum allowable HTST heat treatment given to commercially available raw milk (71.7°C/15 sec) is now generally accepted; however, most fluid milk processors in the United States are pasteurizing milk at 76.7°C for 20 sec, which is well above the minimum requirement established in the Pasteurized Milk Ordinance. This more severe heat treatment markedly extends the shelf life of the finished product by inactivating larger numbers of spoilage organisms than minimal HTST pasteurization. However, the psychrotrophic nature of L. monocytogenes increases the need to prevent introduction of listeriae into the product after pasteurization. Pipeline and Cross-Connections Many large dairy processors have installed up to several miles of pipeline in the factory to handle movement of raw milk from storage tanks to the pasteurizer and pasteurized milk from the pasteurizer to various holding tanks, mixing tanks, and product areas located throughout the factory. Considering the enormous quantities of product (sometimes in excess of 1 million lb) that can be manufactured at such facilities during one production period, careful attention must be given to each stage of manufacture, because an error made during these operations could adversely affect thousands of people, as was true for the 1985 outbreak of milkborne salmonellosis in Chicago. FDA inspections have uncovered numerous violations related to pipelines, including crossconnections between raw and pasteurized milk lines and storage and holding tanks, as well as cross-connections between CIP and product lines and other potentially hazardous circuits. Many of these lines allow easy bypass of raw product around the pasteurizer, thus permitting postpasteurization contamination in the event of equipment failure or operator error. Therefore, factory managers, engineers, or other qualified people need to walk through the factory and construct an up-to-date detailed blueprint of raw and pasteurized product flow throughout the entire factory. Once the blueprint is constructed, any unwanted piping, dead ends, illegal cross-connections, or unauthorized changes made to initial installations should be promptly identified and eliminated. Most important, all pasteurized product lines need to be separated from raw and CIP lines by a physical break. In many plants, pipes are physically labeled with the type of product (raw or pasteurized) that flows through them. Blueprints must be routinely updated and reviewed for accuracy to be of continued use by “walking” the blueprints through the factory. Finally, no piping changes should ever be made without prior review by qualified authorities. Filling and Packaging Postpasteurization contamination frequently occurs during filling and packaging operations when products are exposed to difficult-to-clean surfaces on equipment, the manufacturing environment, and airborne contaminants [92]. Areas associated with product contamination have included mandrels, drip shields, bottom and top breakers, prefilling coding equipment, deflector bars, and cutting blades, as well as overhead shielding, conveyors, conveyor belts, chain rollers, supports, and lubricants. Product extruder heads are particularly prone to contamination and therefore should be sanitized frequently during filling operations. Such practices will lead to the production of safe products with markedly increased shelf lives. Aerosols provide another ready means for disseminating listeriae and other microbial contaminants throughout critical areas of food processing facilities [93], with L. monocytogenes surviving 3.42 h in experimentally produced aerosols of reconstituted skim milk [142]. Therefore, highpressure sprays should never be used in processing and packaging areas for cleaning floors or drains, because both are major sources of listeriae and other microbial contaminants and resulting aerosols can contaminate food contact surfaces of equipment. Operation of unshielded centrifugal pumps in such areas is also discouraged.

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Reclaimed and Reworked Product Salvage programs, by their nature, are high-risk operations that can put an entire company in jeopardy if not done in a sanitary manner. Potential hazards associated with such salvage operations include (1) failure to repasteurize a returned product before reuse; (2) inadvertently pumping returned but not repasteurized product through pasteurized product lines without proper cleaning and sanitizing between use; (3) accidental reuse of outdated product; (4) reuse of product returned from retail stores that may have been temperature abused, tampered with, or exposed to chemical or biological contamination; and (5) the use of product from contaminated, leaking, or otherwise damaged containers. Therefore, any product that left the possession and control of the processor or has been mishandled, inadequately protected from contamination, or exposed to temperatures of >7.2°C (45°F) should be discarded. Dairy processors should also seriously consider confining the use of reclaimed and repasteurized milk to dairy products prepared from non–Grade A milk. The Pasteurized Milk Ordinance indicates that American dairies involved in reclaiming programs must now have separate areas or rooms isolated from Grade A milk operations for receiving, handling, and storing all returned products. Outdated products and those which have left the control of the processor and are later returned to the dairy for disposal should never reenter the factory. Given the isolation of L. monocytogenes and other microbial contaminants from the external surface of cartons containing returned product, along with the proven ability of L. monocytogenes and Salmonella spp. to survive up to 14 days on the external surface of both waxed cardboard and plastic-type milk containers [143], the process of opening containers and emptying reclaimed product into vats for reprocessing will likely introduce many new unwanted microbial contaminants into the factory environment. Therefore, it is imperative that all returned products be handled similar to raw milk and be repasteurized, preferably using times and temperatures well above the required minima. After reprocessing, all equipment, including tanks, pumps, and pipelines used in the reclaiming operation should be thoroughly cleaned and sanitized. In view of the problems associated with salvage operations, each dairy processor needs to reevaluate the advantages and disadvantages involved in reclaiming products and then decide whether or not the monetary benefits gained by such practices will outweigh the potential public health and other risks. Frozen Dairy Products Few bacterial species can grow at temperatures below 0°C, but most microorganisms, including listeriae, can survive for long periods in frozen dairy products such as ice cream, ice cream novelties, and sherbet. Frozen dairy products are particularly susceptible to microbial contamination during freezing and filling operations. All barrel freezers used to make frozen dairy products should be thoroughly sanitized before use, because hand assembly of the many intricate freezer parts is likely to introduce numerous contaminants. The source of air for the barrel freezer is another likely source of contamination. Hence, in addition to maintaining positive air pressure in this area and keeping the surrounding area as clean and sanitary as possible, all air lines connected to the barrel freezer should be equipped with dryers and bacterial filters to prevent airborne contaminants from entering the product. Ingredient feeders are perhaps the greatest source of contaminants in frozen dairy products. Therefore, fruits, nuts, candy, and other ingredients that are added directly to frozen ice cream mix need to be closely monitored for coliforms, pathogens, and other microbial contaminants. Exposure of ingredients to the factory environment should also be minimized. Strict adherence to GMPs is necessary during the production of molded, extruded, or dipped ice cream novelties, as many such products have been recalled because of contamination with L. monocytogenes. Condensate in and around hardening rooms, as well as conveyor belts, appears to be a likely source for such contaminants. Finally, handling of product rerun exiting the freezer needs to be assessed at each factory. Rerun product should never be added directly back to tanks containing unfrozen mix; however, frozen rerun product can be reclaimed by blending it with fresh mix, which is then repasteurized. Any rerun that is not reclaimed should be clearly separated from reclaimable material and properly disposed.

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Fermented Dairy Products Fortunately, the incidence of Listeria contamination in yogurt, cultured cream, cultured buttermilk, and other fermented fluid milk products appears to be quite low, with very few recalls being issued for these products. The species of lactic acid bacteria used in manufacturing these products, as well as the bacteriostatic and bactericidal effects of various organic acids produced during fermentation and the resultant lowering of pH are undoubtedly responsible for the near absence of such recalls. However, because bacterial pathogens (including L. monocytogenes) and various spoilage organisms may inadvertently contaminate fermented milk products during any stage of manufacture, producers of such products need to follow GMPs and be readily aware of potential problems regarding improper cleaning and sanitizing of equipment and the processing-plant environment, as well as potential sources of postpasteurization contamination (e.g., filling and packaging areas) discussed earlier in this chapter. Production of Listeria-free cheese, particularly soft and semisoft varieties surface-ripened with mold (e.g., Brie, Camembert) or bacteria (e.g., brick, Limburger), is difficult, because environmental conditions required for proper cheese ripening also promote growth of L. monocytogenes and other unwanted organisms. Swiss officials who investigated the 1987 listeriosis outbreak involving consumption of Vacherin Mont d’Or soft-ripened cheese eventually isolated the epidemic strain of L. monocytogenes from wooden shelves and cheese hoops found in over half of the caves used to ripen the tainted cheese. Thus, the basic problem associated with soft-cheese manufacture is to prevent postprocessing contamination by eliminating L. monocytogenes from the ripening room and particularly the shelves on which such cheese must be ripened. Considering the ability of L. monocytogenes to grow very rapidly both inside and on the surface of Brie, Camembert, brick, Limburger, and other similar cheeses during ripening, manufacturers of such products should test a portion of each lot for listeriae before releasing the product for sale. In addition to these concerns, several studies have demonstrated that L. monocytogenes can survive well beyond 60 days in brick, Cheddar, and other varieties of cheese that were prepared from pasteurized milk inoculated with the pathogen. Certain cheeses, primarily hard and semihard varieties, can be manufactured from raw milk in the United States and elsewhere if the finished product is aged a minimum of 60 days at or above 1.7°C (35°F) to eliminate pathogenic microorganisms. However, since experimental evidence has indicated that this process is inadequate to free contaminated cheese from viable cells of L. monocytogenes, cheesemakers should consider preparing cheese from pasteurized milk whenever possible.

MEAT INDUSTRY Because Listeria spp., including L. monocytogenes, are virtually endemic to slaughterhouse environments, meat processors are faced with an almost impossible challenge of producing Listeria-free raw meats. Direct application of lactic or acetic acid to animal carcasses is one of the few economically feasible means by which meat processors can effectively reduce populations of listeriae and other surface contaminants, including common spoilage organisms [15,22,110,116]. Nevertheless, although adopting this procedure and following the general guidelines for controlling listeriae in food processing establishments will benefit slaughterhouse operators, it appears unlikely that rigid enforcement of even the most stringent slaughter, dressing, cleaning, and sanitizing procedures will completely eliminate L. monocytogenes from wholesale and retail cuts of raw beef, pork, and lamb. Therefore, consumers of such products need to understand the potential health hazards associated with consumption of less-than-thoroughly cooked meats and must also follow appropriate hygienic practices in the kitchen to prevent the spread of listeriae from raw meats to RTE foods. Firms producing processed meat products must assume that all incoming raw meat is potentially contaminated with listeriae, including L. monocytogenes. Because most Listeria contamination of finished product appears to result from postprocessing contamination rather than from

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the organism surviving various processing treatments, it is essential to segregate raw and finished products, as well as employees working in raw and finished-product areas of the factory. There is no “magic bullet” for Listeria control; however, the incidence of listeriae in all areas of the factory can be greatly reduced through conscientious enforcement of a stringent cleaning and sanitation program. One six-step program that has been recommended for cleaning food contact surfaces [7] includes (1) an initial dry cleanup step to remove as much product residue as possible, followed by (2) a warm water rinse (with minimum splashing) to mobilize fat and remove product, (3) cleaning with an appropriate foaming detergent, (4) warm or hot water rinse with minimum splashing, (5) disinfecting with an appropriate sanitizing agent (i.e., chlorine or quaternary ammonium compound), and finally (6) thorough drying of the cleaned and sanitized area (see also the section entitled “Control of Food Contamination Through Effective Cleaning and Sanitation” in this chapter). According to Boyle et al. [33,34], L. monocytogenes populations in inoculated samples of carcass-rinse fluid, Hobart meat-grinder-rinse fluid, and floor-drain waste water obtained from a beef and lamb processing facility increased one to four orders of magnitude during 24 h of incubation at 8 and 35°C, with the pathogen exhibiting shorter generation times in waste fluids containing 3.1 rather than <1.4% protein. The procedure just described may seem adequate; however, routine random testing for Listeria and coliforms, as well as an estimation of the general microbial load on cleaned and sanitized food contact surfaces, should be done as an integral part of any sanitation program. In 1987, the American Meat Institute published some interim guidelines for controlling the incidence of listeriae and other pathogenic and nonpathogenic microbial contaminants during production of RTE meat products [3]. Since then, others have published similar guidelines [150,151]. The recommendations in these reports regarding facility requirements, factory environment, food contact and non-food-contact surfaces, cross-contamination, airborne contamination, condensation control, cleaning and sanitizing, traffic patterns, and personnel cleanliness are generally similar to those already presented in this chapter as general guidelines; however, this report also outlined some of the critical operations associated with production of specific categories of RTE meat products. Roast Beef, Corned Beef, and Other Rebagged Products Products such as roast beef and corned beef that are repackaged after cooking are particularly prone to contamination with listeriae and other microorganisms. Therefore, attention must be given to proper sanitation and prevention of cross-contamination when these products are removed from bags in which they were cooked. The outside surface of all bags should be thoroughly washed and sanitized before the bags are opened. In addition to a sanitary working environment, repackaging of cooked product requires use of clean clothing, as well as frequently sanitized utensils and gloves. Trimming and cutting of cooked product just before rebagging are two additional critical steps where listeriae and other contaminants can enter and compromise the integrity of the final product. Therefore, contact between cooked product and unsanitized surfaces must be avoided during rebagging operations. As repackaging is by nature a wet process, this operation also needs to be well isolated from other processing areas to reduce cross-contamination. Frankfurters and Other Link Products Sausages such as frankfurters and other link varieties are typically prepared from a finely ground mixture (or emulsion) of beef or pork, which is stuffed into artificial or natural casings. After twisting the casing at approximately 6-in. intervals, the links are cooked using steam or hot water and then hung for smoking. To obtain skinless frankfurters, the artificial casing must be mechanically peeled from the congealed meat mixture. Prompt attention to cleaning, sanitizing, and crosscontamination problems is required during all stages of frankfurter production; however, the product

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is particularly vulnerable to contamination with listeriae and other microorganisms during the peeling process. It is imperative to keep the area around peeling machines as dry and free from meat scraps and juices as possible. Peeling machine operators also need to change protective garments and gloves frequently. Hoods on peeler machines have been cited as a source of listeriae and should therefore be eliminated, if possible. Manufacturing practices should also be reviewed to ensure that losses from floor contamination and reworked product are minimized. Unpeeled frankfurters that touch the floor or other unclean surfaces can be reworked (i.e., appropriately washed and peeled after all other frankfurters have been peeled). However, any peeled frankfurters that come in contact with the floor or other unclean surfaces should be destroyed. This latter recommendation is supported by data indicating that L. monocytogenes is difficult to destroy on the surface of frankfurters during cooking without making the product organoleptically unacceptable [13]. In addition to these concerns, brine chillers have also been cited as a potential source of listeriae, thus leading to contamination of casings and product surfaces. Some studies have suggested that reformulation of frankfurters with various antimicrobial agents or immersion in organic acid solutions (i.e., in brine) may be an effective way to reduce Listeria contamination and prevent growth [26]. Finally, all packaging and heat-shrinking equipment should be cleaned and sanitized daily to avoid spreading contaminants from steam and water to packaging lines. Luncheon Meats Concerns regarding control of listeriae and other contaminants in luncheon meats are generally similar to those just discussed for frankfurters and other link products. However, in addition, slicing equipment should be kept dry and free of scraps and juices that may serve as potential nutrients for microbial contaminants, including listeriae. A variety of technologies have been studied to reduce or control listeriae on RTE meats, including chemical-antimicrobial treatments, irradiation (not yet approved for RTE meats), and thermal processes such as pre- and postpackage thermal treatments [71].

POULTRY INDUSTRY Potential sources of listeriae contamination during processing of raw poultry are in many ways similar to those just discussed for the meat industry. Since a substantial percentage of birds harbor Listeria spp. (including L. monocytogenes) and Salmonella in their intestinal tract, enforcement of proper cleanup (i.e., elimination of water, condensate, and waste) and cleaning and sanitizing programs will likely decrease the incidence of contamination but will never completely eliminate these pathogens from raw poultry processing facilities or the raw product. Most modern poultry processing facilities are continuous line operations in which incoming birds are shackled, electrically stunned, bled, scalded to facilitate feather removal, plucked of feathers, eviscerated, inspected, washed, chilled, dried, and packaged for sale. Processing steps during which L. monocytogenes, Salmonella spp., and other pathogens are most likely to contaminate the product include scalding, defeathering, evisceration, and chilling [120,121]. USDA officials have proposed processing changes that may be helpful in decreasing the incidence of Salmonella (and presumably Listeria) in raw poultry [18]. These changes included (1) segregating and processing pathogen-infected flocks at different times from noninfected flocks; (2) examining the potential benefits of adding bactericidal concentrations of organic acids or other approved antimicrobials to chill water tanks; (3) experimentation with different scalding methods (e.g., hot-water sprays, steam scalders, or scald additives); (4) routine sanitizing of all equipment and utensils with hot water or bactericidal agents; (5) reemphasis of employee hygiene programs with routine hand washing and sanitizing required by all evisceration line workers; (6) elimination of offline processing; and (7) installation of equipment designed automatically to transfer carcasses from the picking line to the evisceration line. Additional work is needed to streamline further processing of poultry carcasses and minimize cross-contamination during their processing.

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Increasing consumption of poultry both in and outside the home mandates that persons preparing these products must take special precautions to prevent the spread of L. monocytogenes, Salmonella spp., and other foodborne pathogens from raw poultry to other products (e.g., fruits and vegetables) that are frequently consumed without heating. The common practice of washing and rinsing raw poultry before cooking has been questioned, because this step fails to markedly reduce microbial populations on poultry skin and also leads to increased contamination of kitchen sinks, faucets, and other food preparation areas [159]. As all foodborne pathogens commonly associated with raw poultry (including L. monocytogenes) are readily susceptible to heat, thorough cooking appears to be the best means of ensuring that such products are free of hazardous microorganisms. An Oklahoma breast cancer patient contracted listeriosis in December 1988 after consuming Listeria-contaminated turkey frankfurters. Thus, producers of processed poultry products (e.g., turkey and chicken frankfurters and rolls) need to take precautions similar to those previously described for the manufacture of roast beef, corned beef, frankfurters, link sausage, and luncheon meats with special attention being given to the cleanliness of rebagging operations and sausage peelers.

EGG INDUSTRY As stated earlier, the contents of intact whole eggs are normally sterile unless the laying hen infects the yolk with Salmonella enteritidis. Foodborne pathogens, including L. monocytogenes and S. enteritidis, have frequently been isolated from commercially broken, raw liquid whole egg, with contamination most likely coming from the manufacturing environment or the eggshells. Pasteurization as required for commercially broken, raw liquid whole egg is likely sufficient to eliminate normally encountered populations of L. monocytogenes and salmonellae in raw liquid egg; however, all egg breaking operations need to be well isolated from pasteurization and filling and packaging areas to minimize recontamination of the finished product. L. monocytogenes and other foodborne pathogens probably enter egg processing facilities as eggshell contaminants; therefore, egg receiving and washing sections of the factory also should be segregated from other processing areas. Considering the potential for postpasteurization contamination, many of the previously described guidelines for cleaning and sanitizing dairy factories also appear to be applicable to manufacturers of pasteurized liquid egg products.

FISH

AND

SEAFOOD INDUSTRY

L. monocytogenes and other foodborne pathogens such as Vibrio, Salmonella, Shigella, Staphylococcus aureus, Clostridium botulinum, Aeromonas hydrophila, and certain strains of Escherichia coli have been isolated from raw or cooked finfish, shrimp, crab, lobster, oysters, and scallops. An integrated approach to product safety is needed to minimize contamination of seafood from harvest to the time of consumption. The FDA adopted final regulations to ensure the safe and sanitary processing of fish and fishery products on December 18, 1997 [154]. These regulations mandate the application of HACCP principles to the processing of seafood. Seafood processors and importers need to evaluate the kinds of hazards that could affect their products, institute controls to keep these hazards from occurring, or significantly minimize their occurrence, monitor the performance of those controls, and maintain records of this monitoring as a matter of routine practice. Limiting postharvest contamination of freshly caught fish and seafood is the first step toward producing a safe, high-quality end product. Adherence to good sanitation and hygienic practices aboard fishing vessels is imperative. Contact between freshly caught seafood and waterfowl, such as pelicans and seagulls, should be minimized because these birds are intestinal carriers of L. monocytogenes and other foodborne pathogens. All seafood should be either frozen or refrigerated immediately after harvest to stop or retard growth of microbial contaminants, including spoilage organisms.

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The general guidelines that were discussed previously regarding factory design, processing environment, proper cleaning and sanitizing, employee traffic patterns, and personnel cleanliness are also valid for the seafood industry. In addition to these recommendations, seafood processors are also urged to: (1) eliminate processing waste, pooled water, and condensate from walls, floors, and ceilings, as well as from processing and refrigerated areas; (2) eliminate use of high-pressure sprays; (3) reduce airborne contamination; (4) cover outside dumpsters to decrease problems involving seagulls and other wildlife; (5) assign specific equipment (i.e., product totes) for use in either raw or cooked product areas of the factory; and (6) if possible, replace wooden totes with fiber totes, which can be easily cleaned and sanitized. Listeria spp., including L. monocytogenes, have been isolated most frequently from crabmeat and cooked and peeled shrimp, as stated earlier in this book. This observation is not surprising if one considers how these products are processed and packaged for the consumer. Processing of Dungeness crab generally begins by immersing and cooking either sections of or the entire crab in boiling water for approximately 7–9 or 17–20 min, respectively. Current information indicates that such a heat treatment is sufficient to destroy listeriae [66]; however, underprocessing may lead to survivors. After cooking, the crab is cooled in a water bath and either “picked” immediately or iced and refrigerated in a walk-in cooler until the meat can be hand picked from the shell. Extensive handling of the product by workers during picking, subsequent inspection, and packaging affords many opportunities for postprocessing contamination. Lactic acid dips appear to be somewhat useful in reducing populations of L. monocytogenes and other microorganisms on the surface of crabmeat and fresh and frozen shrimp; however, such treatments will not completely eliminate listeriae from the finished product [66]. Therefore, strict adherence to GMPs, which include proper employee hygiene and cleaning and sanitizing of picking equipment, must be observed in and among picking areas to avoid negating the benefits of cooking. Unfortunately, crab processing varies widely with the species of crab—Dungeness, blue, stone, king, and golden crab. Hence, some of the critical control points discussed for Dungeness crab are not applicable to other species. For example, blue crabmeat is typically removed from the animal in the raw state, placed in sealed containers, and then pasteurized (85°C/1 min) to eliminate L. monocytogenes and other microbial pathogens. As pasteurization of blue crabmeat becomes a critical control point in processing, it may be prudent to certify or license crabmeat pasteurization operators or their supervisors, as has been required for operators of retorts in the canning industry for many years. Problems regarding postprocessing contamination are also encountered during production of cooked and peeled shrimp. After shrimp are cooked, those destined for breading are mechanically peeled and sometimes deveined by splitting and removing the veinlike intestine. Unfortunately, many mechanical shrimp peelers have design flaws which necessitate almost continuous movement of the operator between both raw and cooked sides of the equipment, thus affording ample opportunity for postprocessing contamination. Proper cleaning and sanitizing of the equipment (particularly protective covers over flumes and gutters) and the surrounding area are essential for producing high-quality microbiologically safe products. Raw seafood such as crab, shrimp, lobster, clams, oysters, and the myriad of finfish currently available to consumers will probably never be completely free of L. monocytogenes or other foodborne pathogens, even when handled under the best possible conditions. Considering that many individuals are not “seafood smart,” processors and marketers of seafood have an obligation to educate the general public and provide consumers with proper handling and cooking instructions. Individuals who insist on consuming unprocessed fish (e.g., sushi) and seafood (e.g., oysters) should also be made aware of potential health problems associated with consumption of such products.

FRUIT

AND

VEGETABLE INDUSTRY

There is limited information concerning the incidence of L. monocytogenes in fruits; however, it has been widely recovered from plant material and vegetables, including cabbage, cucumbers,

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mushrooms, potatoes, leafy vegetables, bean sprouts, and radishes [32]. Well-documented outbreaks from fruits and vegetables have been limited. In addition to the 1981 Canadian listeriosis outbreak involving coleslaw [134], one isolated case in Finland was linked to consumption of raw salted mushrooms. Evidence suggests the implication of raw vegetables (including salads) in other outbreaks. L. monocytogenes can persist on endive, lettuce, tomatoes, asparagus, broccoli, cauliflower, and cabbage [31]. Historically, the scientific community and the public at large have been somewhat less concerned about Listeria contamination in vegetables than in dairy, meat, poultry, and seafood products; however, this may be changing as our culture moves to consumption of more raw fruits and vegetables. Routine examination of raw vegetables for L. monocytogenes and other foodborne pathogens is unlikely to reduce the risk of foodborne illness to any great extent. However, because raw sheep manure was the probable source of L. monocytogenes in the Canadian coleslaw outbreak, vegetable processors should have some assurance that incoming raw vegetables have been grown, irrigated, fertilized, harvested, packaged, and transported to the firm using good agricultural practices. Consumption of vegetables that will be adequately cooked before eating is of little concern, because L. monocytogenes and other non-spore-forming pathogens are destroyed during cooking. A wide range of produce sanitizers (e.g., chlorine, peroxyacetic acid, ozone, chlorine dioxide) is available for commercial disinfection of fresh produce, but their effectiveness varies and none is able to ensure complete destruction of pathogens. Thus good hygienic practices (GHPs) and HACCP should be implemented along the entire process from production to consumption of raw vegetables. Vegetable processors should consider rejecting raw vegetables that are likely to be consumed without cooking if the grower clearly fails to demonstrate the use of good agricultural practices [135]. Routine washing of raw vegetables in potable water is recommended for commercial establishments and homes; however, this practice generally fails to reduce the microbial load on raw vegetables by more than 10-fold. Therefore, persons handling and preparing raw produce and salad vegetables should follow good hygienic practices during slicing, dicing, chopping, and grating operations to prevent the spread of potentially hazardous microorganisms to other foods. Finally, all knives, cutting boards, and other food contact surfaces should be thoroughly cleaned and sanitized after use to inactivate organisms inadvertently introduced into the kitchen environment during preparation of raw produce.

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134. Schlech W.F., P.M. Lavigne, R.A. Bostoulussi, A.C. Allen, E.V. Haldane, A.J. Wort, A.W. Hightower, S.E. Johnson, S.H. King, E.S. Nicholls, and C.V. Broome. 1983. Epidemic listeriosis—evidence for transmission by food. N. Engl. J. Med. 308: 203–206. 135. Scientific Committee on Food. 2002. Risk Profile of Microbiological Contamination of Fruits and Vegetables Eaten Raw. Report of April 29. European Commission, Health & Consumer Protection Directorate-General. Brussels, Belgium. http://europa.eu.int/comm/food/fs/sc/scf/out125_en.pdf. 136. Siegel, J. and I. Walker. 2001. Deposition of biological aerosols on HVAC heat exchangers. ASHRAE IAQ 2001: Moisture, Microbes, and and Health Effects: Indoor Air Quality and Moisture in Buildings, 1–9. eScholarship Repository, University of California, Berkley. http://repositories.cdlib.org/cgi/ viewcontent.cgi?article=2007&context=lbnl. 137. Silliker, J.H. and D.A. Gabis. 1975. A cellulose sponge sampling technique for surfaces. J. Milk Food Technol. 38: 504. 138. Silverside, D. and M. Jones. 1992. Health, hygiene and routine maintenance. In FAO (Ed.), FAO Animal Production and Health Paper 98, “Small-Scale Poultry Processing.” Rome, chap. 4. http://www.fao.org/docrep/003/t0561e/T0561E00.htm#TOC. 139. Smoot, L.M. and M.D. Pierson. 1997. Indicator Microorganisms and Microbiological Criteria. In M. Doyle, L.R. Beuchat, and T.J. Montville (Eds.). Food Microbiology: Fundamentals and Frontiers. ASM Press, Washington, DC, Chapter 4. 140. Sperber, W.H. 1999. Thoughts on today’s food safety—the role of validation in HACCP plans. Dairy Food Environ. Sanit. 19: 920. 141. Sperber, W.H., K.E. Stevenson, D.T. Bernard, K.E. Deibel, L.J. Moberg, L.R. Hontz, and V.N. Scott. 1998. The role of prerequisite programs in managing a HACCP system. Dairy Food Environ. Sanit. 18: 418–423. 142. Spurlock, A.T., E.A. Zottola, and R.K.L. Petran. 1989. The survival of Listeria monocytogenes in aerosols. J. Food Prot. 52: 751 (abstr.). 143. Stanfield, J.T., C.R. Wilson, W.H. Andrews, and G.J. Jackson. 1987. Potential role of refrigerated milk packaging in the transmission of listeriosis and salmonellosis. J. Food Prot. 50: 730–732. 144. Surak, J.G. and S.F. Barefoot. 1987. Control of Listeria in the dairy plant. Vet. Hum. Toxicol. 29: 247–249. 145. Sutherland, P. and R. Porritt. 1995. Dissemination and ecology of Listeria monocytogenes in Australian dairy factory environments. Proceedings of the XII International Symposium on Problems of Listeriosis, Perth, Australia, pp. 291–297. 146. Terplan, G. 1988. Listeria in the dairy industry—situation and problems in the Federal Republic of Germany. Foodborne Listeriosis—Proceedings of a Symposium, Wiesbaden, West Germany, September 7, pp. 52–70. 147. Thimothe, J., J. Walker, and V. Suvanich. 2002. Detection of listeria in crawfish processing plants and in raw whole crawfish and processed crawfish. J. Food Prot. 65: 1735–1739. 148. Thimothe, J., K.K. Nightingale, K. Gall, V.N. Scott, and M. Wiedmann. Tracking of Listeria monocytogenes in smoked fish processing plants. J. Food Prot. 67: 328–341. 149. Tompkin, R.B., L.N. Christiansen, A.B. Shaparis, R.L. Baker, and J.M. Schroeder. 1992. Control of Listeria monocytogenes in processed meats. Food Aust. 44: 370–376. 150. Tompkin, R.B., V.N. Scott, D.T. Bernard, W.H. Sveum, and K.S. Gombas. 1999. Guidelines to prevent post-processing contamination from Listeria monocytogenes. Dairy Food Environ. Sanit. 19(8): 551–562. 151. Tompkin, R.B. 2002. Control of Listeria in the food-processing environment. J. Food Prot. 65(4): 709–723. 152. Tybor, P.T. and W.D. Gilson. 1989. Bulletin1025/Revised 1989. Cooperative extension of the University of Georgia and Ft. Valley State College, and the U.S. Department of Agriculture. 153. USDA/FSIS. Control of Listeria monocytogenes in Ready-to-Eat Meat and Poultry Products; Final Rule. Federal Register: June 6, 2003. 68(109): 34207–34254, 9 CFR Part 430. 154. U.S. Food and Drug Administration. 1995. Procedures for the safe and sanitary processing and importing of fish and fishery products. Final Rule 21 CFR 123 and 1240. Federal Register 60 CFR, pp. 65095–65202. 155. Venables, L.J. 1989. Listeria monocytogenes in dairy products—the Victorian experience. Food Aust. 41: 942–943.

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156. Waak, E., W. Tham, and M.L. Danielsson-Tham. 2002. Prevalence and fingerprinting of Listeria monocytogenes strains isolated from raw whole milk in farm bulk tanks and in dairy plant receiving tanks. Appl. Environ. Microbiol. 68: 3366–3370. 157. Wagner, L.R. van. 1989. FDA takes action to combat seafood contamination. Food Process. 50(2): 8–12. 158. Walker, R.L., L.H. Jensen, H. Kinde, A.V. Alexander, and L.S. Owens. 1991. Environmental survey for Listeria species in frozen milk products plants in California. J. Food Prot. 54: 178–182. 159. Woodburn, M. 1989. Myth: wash poultry before cooking. Dairy Food Environ. Sanit. 9: 65–66. 160. Yan, Z., J.L. Kornacki, C.M. Lin, and M. Doyle. 2004. Fate of Aersolized Listeria monocytogenes in a Closed Bioaersol Chamber. International Association for Food Protection. Annual Meeting. Abstract P058.

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Risk Assessment, 18 Listeria: Regulatory Control, and Economic Impact Ewen C.D. Todd CONTENTS Risk Analysis..................................................................................................................................768 Definitions.............................................................................................................................770 The Risk Assessment Process ..............................................................................................771 Risk Assessments on a Country or International Basis ................................................................773 Australia................................................................................................................................773 Canada ..................................................................................................................................775 China (Hong Kong) ..............................................................................................................775 France ...................................................................................................................................775 Germany ...............................................................................................................................777 The Netherlands....................................................................................................................777 Sweden..................................................................................................................................777 United States.........................................................................................................................778 International..........................................................................................................................781 Codex Alimentarius Commission.............................................................................783 Standards and Criteria....................................................................................................................790 Australia and New Zealand..................................................................................................791 North America ......................................................................................................................793 United States.............................................................................................................793 Canada ......................................................................................................................794 European Countries ..............................................................................................................796 Austria.......................................................................................................................796 Denmark ...................................................................................................................796 France .......................................................................................................................797 Germany ...................................................................................................................797 Italy ...........................................................................................................................798 The Netherlands........................................................................................................798 Slovenia ....................................................................................................................798 United Kingdom .......................................................................................................798 European Union (EU) ..........................................................................................................799 International Activities .........................................................................................................800 Costs .....................................................................................................................................801 References ......................................................................................................................................807

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RISK ANALYSIS The significant increase in listeriosis, particularly outbreaks from contamination of ready-to-eat (RTE) processed food, over the last 20 years has indicated the need for better control using more effective risk management methods. Currently, there is no international agreement on the acceptable levels for Listeria monocytogenes in foods, and the importance of harmonizing microbiological criteria for L. monocytogenes in foods for trade at the international level based on principles of risk assessment is seen as critical. Several countries have begun developing risk-based approaches to manage L. monocytogenes better, along with initiatives at the international level, supported by these countries. The establishment of the World Trade Organization (WTO) in 1995 and the related Sanitary and Phytosanitary Agreement (SPS) between member countries have given new impetus to the Codex Alimentarius Commission (CAC) to develop international standards for all foodborne hazards. The primary purpose of the Joint FAO/WHO Food Standards Programme of the CAC is to protect the health of consumers and ensure fair practice in the food trade. CAC formally adopts Codex standards, guidelines, and other recommendations that have been developed by its subsidiary bodies, such as the Codex Committee on Food Hygiene (CCFH). In addition, CAC provides guidance to these subsidiary bodies, including that related to risk management. The work of these committees is supported by expert advisory groups, such as the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the Joint FAO/WHO Meeting on Pesticide Residues (JMPR), as well as other expert bodies, such as the International Commission on Microbiological Specifications for Food [68]. However, no specific advisory committee had been established for microbiological issues until the Joint FAO/WHO Expert Meeting on Microbiological Risk Assessment (JEMRA) was formed in 2000. Food safety risk analysis is an emerging discipline, particularly for microbial hazards, and the methodological basis for assessing and managing risks associated with food hazards is still developing. Risk assessment is one method by which science can be used to address food safety, but this is only the first of several key steps in managing hazards in foods. Risk assessment is a part of risk analysis, which includes two other components: risk management and risk communication [33] (Figure 18.1). However, the managers decide when and how a risk assessment is to be done, and they use the risk characterization results from the risk assessment process to strategize the best management options. Most government agencies try and keep the risk assessment process separate from the management, but frequent consultation is necessary. In addition to data arising from risk characterization, risk management also includes recommendations for Good Manufacturing Practices (GMPs) and Good Hygienic Practices (GHPs), which may result in criteria for microbial hazards in food. An overall framework is needed to help managers develop a consistent approach that will lead to all relevant inputs and uncertainties, and appropriate decisions. Regulatory authorities need to better balance risks and benefits across a society in an acceptable way, and to make the decisionmaking process more explicit by being more open and transparent. The FAO/WHO approach is to have four steps: risk evaluation, option assessment, option implementation, and monitoring and review [33]. Another such framework prepared by the Presidential/Congressional Commission on environmental health risks in the United States [26] consists of a series of six interconnected and interrelated steps. These are listed as follows, with issues to be considered in each step: 1. Defining the problem and putting it in context: a. Characterization of the problem or a potential problem caused by microorganisms or their toxins. b. Identification of the appropriate risk managers with the authority and involvement of stakeholders.

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Risk Assessment (science based) • Hazard Identification • Hazard Characterization • Exposure Assessment • Risk Characterization

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Risk Management (policy based) • Risk Evaluation • Option Assessment • Option Implementation • Monitoring and Review

Risk Communication (Interactive exchange of information and opinions concerning risk)

FIGURE 18.1 Example of a schematic diagram of the interactions between risk assessment, risk management, and risk communication. (From FAO. 1997 Risk management and food safety—FAO Food and Nutrition Paper 65, Report of a Joint FAO/WHO Consultation, Rome, Italy, January 27–31. http://www.fao.org/docrep/ W4982E/w4982e00.htm. Accessed August 7, 2006.)

2. Analyzing the risks associated with the problem in context: a. Risk assessment with hazard identification, dose response assessment, exposure assessment, and risk characterization. b. Risk perceptions and local or regional information, if the problem is specific to one area, need to be considered. c. This process should generate enough information for managers to make decisions. 3. Examining options for addressing the risks: a. Considering regulatory or other options for managing the risk. b. Exploring the feasibility of carrying out these options. c. Analyses for cost effectiveness and cost distributions among the affected parties. d. Determination of any possible new risks that might arise from the proposed management actions. 4. Making decisions about which options to implement: a. Decisions are based on the best scientific, economic, and other technical information. b. Actions take into account all sources of the hazard and impacts of the problem to be solved. c. Management options are chosen that are feasible and have benefits that are reasonably related to costs. d. A prioritization of options preventing or reducing risks, not just controlling them. e. Alternatives to the command and control approach to regulation are important.

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f. Political, social, legal, and cultural considerations have to be considered. g. Incentives for innovation, evaluation, and research are long-term options. 5. Taking actions to implement the decisions: a. Implementation is carried out by one group of managers in one agency but coordinating as required with other agencies. b. Compliance will be better if all stakeholders agree with the decision-making process and actions taken. 6. Conducting an evaluation of the actions’ results to determine the following: a. Whether decisions resulted in intended actions, whether cost-benefit estimates were accurate. b. What information gaps prevented full implementation of the actions. c. Whether new information has emerged during the process that would be helpful in improving the decision-making process. d. Whether the risk management framework and policy were effective or needed to be changed. e. How stakeholders reacted to the process and how valuable was their input. f. What overall lessons were learned to help other risk management decisions. To these can be added less measurable parameters that are critical for decision makers to be aware of in any management process, which include benefits and constraints of taking action or not, risk perception by stakeholders, involving a variety of value judgments, and the contentious precautionary principle in which a very conservative approach to management is adopted. Although this management structure has not been universally adopted, it is clear that risk assessment is only a part of the process, but a critical one. Risk communication is probably the least understood of the three components of risk analysis because it is difficult to incorporate into a structure. Latest WHO thinking is that risk communication encompasses both risk assessment and risk management and is therefore broader in scope than simply relating results of the assessment and management processes. In any new policy on Listeria, risk communication strategies will be essential to explain what the impact and benefits will be. Even explaining what is a “zero-tolerance policy” is not an easy task (it is certainly not zero tolerance in a total sense, but only absence, by the analytical methods used, in a portion of the food). To this end, the WTO encouraged the CAC to establish quantitative risk assessments regarding pathogenic microorganisms and develop appropriate tools.

DEFINITIONS In any risk assessment process, definitions of terms are important. The following are definitions listed in Principles and Guidelines for the Conduct of Microbiological Risk Assessment [34]. A hazard is a biological, chemical, or physical agent in, or condition of, food with the potential to cause an adverse health effect (harm). In contrast, risk is a function of the probability of an adverse health effect and the severity of that effect, consequential to hazards in food. Understanding the association between a reduction in hazards that may be associated with a food and reduction in the risk to consumers of adverse health effects is of particular importance in development of appropriate food safety controls [115]. Risk analysis—A process consisting of three components: risk assessment, risk management, and risk communication. Risk assessment—A scientifically based process consisting of the following steps: (1) hazard identification, (2) hazard characterization, (3) exposure assessment, and (4) risk characterization. Quantitative risk assessment—A risk assessment that provides numerical expressions of risk and indication of the attendant uncertainties.

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Qualitative risk assessment—A risk assessment based on data, which, although forming an inadequate basis for numerical risk estimations, permits risk ranking or separation into descriptive categories of risk when conditioned by prior expert knowledge and identification of attendant uncertainties. Hazard identification—The identification of biological, chemical, and physical agents that are capable of causing adverse health effects and may be present in a particular food or group of foods. Hazard characterization—The qualitative and/or quantitative evaluation of the nature of adverse health effects associated with the hazard, and this can include a dose–response assessment. For the purpose of microbiological risk assessment (MRA) the concerns relate to microorganisms and/or their toxins. Dose–response assessment—The determination of the relationship between the magnitude of exposure (dose) to a chemical, biological, or physical agent and severity or frequency of associated adverse health effects (response). Exposure assessment—The qualitative and/or quantitative evaluation of the likely intake of biological, chemical, and physical agents via food as well as exposures from other sources if relevant. Risk characterization—The process of determining qualitative and quantitative estimation, including attendant uncertainties, of the probability of occurrence and severity of known or potential adverse health effects in a given population based on hazard identification, hazard characterization, and exposure assessment. Risk estimate—Output of risk characterization. Risk management—The process of weighing policy alternatives in light of results from the risk assessment and, if required, selecting and implementing appropriate control options, including regulatory measures. Risk communication—The interactive exchange of information and opinions concerning risk and risk management among risk assessors, risk managers, consumers, and other interested parties. Sensitivity analysis—A method used to examine the behavior of a model by measuring the variation in its outputs resulting from changes to its inputs. Transparent—Characteristics of a process in which rationale, logic of development, constraints, assumptions, value judgments, decisions, limitations, and uncertainties of the expressed determination are fully and systematically stated, and documented, and are accessible for review. Uncertainty analysis—A method used to estimate uncertainty associated with model inputs, assumptions, and structure/form.

THE RISK ASSESSMENT PROCESS Control of L. monocytogenes in foods provides an example of the need to consider a structured risk management approach. Listeria is frequently consumed in small amounts by the general population without apparent ill effects. It is believed by many that only higher levels of Listeria have caused severe disease problems. It is also believed that Listeria is a pathogen that will always be present in the environment. Therefore, the critical issue may be how to control its survival and growth to minimize the potential risk, rather than how to prevent Listeria from contaminating foods. Despite a zero-tolerance policy in a few countries, many CAC members feel that the complete absence of Listeria in all RTE foods is unattainable, and trying to achieve this goal can limit trade without having any appreciable benefit to public health. One risk management option, therefore, is to focus on foods that have historically been associated with listeriosis, and foods that support growth of Listeria to high levels, rather than those that do not. Thus, establishment of tolerable low levels of Listeria in specific foods may be one food safety objective established by risk managers after a rigorous and transparent risk analysis. Such an approach is now being considered by CAC and its committees [33–35].

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In its general principles of microbiological risk assessment document [34], the CAC so far has preferred a quantitative approach to microbiological risk assessment, although a qualitative approach may be valid depending on the manager’s needs. This is mainly because no expert committee has tackled the various issues of when and how qualitative assessments should be considered. Not all the published assessments adhere to the following principles and guidelines completely, partly because they are more generic models than those used for a particular Listeria problem: 1. Microbiological risk assessment should be soundly based on science. 2. There should be a functional separation between risk assessment and risk management. 3. A microbiological risk assessment should be conducted according to a structured approach that includes hazard identification, hazard characterization, exposure assessment, and risk characterization. 4. A microbiological risk assessment should clearly state the purpose of the exercise, including the form of risk estimate that will be the output. 5. The conduct of a microbiological risk assessment should be transparent. 6. Any constraints that impact the risk assessment, such as cost, resources, or time, should be identified and their possible consequences described. 7. The risk estimate should contain a description of uncertainty in the estimate and where the uncertainty arose during the risk assessment process. 8. Data should be such that uncertainty in the risk estimate can be determined; data and data collection systems should, as far as possible, be of sufficient quality and precision that uncertainty in the risk estimate is minimized. 9. A microbiological risk assessment should explicitly consider the dynamics of microbiological growth, survival, and death in foods and the complexity of the interaction (including sequelae) between human and agent following consumption as well as the potential for further spread. 10. Wherever possible, risk estimates should be reassessed over time by comparison with independent human illness data. 11. A microbiological risk assessment may need reevaluation, as new relevant information becomes available. Risk assessments can be used to rank or measure hazards, assess exposure, and characterize risks involved with any food–pathogen combination, or even a multiple food or pathogen situation. For L. monocytogenes, most risk assessments follow the four stages of hazard identification, hazard characterization, exposure assessment, and risk characterization. The purpose of hazard identification is to identify the types of adverse health effects that may occur from its transmission through one or more foods. Information can be obtained from published and unpublished scientific literature, including food industry, government, and international organization sources, and through solicitation of expert opinions. Relevant information includes data in areas such as clinical studies, epidemiological studies and surveillance, laboratory animal studies, investigations of the characteristics of microorganisms, interaction between microorganisms and their environment through the food chain from primary production up to and including consumption, and studies on analogous microorganisms and situations. An ideal hazard characterization should include a dose–response assessment with end points such as infection or illness; these may use animal models and human outbreak scenarios. The effects on different populations, particularly those at most risk, should be attempted. Factors such as virulence, possibility of secondary transmission (less likely for L. monocytogenes), and attributes of a food that may alter microbial pathogenicity, e.g., high fat content of a food vehicle, are important to identify. Where data are missing, experts may be able to devise ranking systems so that they can be used to characterize severity and/or duration of disease. For L. monocytogenes, several different dose–response assessments have been derived, so that any risk characterization

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process becomes much more uncertain. For instance, from a manager’s point of view, is one dose–response assessment chosen, a combination for different populations at risk, or a median value of several assessments, if such exist? Exposure assessments estimate the level, within various levels of uncertainty, of L. monocytogenes in specific foods at the time of consumption, usually on a serving size or per-gram basis. The assessment can start with data on all ingredients going into the food, or later in the process. With Listeria there is the complication that different strains of the pathogen may enter at different steps in the process, and it may be the rare final contamination in the processing plant that is the most critical. Most assessments have started at the processing stage, partly because of lack of data at the earlier steps. Ideally, assessors attempt to model changes in populations as they go through production and processing, with some kind of measures for growth, decline, and recontamination. Consumer practices are important since long-term storage in refrigerators can greatly impact growth of the pathogen at home. Also, consumption patterns need to be estimated that vary by gender, age, nationality, season, and consumer preferences. Such consumption data are usually limited with expert opinions often used, although some national surveys can prove valuable for some foods. Risk characterization brings together all the qualitative or quantitative information of the previous steps to provide an estimate of risk for a given population. If there is not enough information following the integration of quantitative and qualitative data, only a qualitative estimate of risk may be possible. Typically, in a quantitative risk assessment, an estimate will be expressed in risks per serving (meal size) or per population (region or country), but both of these should be accompanied by some estimate of uncertainty.

RISK ASSESSMENTS ON A COUNTRY OR INTERNATIONAL BASIS AUSTRALIA The Government of New South Wales has outlined its use of risk analysis in managing food safety risks [84]. Safefood NSW was established in 1998 and it became the NSW Food Authority in April 2004 [85]. Safe food legislation [46] requires that a food safety scheme be based on risk assessment to ensure that all known hazards are addressed and that appropriate resources are allocated. Moreover, it was agreed that an assessment of food safety risks in the food industry should be in accordance with national and international standards of risk assessment. The principles that should guide the achievement of these objectives are that (1) protection of public health is paramount; (2) risk assessment principles should be used to systematically identify public health issues of concern and develop effective solutions; (3) implementing reforms should involve government, industry, and consumers in partnership; and (4) although efficiency is to be promoted, costs and benefits should be taken into account [84]. Assessments have been carried out for seafood and meat products. Sumner and Gallagher [94] prepared a semiquantitative risk assessment of ten seafood hazard–product combinations to generate a risk estimate which was scaled from 0 to 100, where 0 represents no risk and 100 represents all meals containing a lethal dose of the hazard. The highest ranking (>48) was for seafood toxins (ciguatera, scombroid, and algal toxins in bivalve mollusks) and viruses in oysters. Risks for listeriosis arising from RTE cold-smoked fish products had a score of 39–45, depending on the population affected. Another unpublished study was to assess the public health risk to Australian consumers caused by L. monocytogenes in Australian-made processed meat products [90]. The project aimed to (1) characterize the nature and size of the microbial food safety risk from Listeria in processed red meat products relative to that of other foodborne pathogens; (2) identify areas in which critical data and/or knowledge are lacking; (3) characterize factors that contribute most significantly to the risk; and (4) recommend management strategies to reduce the microbial food safety risk. No specific constraints on performance of the assessment were identified. The assessment considered processed meats in

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three categories, including luncheon meats (hams, etc.), pâtés, and cooked sausages intended to be reheated. The risk from fermented meats was demonstrated to be insignificantly small and was not of further concern because no additional risk management actions were considered necessary. With cooked products, it was assumed that the cooking step was completely listericidal and that contamination arose after processing, which was the starting point for the risk assessment. The effects of all storage and transport steps on concentration and prevalence of L. monocytogenes in RTE processed meat products were considered, including consumer handling. The effect of contamination at various steps in the factory-to-fork chain was also considered, although quantitative data to describe the numbers of L. monocytogenes transferred were almost nonexistent. Prevalence and concentration data were obtained from published sources, government surveys, and industry-funded studies. In addition, total count data at the point of production were obtained for inclusion in the model to account for interactions affecting the growth potential of L. monocytogenes during the life of the product. Large amounts of data were obtained from producers. Serving sizes and frequencies were determined from several government-sponsored food consumption studies. However, the product groupings in these studies were not fully relevant to product categories described in the assessment. To estimate the amount of each product consumed within the product groupings used in the nutrition surveys, national sales volumes for individual products were obtained from a large supermarket chain. From these data, the proportions of each product type in the nutrition survey groupings were calculated. Production volumes were also estimated from published data as an additional verification that the overall consumption estimates were realistic. The demography of the Australian population was considered. The age and gender structure of the population was determined from 0 to 6 months, and in 1-year age intervals thereafter. Surveys also identified consumption by age and gender. The proportion of pregnant women estimated by two methods was 1.3%. For some product categories, reduced consumption was evident in women of childbearing age and in the elderly. The number of cancer, transplant, AIDS, and diabetes sufferers was also determined. Relative susceptibility by age was determined from several sources of U.S. data for the age of listeriosis victims divided by the proportion of the population in those age classes. Susceptibility was expressed relative to adolescence, which experience the lowest relative risk. Apart from the age and gender data, no differentiation of consumption rate and frequency for the other at-risk groups could be determined. Time and temperature data for transport and storage of meat products were obtained from Australian surveys, but also compared to U.S. data. These data included retail display and domestic refrigeration and transport. Distributions of average temperatures and times during these stages were determined and used in subsequent modeling. Predictive microbiology models for growth rate of L. monocytogenes, including generic and product-specific models, were collated. Where possible, performance and applicability of the models were assessed, including accounting for lag times, with suitable models selected for inclusion in the exposure assessment model. Interactions between the background microbial flora and its effect on potential growth of L. monocytogenes (i.e., the socalled Jameson effect) were also modeled. These data and models were integrated into a process risk model that was used to assess the effectiveness of several putative risk management options and to assess the relative contribution of contamination at the plant compared to contamination at retail. Variability and uncertainty were identified and their effects discussed. In addition, the Monte Carlo simulation model was run several times with alternative assumptions in which uncertainty existed. From this method, the importance of uncertain values was estimated. The exposure assessment was relatively complex, and was a major component of the overall risk assessment, modeling the prevalence and concentration of L. monocytogenes from production to consumption. The effects of temperature, time, product formulation, and competing microorganisms in packages are all considered. Some differentiation of exposure levels among at-risk population subgroups was possible. The dose–response model developed by FAO/WHO [37] was incorporated into the risk assessment without change. The final risk characterization was developed but the results only privy to the meat industry, which commissioned the study.

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CANADA Farber et al. [39] published an assessment focused on RTE foods, particularly pâté and soft cheese, consumed in Canada. The authors recognized that L. monocytogenes may grow or be inactivated, and from a predictive model, they calculated that levels could increase from 1 CFU to 105 CFU in about 40 days at 4°C or 15 days at 8°C. Although it was noted that postprocess contamination of cooked meat products probably results in higher levels of L. monocytogenes at the surface of the product, and also higher levels in or near the rind of soft cheeses because of the higher pH, this information was not used in the assessment. The exposure assessment was limited because there were no quantitative links developed between the different steps in food production, retailing, and storage by the consumer. Unlike other L. monocytogenes risk assessments, the Weibull-Gamma model was chosen for the hazard characterization step. In this model, they used ID10 and ID90 values (doses causing illness in 10 and 90% of the population, respectively), i.e., 107 and 109 L. monocytogenes for normal individuals, and 105 and 107 for high-risk persons. The purpose of the assessment was to help Canadian heath officials set policy direction, allocate resources, and determine research and regulatory surveillance priorities. Todd et al. [99] developed a quantitative risk assessment for L. monocytogenes in cabbage in Canada. The objective, addressed in the extensive production module, was to show the prevalence and concentration of L. monocytogenes in the product after harvest, processing, and packaging under a modified atmosphere, during transportation, and retail and consumer storage. Although worldwide data were used for prevalence and concentration estimates, the geographic range of the assessment was limited to Canada. Distributions were used for all the prevalence, concentration, growth, and reduction scenarios. Consumption characteristics were derived from Canadian surveys. Uncertainty and variation were addressed by use of Monte Carlo simulations. In conclusion, this exposure assessment was relatively complex and modeled the prevalence and concentration of L. monocytogenes from harvest to consumption. The effects of temperature, time, disinfection, growth, pH, and atmosphere in packages were all considered, along with serving volumes and sizes at home and away from home. These were incorporated into the dose–response assessment step using both the Farber et al. [39] Weibull-Gamma and the Buchanan et al. [13] exponential models to determine the probability of infection and the number of illnesses in Canada for normal and susceptible (high-risk) populations. From results of the risk characterization, the likelihood of listeriosis from consumption of shredded cabbage in Canada was considered to be very low. For individuals in the high-risk group, the median probability of illness was 10 −8, with 5th and 95th percentiles 2 × 10 −13 and 9 × 10 −4, giving an estimate of one case every 5 years.

CHINA (HONG KONG) One report of a microbiological risk assessment for salads in Hong Kong is really more of a risk profile than a risk assessment, with several microbiological hazards and indicator organisms examined [67]. L. monocytogenes was only occasionally found in routine surveillance studies of restaurants and other premises (1 in 169 samples in 1999, 6 in 194 samples in 2000, and none in 210 samples in 2001). No specific determination of risk was made, but general hygienic recommendations were given for reducing contamination. The elderly, children, pregnant women, and persons with lowered immunity were advised to be careful choosing salads, considered a high-risk food.

FRANCE Bemrah et al. [11] undertook a quantitative risk assessment of L. monocytogenes from consumption of a generic soft cheese made from unpasteurized milk. L. monocytogenes may be introduced into raw milk either from environmental contamination or from mastitic cows. Although subsequent contamination was recognized, this was not included in the modeling. A key aim was to use the

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QRA model to simulate the consequences of alternative risk management options on the risk of illness from consumption of raw-milk soft cheese. The authors constructed a process risk model from milk production and storage through transport, storage, and cheese production to consumption with data on the prevalence and concentration of L. monocytogenes at the different stages, mainly from French sources. Time and temperature parameters were used to estimate growth in the milk, but it was assumed that no growth would take place after production. The authors assumed a uniform distribution of the pathogen in the cheese, although they recognized that there would likely be a lack of homogeneity. The probability of consuming a contaminated cheese serving was estimated to be 0.653, with concentrations in contaminated servings of 100 CFU (median) with 5 and 95 percentiles of 0 and 450 CFU. The authors noted that assessment was limited by the lack of useful available data. The assessment approach is consistent with the CAC framework for microbial risk assessment [115] and uses a probabilistic approach using distributions and Monte Carlo techniques to produce the model. Environmental contamination resulted in an average of 2.25 L. monocytogenes CFU/mL milk. In addition, individual cows’ milk contaminated at higher levels (104 CFU/mL) could come from infected udders, yet this event occurred in only about 1 of 2000 infected cows. Assumptions were made that (1) there was no net growth of L. monocytogenes during cheese ripening and distribution, (2) the dose–response relationships chosen for normal and at-risk populations were those of Farber et al. [39], and (3) the per capita yearly consumption of such soft cheeses was 50 servings of 31 g each. A Monte Carlo simulation on these data gave an annual cumulative risk of listeriosis ranging from 1.97 × 10 −9 to 6.4 × 10 −8 in the low-risk population, and 1.04 × 10 −6 to 7.19 × 10 −5 in a high-risk population. This is the equivalent to 74 cases of severe listeriosis per year in a population of 50 million, with 20% of these being susceptible individuals (mean 50) at the 99th percentile of incidence. If milk from excreting mastitic cows is not used for cheese production, this percentile could be reduced by a factor of 3.7 (mean could be reduced by 5.2). Model simulation showed that this measure had almost no influence on the proportion of servings with low contamination level (i.e., <10 L. monocytogenes CFU/g). Thus, the risk assessment provided useful information for managing of the risk of listeriosis from raw-milk cheese. A more specific assessment was built on the work of Bemrah et al. [11], being a quantitative risk assessment of human listeriosis associated with consumption of Camembert from Normandy and Brie of Meaux, both soft cheeses made from raw milk in France [91]. The estimated L. monocytogenes concentration in raw milk was on average 0.8 and 0.3 CFU/L, respectively, where Camembert and Brie were produced. A Monte Carlo simulation was used to account for the time–temperature history of the milk and cheeses from farm to table. It was assumed that viable cells would not spread within the solid cheese matrix. Cheese rind was assumed to represent 10% of the cheese volume, and the L. monocytogenes populations in the rind and core were modeled separately. Interaction between pH and temperature was included in the growth model. The simulated proportion of servings with no L. monocytogenes cells was 88% for Brie and 82% for Camembert. The 99th percentile of L. monocytogenes cell numbers in 27-g servings was 131 for Brie and 77 for Camembert at the time of consumption, corresponding, respectively, to 3–5 L. monocytogenes CFU/g. For the 17 million and 480 million annual servings of Brie and Camembert consumed in France, the number of expected listeriosis cases would be ≤5.10 × 10−4 and ≤2.5 × 10−3 per year, respectively, based on an exponential dose–response model. These low probabilities are borne out by effective monitoring throughout the process. The authors argue that improved hygiene at the farms has demonstrated low prevalence levels in raw milk, and heightened testing by cheese producers has resulted in very few milk batches containing the pathogen. Cheese samples are also tested for L. monocytogenes at the end of shelf life in the plant when cheeses are stored at 8°C. Other sampling is done at retail. For example, in 2000, none of 850 cheeses analyzed 21 days after milk coagulation, and none of 450 cheeses kept for 70 days at 8°C in the Brie cheese-making plant was positive, with a detection threshold of 10 L. monocytogenes CFU/g. Similarly, no positive samples were found in 2000 at the Camembert cheese-making plant. They also argue that the predicted probability of severe listeriosis from the two French cheeses made from raw milk does appear lower

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than the probability of listeriosis cases from soft cheeses made from pasteurized milk consumed in the United States based on data in the FDA/FSIS/CDC [42] draft relative risk ranking.

GERMANY Buchanan et al. [13] derived a conservative dose–response relationship for L. monocytogenes based on data from Van Schothorst [110,111]. The exposure assessment in each instance was based on German data from a 1991–1992 retail survey for the presence of L. monocytogenes in 25-g and 1-g samples. Consumption frequency and meal size estimates were combined with the contamination and prevalence survey data to estimate the annual number of exposures to 100-g servings of RTE meat and smoked fish containing between 104 and 106 L. monocytogenes CFU/g and those containing greater than 106 L. monocytogenes CFU/g. The Buchanan et al. [13] study only used the smoked-salmon data with about 20 servings per capita per year in Germany. No attempt was made to model the change in numbers between the time of sampling and consumption. These studies were useful to help prepare later assessments, e.g., in Sweden.

THE NETHERLANDS Notermans et al. [83] used mice to develop dose–response data. The oral ID50 for nonprotected mice was 6.5 log cells compared with >9.0 log cells for immunologically protected mice with a 1–2 log difference between the ID50 and LD50. The dose for infection through intravenous injection was lower. The results showed that both the intestinal barrier and the specific immune defense mechanism were highly effective in preventing infection of mice orally exposed to L. monocytogenes and support the epidemiological findings that listeriosis is a rare disease in humans, despite frequent exposure to the organism.

SWEDEN Gravad or hot- or cold-smoked salmon and rainbow trout are popular RTE foods in Sweden. After processing, the fish is packaged and stored for varying times at refrigerator temperatures before consumption without prior heat treatment. Packaged products made from both types of fish have been identified as potential sources of human listeriosis. A quantitative risk assessment model developed by Lindqvist and Westöö [78] showed the risk per serving and the annual cumulative individual risk based on number of exposures and two different dose–response relationships from the literature. Growth or inactivation was not included in the model. The constant r is specific for each pathogen and helps define the shape of the dose–response curve. In one dose–response study, with an exponential dose–response model, a conservative r value for L. monocytogenes was estimated from the levels in smoked fish and epidemiological data in Germany (GR) [13]. The authors made the assumption that foods with a concentration over 104 L. monocytogenes CFU/g cause illness only in the high-risk population. The second dose–response model chosen was the flexible WeibullGamma (WG) model suggested by Farber et al. [39], which was less conservative. Using these relationships the estimated number of cases in the population was calculated and compared to the annually reported number of listeriosis cases in Sweden. The estimated mean risk per serving was 2.8 × 10 −5 (GR, high-risk group), 2.0 × 10−3 (WG, low-risk group), and 1.6 × 10−2 (WG, high-risk group), respectively. The average number of reported listeriosis cases in Sweden is 37 per year. In comparison, the mean number of annual cases predicted by the risk assessment model was 168 (range 47 to 2800, GR, high-risk group), and 95,000 (range 3.4 × 104 to 1.6 × 10 6, WG, high-risk group), respectively. If 1 to 10% (uniform distribution) of strains, instead of all, were considered virulent, the mean number of predicted cases would decrease to 9 (GR) and 5200 (WG), respectively. Even with underreporting of actual cases, this assessment shows the difficulty in estimating a reasonable number compared to what is expected. The WG model predicted far greater risks than

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the exponential model, and thus, choosing the dose–response model that best fits the data is critical. However, that model may not yet exist. Lindqvist and Westöö [78] stated that apart from dose–response issues, the most important parameter determining the risk was the concentration of L. monocytogenes at the time of consumption.

UNITED STATES Peeler and Bunning’s [88] assessment of L. monocytogenes during processing of bovine milk was one of the earliest publications in microbial risk assessment. However, this study was more a limited exposure assessment rather than a complete risk characterization setup to help establish a hazard analysis and critical control point (HACCP) plan for bovine milk up to and including pasteurization. The authors aimed to demonstrate how determination of critical control points could be more objective using quantitative modeling methods. Survey or experimental data were used wherever possible to model pathogen incidence, potential growth during storage and transport, and death during pasteurization. In comparing their predictions to available data for the probability of contamination of retail units of pasteurized milk in North America, the authors found that the incidence of contamination was much higher than their model predicted, probably because of contamination after pasteurization. Cassin et al. [20] considered that this assessment had overly compounded conservatism into the model. Hitchins [66] used survey data on the frequency of foodborne occurrence and dietary exposure to L. monocytogenes to estimate the minimal mean per person annual rate of exposure in the United States during the late 1980s. The mean amount of each food type per L. monocytogenes occurrence was calculated for about 100 sources, and dietary intake data from published U.S. literature were used to calculate the mean number of occurrences of L. monocytogenes consumption per person per year. The mean number of occurrences of exposure to L. monocytogenes consumed annually per person was determined to be 10 to 100, when the proportion of RTE food of the total dietary intake was estimated at 2 to 20%, respectively. The FDA conducted a risk ranking for L. monocytogenes in collaboration with USDA and CDC [42]. This draft risk assessment compared the risks of illness and death arising from L. monocytogenes in 20 RTE categories. Risk ranking assessments are useful to estimate which foods present the greatest and the least risks per serving or in a population to help prioritize control actions. They are most useful not only to guide regulatory activities, but also to determine data gaps, research needs, educational activities, and predominant risk factors. The working assumption used was that all listeriosis cases were attributed to different categories of foods, so that the risk assessment could be “anchored” to the U.S. public health statistics. A newer version of the risk assessment incorporated changes in response to the submitted comments and newly available data for 23 RTE food categories, including data submitted by the National Food Processors Association (NFPA, see the following text) [43]. This version also contained improved modeling techniques, as well as changes to the model inputs and the use of “what if” scenarios. The assessment was based on foods at the retail level and the overall burden of listeriosis on public health and considered three age-based groups of people (perinatal, i.e., fetuses and newborns, elderly and intermediate age, i.e., remaining population, mostly healthy). The risk assessment used 2078 cases of listeriosis and 390 fatalities as the baseline values for the national annual public health impact of the disease, based on values projected from an average of CDC’s surveillance data from 1997 to 2000. An assumption made was that there were 2460 cases of listeriosis in the United States every year, and it was the rankings within this by food category that were the most important outputs. Much of the exposure assessment modeled changes in contamination levels during refrigerated storage and reheating in the home. The dose–response model was internally designed by FDA. The results of the exposure and dose–response assessments were combined to provide an estimate of risk on a per-serving and annual basis. Although the analysis contained considerable variability and uncertainty, this risk assessment indicates that in spite of consumers being regularly exposed to low levels of L. monocytogenes, relatively few people become seriously ill.

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For the total U.S. population the predicted median risk per serving for foods ranged from a high of 7.7 × 10 −8 (deli meats) to a low of 4.5 × 10−15 (aged hard cheese). The total predicted median number of annual cases in the United States was 1598.7 for deli meats, 90.8 for pasteurized milk, 56.4 for highfat and other dairy products, 30.5 for frankfurters not reheated, 7.7 for soft unripened cheeses, 3.8 for pâté and meat spreads, 3.1 for unpasteurized milk, 2.8 for cooked RTE crustaceans, 1.3 for smoked seafood, and less than 1 for all other categories. The differential between per-serving risks associated with deli meats (relative risk rank of 1) and hard cheeses (relative risk rank of 23) is almost 10,000,000fold. Values were also determined for each subpopulation (perinatal, elderly, and intermediate age). Scenarios were also run to allow comparison of baseline calculations to new situations that might arise as a result of potential risk reduction strategies. The predicted annual number of listeriosis cases would be reduced from 2105 to 656 (69%) if all home refrigerators operated at 45°F, and to 28 (>98%) annually if temperatures were at 41°F. If the storage time for deli meats was reduced from the maximum of 28 days to 14 days, the median number of listeriosis cases in the elderly population would drop from 228 to 197 (13.6% reduction), and a further benefit (total of 154 cases) would result if the storage time was decreased to 10 days (32.5% reduction). Conversely, increasing the maximum storage time for smoked seafood from 30 to 45 days more than doubled the predicted median number of listeriosis cases for the elderly subpopulation (0.8 vs. 2.1). The “what if” scenarios also predict that inclusion of any treatment resulting in a 1-log reduction in contamination of deli meats at retail would reduce the number of predicted cases in the elderly population by 50%, from 227 to 120, and a 2-log treatment would result in a 74% reduction. Data incorporated into the risk assessment largely came from commercially produced Hispanic soft cheeses, whereas the cheeses linked to the disease have most often been associated with noncommercially produced cheese made from raw milk. Consequently, when the exposure model was constructed using contamination data representative of raw milk, the estimated risk per serving was 43 times greater for the neonatal population and 36 times greater for the elderly population when these cheeses were made from unpasteurized as opposed to pasteurized milk. The highest risk categories on both per-serving and per-annum bases were deli meats and frankfurters that have not been reheated. This is because they have relatively high rates of contamination, support the relatively rapid growth of L. monocytogenes under refrigerated storage, are stored for extended periods, and are widely consumed. These products also have been directly linked to outbreaks of listeriosis. They should be given particular attention for development of new control strategies and consumer education programs to help achieve the national goal for reducing the incidence of foodborne listeriosis. Pâté and meat spreads, smoked seafood, and unpasteurized fluid milk have relatively high rates of contamination and thus high predicted per-serving relative risks, but are consumed in small quantities by a relatively small portion of the population. However, all three products have been associated with outbreaks or sporadic cases, which justifies new control measures or recommendations for avoidance. The assessment made some general conclusions: •

•

•

The risk assessment reinforces past epidemiological conclusions that foodborne listeriosis is a moderately rare, although severe, disease. U.S. consumers are exposed to low to moderate levels of L. monocytogenes on a regular basis. The risk assessment supports the findings of epidemiological investigations of both sporadic illness and outbreaks of listeriosis that certain foods are more likely to be vehicles for L. monocytogenes. Three dose–response models were developed that relate exposure to different levels of L. monocytogenes in three age-based subpopulations (i.e., perinatal [fetuses and newborns], elderly, and intermediate age) with the predicted number of fatalities. These models were used to describe the relationship between levels of L. monocytogenes ingested and the incidence of listeriosis. The dose of L. monocytogenes necessary to cause listeriosis depends greatly upon the immune status of the individual, and strategies for control and prevention need to be designed for these populations.

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•

•

The dose–response models developed for this risk assessment considered, for the first time, the range of virulence observed among different isolates of L. monocytogenes. The dose–response curves suggest that the relative risk of contracting listeriosis from lowdose exposures could be less than previously estimated. The exposure models and the accompanying “what-if” scenarios identify five broad factors that affect consumer exposure to L. monocytogenes at the time of food consumption.

These five factors affecting consumer exposure to L. monocytogenes at the time of food consumption are (1) amount and frequency of food consumption, (2) frequency of contamination and levels of L. monocytogenes in RTE food, (3) potential of the food to support growth of L. monocytogenes during refrigerated storage, (4) refrigerated storage temperature, and (5) duration of refrigerated storage before consumption. Any of these factors can affect potential exposure to L. monocytogenes from a food category. These factors are “additive” in the sense that foods in which multiple factors favor high levels of L. monocytogenes at the time of consumption are typically more likely to be riskier than foods in which a single factor is high. These factors also suggest that several broad control strategies could be used to reduce the risk of foodborne listeriosis, such as product reformulation to reduce the potential for L. monocytogenes growth or encouraging consumers to keep refrigerator temperatures at or below 40°F and reduce refrigerated storage times. To eliminate some of the data gaps in the FDA/FSIS/CDC [42] assessment, the NFPA conducted a survey of over 31,000 RTE retail food samples, representing eight product categories, that showed an overall prevalence rate of 1.82%, with these data then used to conduct a risk assessment [24,61]. The rationale for this risk assessment was to develop more effective management strategies for the pathogen because the existing zero-tolerance policy makes no distinction between foods containing high and low levels. The confidence interval for prevalence of L. monocytogenes was 1.68 to 1.97%, which ranged from 2 to 6 log CFU/g, and could be fitted to a beta distribution (0.29, 2.68, 1.69, 6.1). An exponential model with an r value (the probability of a single cell causing illness) of 1.76 × 10−10 for the population at highest risk was used for the dose–response assessment. Based on this assessment, the NFPA (now Food Products Association) argued that management strategies focusing on levels of L. monocytogenes rather than its presence alone should have a greater impact on improving public health by facilitating development of control measures to limit maximum levels of L. monocytogenes in foods. The Food Safety and Inspection Service (FSIS) risk assessment [58] was initiated in February 2002 in response to public comments on the FSIS-proposed rule: Performance Standards for the Production of Processed Meat and Poultry Products [57] and incorporated material from the FDA/FSIS/CDC [42] draft risk ranking. This ranking indicated that deli meats posed the greatest public health risk for listeriosis of all the RTE foods. Several comments indicated that the proposal should require testing and sanitation of food contact surfaces for Listeria species, where the relationship between the prevalence and levels of Listeria species in the plant environment and RTE meat and poultry products is not well understood. This model was originally released for public comment and review in January 2001. Based on review and comments, the exposure assessment for deli meats (and hot dogs) and the dose–response relationship were updated. The FSIS Listeria risk assessment was designed to simulate RTE food production within the processing plant and predicts the L. monocytogenes concentrations at retail. The updated FDA/FSIS exposure assessment for deli meats and the updated dose–response relationship were used to model the distributions of L. monocytogenes concentrations in RTE products at retail through consumption and estimate the subsequent annual number of deaths and illnesses. The model assumes that a Listeria reservoir exists in the plant and is capable of contaminating the food contact surface. These reservoirs include harborage sites, floor drains, air-conditioning ducts, etc. The model assumes that Listeria species move from this reservoir onto the food contact surface during what is termed a contamination event. The key parameters defining a contamination event are as follows: (1) the

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time between initialization of events (i.e., how often is a food contact surface contaminated?), (2) the duration of the event, and (3) the number of Listeria transferred from the in-plant reservoir to various food contact surfaces. Once on the food contact surface, these organisms can be transferred to the lot of RTE product being produced, removed through sanitation after producing each lot of product, or remain on the surface. If the contamination event is continuous, new organisms transferred from the reservoir will be added to those already on the food contact surface. For each lot produced, the food contact surface can also be tested for Listeria species, with various mitigation steps taken if the surface tests positive. A positive food contact surface test can trigger a required lot of RTE product to be tested for L. monocytogenes. It can also trigger more intensive sanitation (i.e., enhanced sanitation) of the food contact surface at the end of production. Some fraction of the Listeria organisms on the food contact surface is transferred to the lot. This fraction is the transfer coefficient, which can range from 0 to 1. A transfer coefficient of 0 indicates that none of the Listeria cells are transferred. A transfer coefficient of 1 indicates that all the organisms present are transferred to the product lot being manufactured. Based on limited data not involving Listeria, transfer coefficients were assumed to be log normally distributed (normally distributed on the log scale) with a mean of –0.14 and a standard deviation of 1. The assessment data generally show that L. monocytogenes concentrations in RTE product decline at retail as food contact surface testing and sanitation efforts increase. The most effective strategy is postprocess intervention or control (90–95% effective) combined with growth-inhibiting packaging or product reformulation (90–95% effective). However, this assumes 100% adoption and compliance by industry. Contamination from the baseline model would drop from 2.06 × 105 to 1.67 × 103 under the most effective scenario. The baseline model was calibrated to 310 deaths per year among the elderly, 67 intermediate age deaths per year, and 16 neonatal (newborn) deaths per year in the U.S. population. These would drop to 59, 13, and 3.3, respectively, if the best intervention and control practices were introduced. The main findings of the assessment were as follows: 1. Food contact surfaces found to be positive for Listeria species greatly increased the likelihood of finding positive lots for L. monocytogenes. 2. The duration of Listeria contamination on food contact surfaces is about a week. 3. Increased frequency of testing food contact surfaces and subsequent sanitation leads to a proportionally lower risk of listeriosis. 4. A combination of intervention procedures (e.g., sanitation, pre- and postprocessing interventions, and use of growth inhibitors or product reformulation) appears to be more effective than any one intervention alone in reducing L. monocytogenes contamination and the number of illnesses and deaths. A slightly modified draft risk assessment for deli meats was released for comment a few months later [59] to incorporate changes in the final FDA/FSIS/CDC [43] risk assessment compared with the draft FDA/FSIS/CDC [42] report. Transfer coefficients are being generated to improve risk assessments such as those by FSIS and FDA [74,113], which will further improve the model. (See Figure 18.2.)

INTERNATIONAL The Report of the FAO Expert Consultation on the Trade Impact of Listeria in Fish Products [35] was developed to assist in the process of assessing the risks of listeriosis from fishery products, and also identify data gaps relating to L. monocytogenes in fish and fish products. The risk assessment was not considered to be quantitative because of time constraints. Products having the potential to contain and not contain high levels of L. monocytogenes at the time of consumption were included. Growth limits and maximum growth rates under different conditions were tabulated. A predictive mathematical model for growth under conditions representative of fish products was

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FIGURE 18.2 Proposed decision tree for the establishment of L. monocytogenes criteria in foods. (From FAO. 1999. Report of the FAO Expert Consultation on the Trade Impact of Listeria in Fish Products, Amherst, USA, May 2000. FAO Fisheries Report No. 604. (FIIU/ESNS/R604). Food and Agriculture Organization, Rome.)

mentioned with some predictions listed, but the model was not used in the exposure assessment. In fact, many of the data discussed were not used in the assessment or referenced. Missing information for an exposure assessment was clearly noted, especially those from tropical regions. Their calculations suggest that per capita human exposure to L. monocytogenes doses exceeding 1000 CFU is likely to occur several times each year. Despite this exposure, the total incidence of invasive listeriosis is estimated to be only 2–10 cases per million population per annum. The document primarily dealt with risk management issues and recommended that fishery products have a “use by date,” such that the product will contain <100 L. monocytogenes CFU/g at the end of shelf life. Furthermore, health authorities should ensure that the stated shelf life for RTE products that support growth of L. monocytogenes is within safe limits and that highly susceptible individuals are informed and provided with guidelines about safe food handling practices. Because of a lack of EU standards for L. monocytogenes in foods other than dairy products, a study was conducted to evaluate the risks of various RTE foods and reported as Opinion of the Scientific Committee on Veterinary Measures Relating to Public Health on Listeria monocytogenes [28]. This study was considered particularly important because member countries have different or no standards for this pathogen in foods, and this issue has complicated intra-EU trade. Their work was a review of some of the earlier risk assessments which the authors admit did not constitute a full risk assessment. The hazard characterization approach discussed most completely was the

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mouse dose–response model of Notermans et al. [83] where there was a protective effect of the physical barrier in the gastrointestinal tract along with prior immunity. In addition, growth of L. monocytogenes would be different in different RTE foods based on limiting factors of pH, water activity, temperature, salt and other chemical preservatives, and competitive flora. Examples were given using UK Food MicroModel and USDA Pathogen Modeling Program software to predict growth of the pathogen in five RTE foods. Very little consumption data were available for the different EU countries. The report states that L. monocytogenes levels <100 CFU/g represent a very low risk to consumers, but consumption patterns, as yet not known, may influence any management policy. The authors recommend criteria based on groupings of RTE foods with different control potentials (see the section titled “Standards and Criteria”). Codex Alimentarius Commission The CAC requested that FAO and WHO collaborate with world experts to assess a variety of microbial foodborne risks. The FAO/WHO program initiated work on various pathogen–commodity combinations, one of them being L. monocytogenes in RTE foods to respond to CAC and member country needs. This L. monocytogenes assessment resulted in an extensive monograph and interpretive summary [36,37]. The current risk assessment was undertaken, in part, to determine how previously developed risk assessments done at the national level could be adapted or expanded to address concerns related to L. monocytogenes in RTE foods at an international level. In addition, the risk assessors were asked to consider three specific questions related to RTE foods. These were the issues addressed: 1. Estimate the risk from L. monocytogenes in food when the number of organisms ranges from 0 in 25 g to 1000 CFU per g or mL, or does not exceed specified levels at the point of consumption. 2. Estimate the risk for consumers in different susceptible population groups (elderly, infants, pregnant women, and immunocompromised patients) relative to the general population. 3. Estimate the risk from L. monocytogenes in foods that support growth and foods that do not support growth at specific storage and shelf life conditions. RTE Foods Selected Four RTE foods were selected as examples where L. monocytogenes contamination would demonstrate different risks. Pasteurized milk is widely consumed and has very low frequencies and levels of contamination but allows growth during storage. Ice cream is similar to milk but does not permit growth during storage. Fermented dried sausages are often contaminated and are produced without any lethal processing step, but the final composition prevents growth during storage. Cold-smoked fish is frequently contaminated, has no lethal processing step, and permits growth during extended storage. Both fermented meats and smoked fish have low consumption rates compared with milk and ice cream. The four selected foods illustrate how different factors interact to affect the risk of listeriosis per one million servings and the risk per 100,000 people per year. The four foods were modeled with the same general structure: contamination frequency and level at retail; growth or inactivation until consumption using storage temperatures, storage times, exponential growth rates or death rates, lag phases, maximum growth, and consideration of spoilage; frequency and amount of consumption; and dose–response relationship for healthy and susceptible populations. However, available data and approaches to the modeling process were not always the same for each of the four foods. Initial Contamination at Retail Data from published scientific papers, government surveys, and the FDA/FSIS/CDC [42] risk assessment were collected by the risk assessment team. Data from all countries and years that were

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found in the literature were included in this assessment. Most of the data related to prevalence, usually presence or absence of the organism, with determinations based on an analytical sensitivity of 0.04 L. monocytogenes/g (1 microorganism per 25-g sample). An estimate of uncertainties about presence or absence data was made with a Beta distribution, thereby including the effect of the number of samples in a data set. Only a small portion of the available data sets stated levels of L. monocytogenes/g in positive samples. These quantitative data were arrayed as a cumulative frequency distribution. After uncertainty ranges were assigned, these distributions were used for estimating the levels of L. monocytogenes in foods evaluated at the point of purchase. Growth before Consumption A survey of 939 home refrigerators conducted by Audits International [10] in the United States provided data for considering the impact of home storage temperatures on the levels of L. monocytogenes at consumption. A cumulative distribution of the data was used without any model fitting. The 5th, 50th, and 95th percentile temperatures were 0.5, 3.4, and 6.9°C, respectively. Other published home refrigerator temperature studies in the United Kingdom, United States, and Greece were converted to distributions but were not used in the final analysis; all these would generate a warmer range of temperatures, particularly the Greek information, that would increase the potential for growth. Total storage times at retail and in homes were based on expert opinion. Triangular distributions for the variation in storage times were defined by minimum, most frequent, and maximum times, e.g., milk was given values of 1, 5, and 12 days, respectively. To further emphasize and explore the uncertainty associated with storage time values, the most likely and maximum values were given uniform distributions, e.g., for milk, the most likely value was assigned an uncertainty range of 4 to 6 days. Storage times and temperatures were not considered by the risk assessors to be independent, and models allowed spoilage or growth of lactic acid bacteria (in smoked salmon) to be limiting factors. The storage time and temperature data were used in combination with information on growth rates of L. monocytogenes to estimate how levels in foods were likely to change between point of purchase and time of consumption. Most growth rates in the selected foods were from published inoculated pack studies where foods with their normal spoilage flora were inoculated with L. monocytogenes. The inoculated foods were stored at various temperatures and sampled at various times with L. monocytogenes enumerated, and the exponential growth rate determined. Except for ice cream, predictive models were used to estimate growth and inactivation rates, and growth limits for L. monocytogenes in foods. Whenever possible, the growth model also considered the effect of temperature on the maximum growth level. Consumption Unlike prevalence and concentration data, consumption patterns are often regionally specific. Thus, examples of serving size and consumption frequencies were either taken from Canadian Federal-Provincial Nutrition Survey’s databases or were estimated globally from national consumption statistics. Data were used to represent the total population or an estimated susceptible (at-risk) population. Serving frequencies were calculated as both the probability of consumption during a day and the total number of servings per year for 100,000 people. The survey data were not complete for all ages, nor were there any seasonal patterns of consumption. Dose–Response Assessment The outputs from the exposure assessment were fed into the dose–response models. Distribution at consumption was characterized as a cumulative frequency of log10 CFU/serving of contaminated food. Uncertainty estimates accompanied each percentile value to estimate confidence in the accuracy of the percentiles. Other output values were the Beta distribution for contamination

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frequency, the number of servings per year, and size of servings. Twelve dose–response models based on outbreak data, animal dose studies, and expert opinion were reviewed. Most models were exponential, but one was combined with a Beta-Poisson model and another was a WeibullGamma model. Each of these had different r values (see the following text). The exponential dose–response model was chosen because it was a simple single-parameter model, it fitted well when modeling severe listeriosis, and it could be extrapolated to the low-dose ranges of interest. The equation is P=1−e

−r*N

where P is the probability of severe illness, N is the number of L. monocytogenes cells consumed, and r is the parameter that defines the dose–response relation for the population being considered. The r value is considered to be a constant for a specified population, but its accuracy is dependent on size and inclusiveness of the population being considered, accuracy of listeriosis surveillance data, reliability of the frequency, and level of L. monocytogenes contamination in the food. The exponential model is a nonthreshold model that implies that there is no minimum infectious dose. Instead the model assumes that a single L. monocytogenes cell has a very small but finite probability of causing illness. The maximum levels of L. monocytogenes encountered in foods have a large impact on the calculated maximum ingested doses. The r values were estimated for 4-point estimates of the maximum doses of 7.5, 8.5, 9.5, and 10.5 log CFU, respectively. The lower the maximum dose assumed, the larger is the estimated r value. The larger the r value, the greater is the assumed virulence of L. monocytogenes. The available contamination and epidemiological data for L. monocytogenes did not permit a clear choice of the most appropriate r values for different populations. Different r values were chosen to address the questions posed by the CCFH. For CCFH Question 1, an r value of 5.85 × 10 −12 was used for the susceptible population. This was the most “conservative” (i.e., the greatest assumed virulence for L. monocytogenes) dose–response curve used in the current risk assessment and was calculated under the assumption of a maximum dose of 7.5 log CFU per serving. For illustrating how to estimate r values based on relative risks for different susceptible subpopulations in Question 2, an r value of 5.34 × 10 −14 was selected as the reference value for the general healthy population. This r value was calculated based on the assumption of an intermediate maximum dose, 8.5 log10 CFU per serving, in food. For the food examples described in the risk assessment and CCFH Question 3, the r values used were based on the use of Monte Carlo simulation techniques in combination with a discrete uniform distribution wherein the maximum number of L. monocytogenes consumed varied from log10 7.5 to 10.5 CFU per serving. For the median population with increased susceptibility, the median r value used with its distribution was 1.06 × 10−12 and for the healthy population this was 2.37 × 10−14. Risk Characterization Exposure assessment outputs and dose–response relationships were combined in the risk characterization portion of the risk assessment to calculate the probability of contracting listeriosis. Distributions of prevalence and level of L. monocytogenes in contaminated food at consumption and dose–response relationships led to estimates of risk per million servings for healthy and susceptible populations. The risk per serving and number of servings were used to estimate the number of illness per 100,000 people per year (Table 18.1). It is clear that the risks for fermented meats and ice cream are much less than for smoked fish and pasteurized milk. Not only are initial prevalence rates and concentration levels important, but so also are the potential for growth during storage and amounts of these foods typically consumed.

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TABLE 18.1 Estimated Risk of Listeriosis for Selected Foods Using a Range of r Values for Both Healthy and Susceptible Populations Food

Cases of Listeriosis per 10 Million People

Cases of Listeriosis per 1 Million Servings

9.1 0.012 0.00066 0.46

0.005 0.000014 0.0000025 0.021

Pasteurized milk Ice cream Fermented meats Smoked fish

Source: FAO/WHO. 2004. R. Buchanan, R. Lindqvist, T. Ross, M. Smith, E. Todd, and R. Whiting. Risk Assessment of Listeria monocytogenes in Ready-toEat Foods—Technical Report. Microbiological Risk Assessment Series 5. 304 pp. http://www.fao.org/es/esn/food/risk_mra_listeria_report_en.stm. Accessed October 1, 2004.

Response to Specific Codex Questions Codex Question 1: Estimate the risk from L. monocytogenes in food when the number of organisms ranges from 0 in 25 g to 1000 CFU/g or mL or does not exceed specified levels at the point of consumption. The most conservative dose–response model was used; i.e., maximum virulence of L. monocytogenes was assumed. The r value for this relationship was 5.85 × 1012. The dose ingested is a function of the pathogen level in the food (CFU/g) multiplied by the serving size. Thus, the equation for calculating the probability of listeriosis was P=1−e

(5.85 × 10−12) (31.6g × n)

where n is the number of L. monocytogenes CFU/g.

By substituting different values for n, the likelihood of listeriosis at levels between 0.04 and 1000 CFU/g was calculated. The total number of RTE servings was assumed to be 6.41 × 1010, and the corresponding number of listeriosis cases for the susceptible population was considered to be 2130, both based on the FDA/FSIS/CDC [42] draft risk assessment. The results in Table 18.2 show a large number of predicted cases for servings containing higher contamination levels. Another approach would be to employ a known or estimated distribution of L. monocytogenes levels in foods when consumed. The example chosen was the overall distribution of L. monocytogenes levels in 20 classes of RTE foods from the FDA/FSIS/CDC [42] risk assessment. This, then, could be combined with the dose–response assessment to estimate the number of cases (Table 18.3). The large difference in estimated number of cases (shown in Tables 18.2 and 18.3) is not only a factor of the dose–response model chosen, but also of distribution of contaminated servings consumed. As either the frequency of contamination or the level of contamination increases, so does the risk and the predicted number of cases. Thus, if L. monocytogenes increased in all RTE foods from 1 to 1,000 CFU per serving, the risk of listeriosis would increase 1,000-fold if the serving size remained the same.

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TABLE 18.2 Probability of Illness and the Estimated Number of Cases per Year in the United States for Different Levels of L. monocytogenes at the Time of Consumption Level (CFU/g)

Dosea (CFU)

Log10 Dose (log10 CFU/serving)

Probability of Illness per Serving

< 0.04 0.1 1 10 100 1000

1 3 32 316 3160 31600

0 0.5 1.5 2.5 3.5 4.5

7.39 × 1012 1.85 × 1011 1.85 × 1010 1.85 × 109 1.85 × 108 1.85 × 107

Relative Riskb 1 2.5 25 250 2,500 25,000

Estimated Number of Cases per Yearc 0.54 d 1 12 118 1,185 11,850

a

Serving size of 31.6 g. A fixed serving size of 31.6 g was assumed to simplify calculations because it approximates to a typical serving size and because dose levels were estimated in 0.5 log10 increments (100.5 = 3.16). b Using the risk from a dose of 1 CFU as reference. cA total of 6.41 × 1010 serving per year assumed. d<1 per year. Source: From FAO/WHO. 2004. R. Buchanan, R. Lindqvist, T. Ross, M. Smith, E. Todd, and R. Whiting. Risk Assessment of Listeria monocytogenes in Ready-to-Eat Foods—Technical Report. Microbiological Risk Assessment Series 5. 304 pp. http: //www.fao.org/es/esn/food/risk_mra_listeria_report_en.stm. Accessed October 1, 2004.

TABLE 18.3 Predicted Annual Number of Listeriosis Cases When the Level of L. monocytogenes Was Assumed Not to Exceed a Specified Maximum Value and the Levels in L. monocytogenes in the Food Are Distributed as Indicated in Table 18.2 Level (CFU/g)

Maximum Dosea (CFU)

0.04 0.1 1 10 100 1000

1 3 32 316 3,160 31,600

a

Percentage of Servings When at Maximum Levelb 100.0 3.6 1.7 0.8 0.4 0.2

Estimated Number of Listeriosis Cases per Yearc 0.5d 0.5d 0.7d 1.6 5.7 25.4

Serving size of 31.6 g. Number of servings in the highest L. monocytogenes level assumed divided by 6.41 × 1010 times 100. cLevels of L. monocytogenes per serving used to calculate predicted number of cases based on the overall distribution from the FDA/FSIS/CDC. From FDA/FSIS/CDC. 2001. Center for Food Safety and Applied Nutrition, Food and Drug Administration/Food Safety and Inspection Service, USDA and Centers for Disease Control and Prevention. Draft assessment of the relative risk to public health from foodborne Listeria monocytogenes among selected categories of ready-to-eat foods. http: //www.foodsafety.gov/~dms/lmrisk.html. Accessed October 1, 2004) risk assessment. A total of 6.41 × 1010 serving per year was assumed. d <1 case per year. b

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TABLE 18.4 Hypothetical “What-if” Scenario Demonstrating the Effect of Different Defect Rates for Servings on the Number of Predicted Listeriosis Cases Assumed Percentage of Defect Servings a 0.0 0.00001 0.0001 0.001 0.01 0.018 0.1 1.0

Predicted Listeriosis Cases Where the Limit Is 0.04 CFU/g b

Predicted Listeriosis Cases Where the Limit Is 100 CFU/g b

0.5 1.7 12.3 119 1185 2133 11,837 117,300

5.7 6.9 17.4 124 1191 2133 11,848 117,363

a

All defective servings are assumed to contain 10 CFU/g. r value of 5.85 × 1012 and serving size of 31.6 g.

b

Conversely, the effect of introducing into the food supply 10,000 servings contaminated with L. monocytogenes at a level of 1,000 CFU/g would, in theory, be compensated by removing from the food supply a single serving contaminated at a level of 107 CFU/g. These scenarios are made on the assumption that they occur without any deviation from the set limits. In fact, the zero tolerance policy for the United States is equivalent to the first scenario in Table 18.3 (a maximum of 0.04 CFU/g or a dose of 1 CFU). If compliance were 100%, less than one case of listeriosis per year would occur in the United States, compared with the estimated 2,130 baseline level in the FDA/FSIS/CDC [42] risk assessment. If a microbiological limit of 0.04 CFU/g with a defect rate of 0.018% (2,133 cases) is replaced by a 100 CFU/g limit and a 0.001% defect rate (124 cases), the predicted result would reduce the number of listeriosis cases by about 95%. (Table 18.4). Compliance, therefore, is a critical issue in reducing illnesses and is more important than whether the regulatory limit is 0.004 or 100 CFU/g.

Codex Question 2. Estimate the risk for consumers in different susceptible population groups. The basic approach taken to developing the requested dose–response relations was to take advantage of epidemiological estimates of the relative rates of listeriosis for different subpopulations. These relative susceptibility values were generated by taking the total number of listeriosis cases for different subpopulations and dividing them by the estimated number of people in the total population with that condition. This value is then divided by a similar value for the general population. The r values for an exponential dose–response curve can be estimated for a subpopulation using a relative susceptibility ratio and a reference r value for the general population, with epidemiological data from France (transplant, AIDS, dialysis, cancer, liver disease, diabetes, alcoholic, >65-year-old patients, and <65 years, no other condition) and the United States (perinatal, >60 years, general population). The relative susceptibility values and corresponding r values increase as the immune system becomes increasingly compromised. The most compromised group, transplant patients, is 2584 times more susceptible to infection than individuals <65 years old with no other medical conditions (the reference population), with r values of 1.41 × 10 −10 and 5.34 × 10−14, respectively. This is followed by leukemia and AIDS patients, respectively, 1364 and 865 (times more susceptible.) Pregnant women/newborns

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at the perinatal stage were 14 times and the elderly <10 times more susceptible. In the two outbreaks in which r values were able to be determined, the r value for the Los Angeles Hispanic cheese outbreak involving pregnant women was very close to that estimated (3 × 10−11 vs. 4.51 × 10−11). The r value for an outbreak in Finland that was traced to hospitalized transplant patients who consumed butter was much higher than the values for transplant patients (3 × 10−7 vs. 1.41 × 10−10). This was explained by the smaller number of individuals exposed in the outbreak, the extremely compromised and highly variable immunological status of the population, or the involvement of a highly virulent strain of L. monocytogenes.

Codex Question 3. Estimate the risk from L. monocytogenes in foods that support growth and foods that do not support growth at specific storage and shelf-life conditions. The risk characterization has shown that the ability of a product to support growth can increase substantially the risk of its being a vehicle for foodborne listeriosis (Table 18.1). To show how much growth is important, the mathematical models in the exposure assessment for pasteurized milk were modified to ignore any increase in numbers; i.e., the milk was consumed immediately after purchase at retail (Table 18.5). The results suggest an approximately 1,000-fold increase in risk can be attributed to predicted growth of L. monocytogenes in pasteurized milk in risk per 100,000 total population. The healthy population has an increased risk over the susceptible population if growth is permitted, because higher numbers of L. monocytogenes are required to infect healthy people. Another scenario was to let the contamination levels of milk be truncated at 100 CFU/g at retail, but with growth still allowed (this only reduces the incidence of listeriosis by about two-thirds). Two other scenarios relate to growth: increased storage temperature from a median of 3.4 to 6.2°C (mean number of illnesses increased over 10-fold for both populations), and increased storage time from a median of 5.3 to 6.7 days (4.5-fold and 1.2-fold for the healthy and susceptible populations, respectively).

A similar approach was taken with smoked fish for not only time and temperature but also the impact of lactic acid bacteria. The risk per serving and instances per 100,000 population increased 700- to 1000-fold in the 80 to 100% suppression model, and 67- to 85-fold in the 95% suppression model from no L. monocytogenes growth to baseline (growth) scenarios. The predicted effect of reducing the shelf life of smoked fish by 50% (1–28 days to 1–14 days) reduced the predicted increase in risk from growth by 80%.

TABLE 18.5 Three Scenarios That Illustrate the Impact of Growth, Contamination, and Storage of Pasteurized Milk on the Estimated Risks of Listeriosis per 100,000 Population Milk Milk baseline No growth Truncate contamination at 100 CFU/g Increase storage temperature from 3.4 to 6.2°C Increase storage time from 5.3 to 6.7 days

Cases of Listeriosis per 100,000 People (Mean Value) 9.1 × 102 6.7 × 105 2.8 × 102 1.2 × 100 2.0 × 101

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Summary of the Assessment The public health impact of an RTE food can be evaluated by both risk per serving and number of cases per population per year, and risks may be different. Models developed predict that nearly all cases of listeriosis result from consumption of high numbers of the pathogen. There are differences in frequency of contamination and distribution of contamination levels within different types of RTE food, although only four RTE foods were chosen for illustration. Differences in manufacturing, storage practices, and consumption patterns in various countries may affect contamination level and, therefore, risk per serving for a food. High levels of contamination at retail are relatively rare, but cases could be reduced if vigilance in food processing facilities keeps sources of L. monocytogenes entering the manufacturing environment to a minimum and there is effective cleanup in the plant. Control measures that reduce the frequency of contamination will proportionaly reduce the rate of illness, provided the proportion of high contamination is similarly reduced. All cases of foodborne listeriosis in the United States and other countries with a similar zero-tolerance policy occur from foods with more than 1 CFU in 25 g. From this assessment, it would appear that many RTE foods exceed this level at manufacturing, although in many instances not by much, with subsequent growth permitting levels to rise to those sufficient to cause illness. However, it is also clear that raising a zero-tolerance standard to a higher value (e.g., 1 CFU/25 g to 100 CFU/g) would increase the incidence of listeriosis unless new or improved mitigation strategies were effective, such as reducing levels of L. monocytogenes in RTE food servings. In foods that permit growth, control measures, such as better temperature control or limiting the length of the storage period, will reduce the increases in risk from higher levels of L. monocytogenes. Specific populations at more risk than others may need specific control conditions not necessary for the general population.

STANDARDS AND CRITERIA Although food safety is mainly improved through efficient implementation of GMP and HACCP systems, including prerequisite programs, microbiological criteria may be used to help producers verify that the HACCP plan has been correctly carried out. In addition, for trade purposes, the burden of microbial load in foods is often required and this applies particularly to Listeria in RTE foods. Currently there is no international agreement on what numbers of L. monocytogenes in foods are acceptable to protect the consumer. In several countries, different criteria or recommendations for tolerable levels of L. monocytogenes in RTE foods have been established [35]. Some countries like the United States, Austria, Australia, New Zealand, and Italy require absence of L. monocytogenes in ≥25 g of food (referred to as zero tolerance). Other European countries, for example, Germany, The Netherlands, and France, have a tolerance of <100 CFU/g at the point of consumption. Others, such as Canada and Denmark, have a tolerance of <100 CFU/g for some foods and a zero tolerance for other foods, especially those with extended shelf lives that can support growth of L. monocytogenes. In the United Kingdom, the standard only officially applies to dairy products. In addition to differences in criteria for L. monocytogenes, several countries use different sampling plans and methodologies that make comparisons difficult for trade purposes. The EU is at the discussion stage to try and harmonize standards and cover all RTE foods that are potentially hazardous. Therefore, there is agreement for the need to develop microbiological criteria for L. monocytogenes in foods at the international level based on the principles of risk assessment, both for protecting public health and for facilitating trade in various products. For regulatory purposes, it has been assumed explicitly or implicitly that all strains of L. monocytogenes are potentially pathogenic, although this may not be true, and discussions on standards have often focused on the dose–response for individuals both in the general population and those at high risk. At the international level, the CAC is encouraging the CCFH to develop guidelines for L. monocytogenes in RTE foods to be used by member countries [19]. Where standards are applied, there is often a sampling plan and

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recommended analytical method to be used. The two- or three-class sampling plans are usually based on ICMSF [68], where n = the number of sampling units from the lot and c = the maximum allowed number of sample units that exceed the microbiological criterion. The three-class plan is used to designate acceptable, marginally acceptable, or unacceptable food where m = the maximum number or level of L. monocytogenes CFU/g or CFU/mL (threshold value), and M = the maximum number used to separate marginally acceptable and unacceptable quality food (maximum permitted level). In the two-class plan, used to designate a batch or lot of food, either acceptable or unacceptable, m = the maximum level of L. monocytogenes CFU/g or CFU/mL.

AUSTRALIA

AND

NEW ZEALAND

The approach to food safety in both Australia and New Zealand is undergoing change as the two countries try to harmonize their regulations. Although both countries and the individual states in Australia have their own food codes and regulations, the Australia/New Zealand Food Authority (ANZFA), created in 1996 to facilitate trade, is attempting to form a unified food safety program [48]. This authority will develop an agreed, uniform legal framework governing food in Australia and New Zealand. Transitional arrangements were negotiated to cover the period as new standards are produced over time. Food Standards Australia New Zealand [55] now takes over responsibility for ANZFA. The transitional arrangements are as follows: • •

• •

New Zealand’s agreement to adopt Australian standards Ordering the work of the current Australian review of the Australian Food Standards Code (AFSC) to give priority consideration to those standards seen as impeding trade between Australia and New Zealand The adoption by New Zealand of the entire AFSC thereby establishing dual standards for that country Recognition by Australia of New Zealand food regulations for New Zealand food sold in Australia during the period, i.e., dual standards in Australia also

During the review of microbiological criteria in developing the joint Australia New Zealand Food Standards Code (ANZFSC), a number of policy principles were established by ANZFA [3]. It was stated that microbiological criteria should be established and applied only where there is a definite need and where its application is practical. A definite need may be identified through a risk assessment. To fulfill the purposes of a microbiological criterion, consideration should be given to the following: • • • • • • •

Evidence of actual or potential hazards to health Microbiological status of the raw materials Effect of processing on microbiological status of the food Likelihood and consequences of microbial contamination and/or growth during subsequent handling, storage, and use Categories of consumers Cost–benefit ratio associated with application of the criteria Intended use of the food

In Australia, the number of listeriosis cases reported averages about 50–70 per year for persons of all ages. In the Australian Capital Territory, nine cases were reported since the disease became notifiable in June 1992 until 1999 [52]. There have been relatively few incidents of listeriosis in these countries with the exception of pâté. In Western Australia in 1990, there were six stillbirths in a cluster of nine cases after contaminated pâté was eaten. A listeriosis warning was issued in

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1995 by the Health and Community Services Department in Victoria after a middle-aged woman became ill after consuming contaminated pâté [47]. In New Zealand in 1992, delivery of stillborne twins was linked to a mother who consumed contaminated smoked mussels during her pregnancy [52]. The safety of foods imported into Australia is evaluated by laboratory testing for microbiological and chemical hazards under the Imported Food Program. The program, operating under the Imported Food Control Act of 1992, is jointly administered by the Australian Quarantine Inspection Service and ANZFA, now FSANZ [14]. Of the 17,685 microbiological tests done on so-called risk foods from 1995 to 1999, there were 486 failures (2.7%), with individual yearly failure rates ranging from 2.3% to 4.0%. Foods tested for Listeria included smoked vacuum-packed fish, soft cheeses, chicken, and mussels. The percentage of total Listeria failures increased sharply until 1998 and then declined in 1999. Of all risk foods tested, smoked vacuum-packed fish had the highest failure rate of 8.6% for Listeria contamination (388 samples tested). Soft cheeses had a 1.1% failure rate of 1140 samples tested. Thus, there was some concern about the pathogen in both countries. In the AFSC, microbiological standards for L. monocytogenes specify the following: not detected in soft cheeses, cheeses manufactured from thermized milk, smoked fish products, smoked mussels, meat paste, and pâté. These standards apply to products sampled at the processing factory or wholesale level, but do not apply to products at the retail level [4]. Microbiological standards for L. monocytogenes are also contained in Standard 1.6.1 Microbiological Limits for Food [5] and the User Guide [6]. These standards for L. monocytogenes include the following: no detection in five 25-g samples (n = 5, c = 0, m = 0) for unpasteurized milk, butter made from unpasteurized milk and/or unpasteurized milk products, soft and semisoft cheese (moisture content >39%) with pH >5.0, all raw milk cheese (cheese made from milk not pasteurized or thermized), unpasteurized milk, packaged cooked cured/salted meat, packaged heat-treated meat paste and packaged heattreated pâté, cooked crustaceans, and mollusks that have undergone processing other than depuration. For RTE processed finfish other than fully retorted finfish, one out of five samples may contain up to 100 CFU/g of L. monocytogenes (n = 5, c = 1, m = 100 CFU/g). Because of industry concerns, FSANZ undertook to review the L. monocytogenes limits set for cooked crustaceans and RTE processed finfish just described. Based on a scientific risk assessment [7], FSANZ recommended that existing microbiological limits for Listeria in finfish be retained, along with zero tolerance for L. monocytogenes in cooked crustaceans, since these products can potentially compromise public health and safety, particularly for vulnerable subgroups. The implications of a zero tolerance are that any product recall would affect the importer or manufacturer, as well as the entire seafood industry, costing millions of dollars (one submitter estimated this cost to be $10 million in terms of business revenue alone, based on a $1/kg reduction in price). Estimates for the cost of compliance vary from A$200,000 to A$530,000 p.a. for the prawn industry, and $60,000 for the rock lobster industry. ANZFA was aware that this regulation comes with a substantial cost to the food industry. However, the potential benefit is a reduction in the number of listeriosis cases in Australia and New Zealand. ANZFA recognized that it is difficult to compare the costs to industry with potential benefits to the community, particularly when some of the community benefits are less tangible, such as quality of life and longevity. However, when setting regulatory policy, ANZFA took a precautionary approach where the public health and safety consequences are severe. Nevertheless, the exclusion position for crustaceans was upheld in the 2003 review [53,54]. This report claimed that modeling of a worst-case scenario, which assumed that cooked prawns would be stored in a domestic refrigerator for a maximum of 3 days after purchase allowing growth, would yield one case of listeriosis from cooked crustaceans every 2.5 years, a risk considered to be acceptable. The report recommended a variation to Standard 1.6.1 by deleting the microbiological limit for L. monocytogenes in cooked crustaceans, because (1) risk assessment work undertaken for P239 shows that L. monocytogenes in cooked crustaceans presents a low risk to public health and safety, and (2) a stringent microbiological standard requiring an absence of L. monocytogenes in cooked crustaceans cannot be justified on public health and safety grounds given the low risk presented by this pathogen–commodity combination. In a similar assessment ANZFA proposed not to amend the microbiological criteria for

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L. monocytogenes in processed RTE finfish (other than fully retorted finfish) of n = 5, c = 1, m = 100 CFU/g in Standard 1.6.1. The assessors concluded that smoked salmon posed a moderate-to-high risk because the shelf life of smoked salmon is 4 to 6 weeks, allowing growth of the pathogen. These actions show that the ANZFA management policy is based on a risk assessment approach. However, these standards apply to specific product types. For foods not mentioned, recall guidelines are similar to the Canadian policy. Category 1 RTE foods are those requiring refrigerated storage that are able to support growth of L. monocytogenes, and RTE foods that have been implicated in human listeriosis (e.g., soft and semisoft cheeses, pâté, cooked cold chicken, coldsmoked fish) and/or which may be consumed by at-risk groups, especially infants. Although the pathogen should not be detected in 25 g, the ANZFSC has a sampling plan for cold-smoked fish that allows one out of five samples to contain L. monocytogenes at levels up to 100 CFU/g (see preceding text). Category 2 includes all other packaged RTE foods and has a limit of 100 CFU/g. In New Zealand, because Listeria contamination of fishery products is seen as a threat to its continued success both domestically and internationally, standards for monitoring L. monocytogenes in the environment and product were developed under the 1995 Fish Export Processing Regulations [86]. A decision tree approach to finding positive environmental testing applies (positives increase the amount of testing until the pathogen is at acceptable levels). Positive product is reprocessed or destroyed. The requirements of industry-agreed standards and FIICC circulars, the 1981 Meat Act and the Fish (Packing for Export) Regulations [87], are mandatory and must be followed. It is recommended that all fish-processing operations in New Zealand use these guidelines to minimize the risk of Listeria contamination of their products. In addition to the policies with standards, FSANZ and ANZFA have produced an educational brochure on Listeria and pregnancy with updated information over several years [8,52,56] listing which foods are safe to eat and which to avoid.

NORTH AMERICA United States The first large listeriosis outbreak in the United States occurred in 1985 when Mexican-style soft cheese in Los Angeles, California caused over 142 cases with 48 fatalities, mainly in perinatal cases. L. monocytogenes was present in several cheese samples, most likely coming from the raw-milk supply when the pasteurizer was bypassed. Surveys done the same year by the FDA found L. monocytogenes in both imported and domestic soft cheeses. It was recognized that L. monocytogenes has caused foodborne disease from food products regulated by FDA and USDA, or had the potential to do so. Therefore, the FDA established a policy of zero tolerance for L. monocytogenes in RTE foods [93]. This means that detection of any L. monocytogenes in either of two 25-g samples of a food renders the product adulterated as defined by the Federal Food, Drug, and Cosmetic Act. This policy was affirmed in the 1995 U.S. District court decision in United States v. Union Cheese Co. In another decision in 2001, a motion was granted to FDA to enjoin a fish processor from violating adulteration provisions of the Food, Drug, and Cosmetic Act [40]. It reemphasized that the responsibility for protecting public health was on the producer, not consumers. The company challenged the adulteration charge based on the presence of L. monocytogenes in its fish products by arguing that this pathogen is not an added substance and does not ordinarily present a risk to health because it affects only some segments of the population. The processor argued that if the court found L. monocytogenes to be an added substance, the FDA’s zero-tolerance policy violates provisions of the Food, Drug, and Cosmetic Act. The court rejected these arguments, finding that (1) L. monocytogenes is an added substance, (2) L. monocytogenes is injurious to the health of significant populations of consumers, and (3) the FDA is not required to set a tolerance level for L. monocytogenes. The USDA also considers L. monocytogenes to be an adulterant in RTE products and enforces a zero tolerance (no detectable level permitted) for this pathogen in RTE meat and poultry products [105].

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Since 1989, FSIS analyzed approximately 3500 samples for L. monocytogenes each year. The following product categories are included in the monitoring program: (1) beef jerky, (2) roast beef, cooked beef, and cooked corned beef, (3) sliced ham and luncheon meat, (4) small-diameter sausage, (5) large-diameter sausage, (6) cooked, uncured poultry, (7) salads and spreads, and (8) dry and semidry fermented sausage. Of 3547 samples of RTE meat and poultry products analyzed in 1998, approximately 2.5% tested positive for L. monocytogenes. FSIS sampling of hot dogs from 1993 to 1996 showed that approximately 4.4% of the samples were positive for L. monocytogenes. Action by both the FDA and FSIS, together with compliance by industry, seemed to be on track in reducing L. monocytogenes contamination in food and the number of listeriosis cases. However, two major outbreaks have caused renewed concern about this pathogen and its control in the United States. From August 1998 to February 1999 an outbreak, which affected at least 100 people and caused 20 deaths, was associated with the consumption of hot dogs and deli meats produced in a Michigan plant [22,95]. In 2002, another major outbreak occurred with 46 cultureconfirmed cases, seven deaths, and three stillbirths or miscarriages in eight states that had been linked to eating sliceable turkey deli meat [23]. The responsible company in Pennsylvania recalled 27.4 million pounds of cooked sandwich meat [89]. This came less than 3 months after a beef supplier recalled nearly 19 million pounds of ground beef because of an E. coli O157:H7 contamination in Colorado. Government agencies are responding to these concerns by encouraging more research, continuing dialogue with industry, and initiating risk assessments. HACCP and GHPs still seem to be a logical way to reduce contamination, but from the information revealed during the outbreak investigations, it seems that compliance with in-house and government policies still needs more attention. Fifteen trade associations submitted a citizen petition on December 24, 2003, requesting that the FDA amend the regulations for the agency to establish a regulatory limit of 100 CFU/g for L. monocytogenes in foods that do not support growth of the microorganism [41]. The petitioners assert that such a regulatory limit would establish a science-based standard for presence of L. monocytogenes in such foods, noting that their request is based on new and emerging evidence that consumer protection is a function of the organism’s cell numbers in food, and not its mere presence, and that there is general scientific agreement that low levels of L. monocytogenes are not uncommon in the food supply and that such low levels are regularly consumed without apparent harm. The agency is requesting comment on the petition. On December 1, 2004, a report outlining the impact of the interim final rule designed to further reduce the incidence of L. monocytogenes in RTE meat and poultry products and making recommendations for possible future actions was released for public comment by FSIS. The report shows that the overall safety of these products has improved in response to the Listeria interim final rule because establishments have strengthened their control procedures, increased testing, and taken additional steps to eliminate the pathogen [109]. Canada Health Canada developed its policy independently of other countries, and not in response to major outbreak problems, unlike the United States. It is considered a risk-based approach with different requirements for different types of RTE foods. The highest priority is given to those RTE foods that have been linked causally to listeriosis and those with a shelf life greater than 10 days [38]. The policy is based on a combination of inspection, environmental sampling, and product testing. Because of changes in government policy in 2000, the Canadian Food Inspection Agency is now responsible for any regulatory action, and Health Canada is required to set policy and standards, and conduct risk assessments. In October 2004, a revised Policy on Listeria monocytogenes in Ready-To-Eat Foods was developed by Health Canada [64]. RTE foods have been placed in three categories, based upon health risk. In the establishment of the Category 1 food list, considerations were given to RTE

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foods that have been causally linked to documented outbreaks of listeriosis and/or have been placed in the high-risk category in the recent FDA/FSIS/CDC [43] assessment. Products in Category 1 should receive the highest priority for inspection and compliance activities. Presence of L. monocytogenes in these RTE foods will trigger a Health Risk 1 concern with consideration of a public alert. Category 2 contains all other RTE foods that are capable of supporting growth of L. monocytogenes and have a shelf life exceeding 10 days. Presence of L. monocytogenes in these products will trigger a Health Risk 2 concern, with possible consideration of a public alert, and receives the second highest priority in inspection and compliance activity. Category 3 contains two types of RTE food products: those supporting growth with a ≤10-day shelf life and those not supporting growth. These products receive the lowest priority in terms of inspection and compliance action. For Category 3 RTE foods, factors such as adherence to GMPs, levels of L. monocytogenes in the food (action level, 100 CFU/g), and/or a health risk assessment, should all be considered in determining the compliance action taken. RTE foods contaminated with L. monocytogenes produced primarily for consumption by individuals who might be considered as most susceptible to listeriosis (neonates, pregnant women, the elderly, and immunocompromised individuals) would be considered a Health Risk 1 or 2 (Table 18.6), but would not be considered a Health Risk 3, regardless of product type. Health risk 1 represents a situation in which there is a reasonable probability that consumption of or exposure to a food will cause adverse health consequences which are serious or life threatening, or where the probability of a foodborne

TABLE 18.6 Compliance Criteria for L. monocytogenes in Ready-to-Eat (RTE) Foods in Canada Category 1. The list presently includesa soft cheese, liver pâté, unacidified jellied pork tongue, hot dogs/wieners, cold-smoked rainbow trout, and processed deli turkey meat 2. All other RTE foods supporting growth of LM with a refrigerated shelf life >10 days (e.g., vacuum-packaged meats, modifiedatmosphere-packaged sandwiches, refrigerated sauces) 3. RTE foods supporting growth of LM with a refrigerated shelf life of 10 ≤days (e.g., packaged salads) and all RTE foods not supporting growthb (e.g., ice cream, hard cheese, dry salami, salted fish, breakfast, and other cereal products)

Action Level for LM

GMPs Status

Nature of Concern

Detected in 50 g

NA

Health Risk 1

Detected in 25 g

NA

Health Risk 2

Adequate GMPs Inadequate, absent, or no information on GMPs NA

Health Risk 3 Health Risk 2

≤100 CFU/g ≤100 CFU/g

>100 CFU/g

Health Risk 2

Note: LM = L. monocytogenes, NA = not available, GMP = Good Manufacturing Practices a

RTE foods causally linked to documented outbreaks of listeriosis and/or to RTE foods rated as “high risk” in the FDA/FSIS risk assessment (from FDA/FSIS/CDC. 2003. Center for Food Safety and Applied Nutrition, Food and Drug Administration/Food Safety and Inspection Service, USDA and Centers for Disease Control and Prevention. Quantitative assessment of the relative risk to public health from foodborne Listeria monocytogenes among selected categories of ready-to-eat foods. http: //www.foodsafety.gov/~dms/lmr2-toc.html. Accessed October 1, 2004.). bA refrigerated RTE food not supporting growth of L. monocytogenes includes the following: pH 5.0–5.5 and a w <0.95; pH <5.0 regardless of aw; aw ≤0.92 regardless of pH; or frozen foods.

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outbreak situation is considered high. Health Risk 2 represents a situation in which there is a reasonable probability that consumption of or exposure to a food will cause temporary or non–life threatening health consequences, or the probability of serious adverse health consequences is considered remote. Health Risk 3 represents a situation in which no health hazard has been identified and there is a reasonable probability that consumption of or exposure to the food is not likely to result in any adverse health consequence.

EUROPEAN COUNTRIES Austria Austria has a zero-tolerance policy for most foods, such as fish, and not only those that are RTE products [2,35]. This presents some problems for the industry. For instance, all raw meat is required by the existing law to be free of L. monocytogenes in 25 g, which could result in many lots being recalled; therefore, these products are not routinely tested for the pathogen (personal communication, Franz Allerberger, Institute for Hygiene and Social Medicine, Bereich Hygiene, University of Innsbruck, Austria). There are exceptions to this policy, brought about through individual court actions. These are raw sausage, e.g., salami and mettwurst (spreadable, pâté-like), raw pork products cured with nitrate, other meat and fish products with added chemical preservatives, and RTE salads with mayonnaise. All these are permitted to have up to 100 CFU/g. Following an appeal against food standards introduced by the Government of Austria, the European Court decided that European law does not preclude the imposition by a member state of a zero tolerance for L. monocytogenes in fishery products, even if it is not technically possible to eliminate that hazard [44,45]. This would seem to indicate that some fishery products might not be sold in Austria without risking prosecution. Therefore, it appears that the European Court has decided that zero-tolerance standards for L. monocytogenes are lawful. Denmark In 1999, several thousand samples of RTE foods at the retail level were tested and less than 1% were found positive [25,82]. The Danish regulatory policy on L. monocytogenes in foods, built on the principles of HACCP and developed using a health risk assessment approach, is based on a combination of inspection and product testing [35,82]. RTE foods are placed in six categories (I–VI), absence of L. monocytogenes in 25 g being required in the first three. This level is necessary in foods capable of supporting growth, unless there is evidence of a listericidal treatment in category I, not to exceed 100 L. monocytogenes per gram at the point of consumption. In the other three categories, a level between 10 and 100 L. monocytogenes per gram is not satisfactory and a level above 100/g is not acceptable, depending on the sampling scheme. In 1994 and 1995, 1.3% of RTE food samples (heat-treated meat products, preserved meat and fish products) were contaminated with L. monocytogenes at a level above 100 CFU/g. The samples included in this survey were primarily foods produced by approved companies and were comprised mainly of vacuumpacked products or products packed in modified atmosphere and with long shelf lives, typically above several weeks. The corresponding percentages of positive samples primarily processed in the retail outlets (heat-treated meat products, preserved meat and fish products) in 1997 and 1998 were 0.3 and 0.6%, respectively. These results suggest that RTE meat and fish products with extended shelf lives produced by companies are more likely to contain high numbers (>100 CFU/g) of L. monocytogenes than products processed in the retail sector, which often have a shorter shelf life. In 1997, preserved meat and fish products, and to a lesser extent, vegetables and meat or vegetable mayonnaise were more likely to contain >100 L. monocytogenes CFU/g than other food commodities. In 1998, preserved meat products, but also heat-treated meat products, vegetables, and meat or vegetable mayonnaise had the highest frequency of samples with >100 L. monocytogenes

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per gram. In the Danish categorization of foods heat treatment, storage time, and stabilization are key factors. The means of stabilizing foods include freezing, pH < 4.5, and aw < 0.9. The categories are as follows: • •

•

•

•

•

Foods heat treated in the final package (absence in 25 g). Heat-treated foods that are handled after heat treatment. The products support growth of L. monocytogenes during shelf life. Typically, shelf life of these products is above 1 week. Examples of products: packed meat products such as cooked ham and wiener sausages, and hot-smoked fish (absence in 25 g). Lightly preserved non-heat-treated RTE products. The products support growth of L. monocytogenes during shelf life. Typically, shelf life of these products is above 3 weeks. Examples of products: cold-smoked or gravad fish and meat (absence in 25 g). Heat-treated foods handled after heat treatment. The products are stabilized against growth of L. monocytogenes within the shelf life. Products that have a shelf life less than 1 week are regarded as stabilized. Examples of products: packed or unpacked meat products such as cooked ham and wiener sausages, and hot-smoked fish (sampling plan: n = 5, c = 1, m = 100). Lightly preserved non-heat-treated RTE products. The products are stabilized against growth of L. monocytogenes during the shelf life. Products with a shelf life less than 3 weeks are regarded as stabilized. Examples of products: cold-smoked or gravad fish and meat (sampling plan: n = 5, c = 1, m = 100). Raw RTE foods. Examples of products: steak tartar (tatar), sliced vegetables, sprouts (sampling plan: n = 5, c = 1, m = 100).

France Outbreaks have occurred in France in the past, but the incidence is lower today [79,102]. In France, hygiene and sanitary regulations require the absence of any pathogen in foodstuffs for human consumption. For dairy products, the Community Directive 92/46 of 16 June 1992 is more precise because it requires the absence of L. monocytogenes in 25 g of soft cheese, and in 1 g of hard cheese and other dairy products, at the manufacturing stage [79]. France conformed to this directive in 1999 [62]. The CSHPF (French High Council for Public Hygiene) established the criterion of 100 L. monocytogenes per gram as the maximum admissible threshold at consumption, a threshold that applies to all other foodstuffs [79]. Germany Listeriosis in Germany is not a notifiable disease, so the total number of cases is unknown, but the estimate is at least 50–80 cases per year [101]. Germany has developed recommendations to be used by food inspection authorities. Four different categories of food have been developed along with action levels [35]. Group I consists of foods for infants and small children as well as dietary foods, with the action level being the presence of L. monocytogenes in 25 g of product. Group II contains foods such as pasteurized milk and aseptically packaged products, with the same action level as Group I foods. Foods in Group III include frozen meals, cheese (but not raw milk cheese), prepacked sausages, and shrimp, with an action level of >100 CFU/g. Low-level contamination would precipitate follow-up action in the plant, and higher contamination would render a food “unfit for human consumption.” The last group (Group IV) consists of three different food categories that are “otherwise stabilized foods,” such as smoked fish and fermented sausages, raw foods consumed in the raw or unprocessed state, and raw food that is heated before consumption. For Group IV foods, L. monocytogenes levels of <100 CFU/g usually are permissible, but in cases in which higher levels are observed, the food plant or shop would be inspected. The upper limit of

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L. monocytogenes allowable in any food product is 104 CFU/g or CFU/mL (action level, not tolerance level), but authorities are considering lowering this to 103 CFU/g or CFU/mL. Italy A general food law [70] prohibits the marketing of food containing harmful microbes, which is interpreted as zero tolerance for pathogenic organisms. There was also a more specific order of the Ministry of Health [71,73]. This requires that the label must bear “to be consumed after heat treatment.” The order allows different limits in three groupings for all foods except milk and dairy products. •

• •

Raw foods that have not been heat treated, 3 sample units—not more than 11 CFU/g in 1 sample unit and not more than 110 CFU/g in 2 sample units; n = 3, c = 2, m = 11, M = 110 Frozen or quick-frozen foods, 5 sample units—not more than 11 CFU/g in 2 sample units and not more than 110 CFU/g in 3 sample units; n = 5, c = 3, m = 11, M = 110 Precooked or pasteurized foods, 5 sample units—not more than 11 CFU/g in 4 sample units and not more than 110 CFU/g in 1 sample unit; n = 5, c = 1, m = 11, M = 110

In addition, a more recent decree [72] covers dairy products in a similar way to regulations in The Netherlands and the U.K.; no L. monocytogenes in 25 g of soft cheeses (“not hard”), and no L. monocytogenes in 1 g of milk and all other dairy products (c = number of sample units giving values between m and M). The Netherlands Under The Netherlands National Food and Commodities Law, L. monocytogenes should be <100/g for all food types except raw food, based on EU draft regulations [29]. There is, however, absence of specific standards for milk-based products based on EC Council Directive 92/46 [27]: absence of L. monocytogenes in 25 g of cheese other than hard cheese and absence in 1 g of other milkbased products (n = 10, c = 0, m = 0). L. monocytogenes was not identified in HACCP plans of two establishments in which a hazard was likely to occur. Slovenia A recent study in Slovenia showed that L. monocytogenes was found in samples of raw meats, raw sausage, mechanically deboned chicken meat, and fast fermented sausages [80]. Recommendations were to improve hygiene and to ensure that sausages were fully matured before sale, but did not include any proposed regulations. United Kingdom Specific legislation only exists for dairy products [103]. These state that L. monocytogenes must be absent in 25 g cheese other than hard cheese, where n = 5 and c = 0. For all other milk-based products, the pathogen must be absent in 1 g. This legislation is similar to that in The Netherlands except that the sampling plans are different. However, this regulation is broadly interpreted to cover other RTE foods besides dairy products, in which L. monocytogenes may or may not grow. In 2001, 62,209 laboratory-confirmed instances of food poisoning were documented, including 113 from infection by L. monocytogenes [1]. The Food Standards Agency wants to see a 20% decrease in the number of all foodborne illnesses, including listeriosis, by 2006 [50,51]. The agency established a Foodborne Disease Strategy Consultative Group involving a cross-section of stakeholders to help

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achieve its goals in 2002 [51]. Most of the efforts used so far employ farm-to-fork approach, with the aim of reducing contamination of foods during production and processing and of promoting good food hygiene practice in the commercial and home kitchen. It will involve both sector-specific measures and measures that will have impact across all food sectors, including training and education of food handlers, promotion of HACCP, and a media food hygiene campaign. There is nothing specific to Listeria.

EUROPEAN UNION (EU) In Europe, most member states report on human listeriosis, but the disease is only notifiable in Sweden and Denmark, whereas in other member states reporting is based on laboratory findings [28,31]. The EU reported 665 cases of listeriosis in 1999, but with no information on fatalities [30]. There are also animal listeriosis monitoring programs. Many member countries conduct periodic sampling of foods; however; the sampling schemes and analytical methods used are not harmonized. Belgium continually monitors for L. monocytogenes in meats, and The Netherlands monitors its prevalence in cheese. European countries have different policies for Listeria control, including two with strict zero-tolerance policies. In June 2002, a parliamentary question was asked in the EU on zero-tolerance standards in some member states for L. monocytogenes in smoked salmon. It was recognized that elimination of this hazard from this product is not possible with some member states (notably Italy) maintaining a zero tolerance compared to a limit of 100 CFU/g in some other states [44]. The Commission had no plans to harmonize standards and did not appear unduly concerned at the anomaly. However, the European Court decided in late 2002 that zerotolerance standards for L. monocytogenes are lawful, which will probably have repercussions in the form of legal action, because this may conflict with some existing policies [45]. Countries exporting meat to the United States also have periodic inspections of selected establishments. Audit reports have indicated some deficiencies, indicating that even if regulations are promulgated, they are not necessarily followed. For instance, reports in Austria, Germany, Italy, and The Netherlands indicated that there were some areas that needed corrections [104,106–108]. Two establishments were delisted for noncompliance with HACCP by the Government of Austria. Most problems relating to L. monocytogenes involved the lack of a testing schedule, particularly relating to HACCP programs, and improper hygienic controls. Two of these countries (Austria and Italy) have zero-tolerance policies for L. monocytogenes. In the opinion of a scientific committee [28], control of Listeria in Europe must be based on knowledge of the pH, water activity, presence of preservatives, and shelf life of the different RTE products. Food groupings used by Germany, Denmark, and Canada were presented as examples. The opinion suggested that the groupings can be summarized in three areas: (1) the capacity of food technology to kill L. monocytogenes, (2) the capacity of food technology to prevent recontamination with L. monocytogenes, and (3) the potential for L. monocytogenes to grow in the food commodity. The opinion favored a grouping almost identical to that used in Denmark (A–E vs. I–VI). For foods in Groups A, B, and C, L. monocytogenes should not be detected in 25 g at the time of production. For foods in Groups D, E, and F, L. monocytogenes levels should be <100 CFU/g at the time of consumption and, therefore, throughout the shelf life of the commodity. Apart from these microbiological criteria, adoption of HACCP, displaying production dates on all product packages, paying attention to temperature and time of storage, and exploring the potential to limit shelf life are issues that should be considered. Risk communication strategies need to be implemented to advise consumer groups about increased risk. In its opinion of 22 June 2000, The Scientific Committee on Food (SCF) agreed with the opinion that the concentration of L. monocytogenes in food should be kept below 100 CFU/g. Discussions on how microbiological criteria can be derived in the EU are in progress. One possible set of criteria shown in Table 18.7 was presented for consideration of member countries in 2003 [32]. These came into force on January 1, 2006, and should be adopted by all EU member nations.

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TABLE 18.7 Proposed EU Criteria for Listeria monocytogenes in RTE Foods Sampling Plana

Stages at Which the Criterion Applies

Food Categories

n

c

Limit m

RTE foods intended for infants and for special medical purposes RTE foods able to support growth of L. monocytogenes other than those intended for infants and for special medical purposes RTE foods able to support growth of L. monocytogenes other than those intended for infants and for special medical purposes RTE foods unable to support growth of L. monocytogenes other than those intended for infants and for special medical purposes

10

0

Absence in 25 g

5

0

Absence in 25 gb

5

0

<100 CFU/gc

End of the manufacturing process and products on the market

5

0

<100 CFU/g

Products during the shelf life

End of the manufacturing process and products on the market End of the manufacturing process

n = number of units comprising the sample, c = number of sample units giving values over m. This criterion applies if the food manufacturer is not able to demonstrate, to the satisfaction of competent authority, that the product will meet the limit <100 CFU/g throughout shelf life. cThis criterion applies if the food manufacturer is able to demonstrate, to the satisfaction of competent authority, that the product will meet the limit <100 CFU/g throughout shelf life. The exact value of the limit to be implemented depends on characteristics of the product and conditions during shelf life; the limit shall be low enough to guarantee that the limit 100 CFU/g is not exceeded at the end of shelf life. a

b

For all RTE foods, the microbiological quality of the batch tested shall be considered unsatisfactory if L. monocytogenes is present in any one of the sample units for the different limits of m. Where the criteria are exceeded the lot or batch shall not be released to the market or it shall be withdrawn from the market. The aforementioned criteria for L. monocytogenes apply to all RTE foods, but regular testing is usually not useful for foodstuffs such as those that have received heat treatment or other effective processing to eliminate L. monocytogenes, when recontamination is not possible after this treatment, e.g., packaged products and UHT milk; fresh, uncut, and unprocessed vegetables and fruits, excluding sprouted seeds; bread and biscuits and similar products; bottled or packed waters, soft drinks, beer, cider, wine, spirit drinks, and similar products; sugar, honey and confectionery, including cocoa and chocolate products; and live bivalve mollusks. The keys to setting criteria are the analytical methods used and an appropriate sampling plan.

INTERNATIONAL ACTIVITIES The International Commission for Microbiological Specifications for Foods (ICMSF) recommends that some testing is useful as part of the HACCP verification program, and there are specific protocols that apply to L. monocytogenes [35]: • •

In-pack, heat-treated products—no testing is necessary (documentation for the heat treatment process). Raw products and/or products that are to be heat-treated before consumption—no testing is necessary.

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• •

801

RTE products unable to support growth of L. monocytogenes—10 samples should be taken and the lot should be rejected if any sample contains >100 L. monocytogenes/g. RTE products able to support growth of L. monocytogenes—20 samples should be taken and the lot rejected if any sample contains >100 L. monocytogenes/g.

The CAC has also been studying international criteria for L. monocytogenes through a Working Group of the Codex Committee for Food Hygiene [34]. This working group agreed to propose to the Codex Committee on Food Hygiene to recognize that numbers of L. monocytogenes not exceeding 100 CFU/g in food at the time of consumption are of low risk to consumers. Furthermore, with respect to imported foods, it agreed to recommend that lower levels may need to be applied at the port-of-entry for foods that support growth of L. monocytogenes, so as not to exceed 100 CFU/g at the point of consumption. The recent FAO/WHO risk assessment for L. monocytogenes [36,37] is evidence of the need to harmonize the criteria for this pathogen around the world based on sound science. Because L. monocytogenes contamination can come from multiple sources, a comprehensive control program may involve a combination of strategies that involve HACCP and GHP. A decision tree approach was suggested by FAO [35] to discover which category of RTE foods has the commodity of interest fit. There is also an ongoing discussion through working groups in the CCFH to make recommendations on management of processes by adopting GHPs specifically to reduce L. monocytogenes contamination and growth [19], and the International Life Sciences Institute is preparing a report on a risk-based approach to achieving continuous improvement in reduction of foodborne listeriosis [69], which includes education strategies.

COSTS Relatively few economic analyses of foodborne disease for countries or regions have been made, and even fewer for listeriosis, even though these are valuable in three main ways: (1) evaluating the economic impact of foodborne diseases in a country, (2) helping target pathogen reduction efforts toward the most costly diseases, and (3) comparing benefits and costs of control efforts to determine the most cost-effective interventions. These analyses cannot be made without some understanding of the degree of morbidity and mortality of the pathogen, which are not easily available. In the United States, only the more severe hospitalized cases and deaths are estimated, although outbreaks involving milder cases have been investigated and reported. For most other foodborne pathogens, all cases are considered in determining the burden of disease [81]. Attempts at costing all foodborne disease for the United States started in the mid-1980s, although costs of a few individual outbreaks had been published earlier [9,12,60,96–98]. In 1994, the Council for Agriculture and Science Technology [21] reviewed the literature and used expert opinion to indicate that microbial foodborne pathogens cause 6.5–33 million cases of human illnesses and 9000 deaths in the United States each year. Based on these data, the Economic Research Service (ERS) of the USDA estimated that the annual cost of human illnesses for six foodborne bacteria and one parasite from all food sources was $5.6–$9.4 billion [17,18]. Many costs are involved in estimating the impact of a disease (Table 18.8), but most are hard to measure or determine. The ERS approach focuses on the cost-of-illness (COI) and willingness-to-pay (WTP) methods. The COI method measures the combination of medical expenses, lost earnings of affected individuals, and productivity losses to employers of affected individuals on paid sick leave. The WTP method attempts to estimate what individuals place on the value to society of publicly provided risk reduction strategies. The strength of using a COI approach is that it uses available, calculable data, and such measures have been widely used for many years. The weakness is that these costs are a small portion of the many experienced by individuals and society, and exclude, for instance, costs to the industry when an outbreak or recall occurs. The foregone earnings associated with premature deaths, which vary with age and gender, usually dominate COI estimates, and higherpaid members of society will be assigned higher values of life, e.g., a statistical life of $11,867 to

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TABLE 18.8 Societal Costs of Foodborne Illness Costs to individuals/householdsa Human illness costs Medical costs Physician visits Laboratory costs Hospitalization or nursing home Drugs and other medications Ambulance or other travel costs Income or productivity loss for— Ill person or person dying Caregiver for ill person Other illness costs Travel costs to visit ill person Home modifications Vocational/physical rehabilitation Child care costs Special educational programs Institutional care Lost leisure time Psychological (psychic) costs (pain and other psychological suffering) Risk aversion Averting behavior costs Extra cleaning/cooking time costs Extra cost of refrigerator, freezer, etc. Flavor changes from traditional recipes (especially meat, milk, egg dishes) Increased food cost when more expensive but safer foods are purchased Altruism (willingness to pay for others to avoid illness) Industry costsb Costs of food production Morbidity and mortality of animals on farms, loss of crops, etc. Reduced growth rate/feed efficiency and increased time to market for farmed animals Costs of disposal of contaminated animals or crops Illness among workers because of handling contaminated animals or ingredients Control costs for pathogens at all links in the food chain New farm practices (GAPs, HACCP, etc.) New wholesale/retail practices (pathogen tests, employee training, procedures) Risk assessment modeling by industry for all links in the food chain Price incentives for pathogen-reduced product at each link in the food chain Outbreak costs Product recall Plant closings and cleanup Regulatory fines Product liability suits from consumers and other firms Reduced product demand because of outbreak Generic animal product—all firms affected Reduction for specific firm at wholesale or retail level Increased advertising or consumer assurances following outbreak (continued)

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TABLE 18.8 (CONTINUED) Societal Costs of Foodborne Illness Regulatory and public health sector costs for foodborne pathogens Disease surveillance costs to Monitor incidence/severity of human disease by foodborne pathogens Monitor pathogen incidence in the food chain Develop integrated database from farm to table for foodborne pathogens Research to Identify new foodborne pathogens for acute and chronic human illnesses Establish appropriate production and consumption practices for high-risk products Identify which consumers are at high risk for specific pathogens Develop cheaper and faster pathogen tests Risk assessment modeling for all links in the food chain Outbreak costs Costs of investigating outbreak Testing to contain an outbreak Costs of cleanup Legal suits to enforce regulations that may have been violatedc Other considerations Distributional effects in different regions, industries, etc. Equity considerations, such as special concern for children a

WTP estimate for reducing risks of foodborne disease is a comprehensive estimate of all these categories (assuming that individuals have included employer-funded sick leave and medical programs in their estimates). The estimate is comprehensive and covers reduced risks for everyone—those who will become ill as well as those who will not. bSome industry costs may decrease with better pathogen control, such as reduced product spoilage, possible increases in product shelf life, and extended shelf life permitting shipment to more distant markets or lowering shipment costs to nearby markets. cIn adding up costs, care must be taken to ensure that product liability costs to firms are not already counted in the estimated cost of pain and suffering to individuals. However, legal and court expenses incurred by all parties are societal costs. Source: USDA, Economic Research Service, based on Todd, E.C.D. and T. Roberts. 1996. Approaches to estimating the benefits and costs of foodborne disease control choices. In WHO Consultation on Costs and Preharvest Treatment of Animals, June 8–10, 1995, Washington, DC.

$1,584,605 in 1993 dollars (based on Landefeld and Seskin, [76]). This is compared with wage studies suggesting that employed people would be willing to pay between $3 million and $7 million (1990 dollars) to reduce the risks generating each additional death [112]. Because the COI method is likely to give extremely conservative benefit estimates for publicly provided risk reduction, the COI has been recommended as a lower bound estimate for WTP estimates [63]. Kuchler and Golan [75] have reviewed strengths and weaknesses of different methods, with suggestions regarding how the results should be interpreted. Different data sources are used to determine the number (or range) of instances and deaths for COI evaluations of foodborne diseases. According to CAST [21], these sources are: (1) national surveys conducted by national heath departments, (2) passive and active surveillance of foodborne disease, (3) risk models based on prevalence of pathogens in foods, and on infectious doses, (4) case histories of individuals who have been infected, and (5) extrapolations by experts to obtain

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estimates of the total number of instances and the disease severity distributions to account for undiagnosed or unreported instances. Productivity losses include ill workers, poorly performing workers because of discomfort and frequent medical checks, and those who die prematurely. Current legal incentives to produce safer food in the United States are limited, and less than 0.01% of instances are litigated and even fewer are paid compensation [16]. However, a settlement is more likely in situations resulting from outbreaks in which foodborne illness can be more easily traced to individual firms, and some settlements can be high. In 2000, the CDC revised its estimates of foodborne disease, with 76 million cases and 5000 deaths each year in the United States [81]. Of these, 2298 cases requiring hospitalization and 499 deaths were caused by L. monocytogenes, and 62 of the nonfatal cases developed chronic complications. The ERS calculated that costs of these were $2.3 billion annually (Table 18.9). The estimated costs include chronic disability or impairment resulting from congenital and newborn infections, but exclude other chronic complications, less severe cases not requiring hospitalization (the numbers were not estimated by CDC), time lost from work caring for sick children, travel to obtain medical care, lost leisure time, and pain and suffering. Thus, $2.3 billion can be considered a conservative estimate, and the annual costs from foodborne listeriosis would be substantially increased if WTP to avoid disability, pain, and suffering were also taken into account. The risk of contamination of RTE food by L. monocytogenes is of great concern to the food industry, not only because of possible legal suits but because of recalls following the isolation of the pathogen at any level by a regulatory agency. However, Hewitt [65] argues that there are weak economic incentives for industry to improve because information on pathogen contamination or its likelihood is hidden from the purchasing public. Because industry cannot earn a monetary premium for safer products, food producers have little incentive to conduct research and development that might enhance safety. Hewitt [65] suggests some economic incentives to reduce the incidence of foodborne disease such as listeriosis: 1. Publishing more information on the inspection history and pathogen levels by plant. 2. Creating a consumer label for use on products produced meeting superior “pathogen control standards.” This could be implemented by a joint industry–government body that oversees approval and enforcement. 3. Creating special tax breaks for industry investing in new food safety inventions, or for their adoption. 4. Increasing funding of epidemiological research to discover risks associated with various production and consumption practices and behaviors. 5. Creating a mechanism for industry to have an incentive to share food safety information with researchers. This might be done through an insurance mechanism that protects industry from costs associated with an outbreak. Plants that share auditing information and pathogen test results with researchers could participate in the insurance program at a lower cost than plants that do not share information. 6. Better enforcement, higher fines, and more frequent pathogen testing may increase the economic incentives to reduce the incidence of foodborne disease. Items 1, 2, and 6 are already being carried out to some extent. National governments occasionally carry out surveys of food products or processing plants and may release information on prevalence levels of pathogens, but quantitative data are rarely published. One study by Gombas et al. [61] does give such information, which was generated to provide input to a quantitative risk assessment for L. monocytogenes in RTE products. So-called superior pathogen control standards could be built upon HACCP plans, and performance criteria and food safety objectives established [68]. Fines are probably less of an incentive compared with costs of a recall triggered by detection of L. monocytogenes in a food product.

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TABLE 18.9 Cost Estimates for Listeriosis

Acute a Medical No medical care Physician visit Hospitalized and survived Maternal Newborn/fetal Other adult Moderate illness Severe illness Hospitalized and died Maternal Newborn/fetal Other adult Subtotal Productivity loss/premature deaths No medical care Physician visit Hospitalized and survived Maternal Newborn/fetal Other adult Moderate disability Severe disability Hospitalized and diedb Maternal Newborn/fetal Other adult Subtotal Subtotal—Acute Chronic (all newborn/fetal)c Medical Hospitalized and survived Mild disability Moderate disability Severe disability Hospitalized and died Subtotal Productivity loss/premature deaths Hospitalized and survived Mild disability Moderate disability Severe disability

Cases (Number)

Estimated Annual Costs (Million U.S. Dollars)

Unknown Unknown — 311 368 — 58 1,062 — 0 77 422 2,298

Not estimated Not estimated — 3 14.1 — 0.6 30.6 — 0 0.1 12.1 60.5

Unknown Unknown — 311 0 — 58 1,062 — 0 77 422 1,930 2,298 —

Not estimated Not estimated — 0.6 0 — 0.1 2.2 — 0 669.9 1,514.30 2,187.10 2,247.60 —

— — 11 34 11 0 56

— — 0.5 4.3 6.5 0 11.3

— 11 34 11

— 4.8 51.5 17.9 (continued)

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TABLE 18.9 (CONTINUED) Cost Estimates for Listeriosis Cases Hospitalized and died Subtotal Subtotal—Chronic Total

Estimated Annual Costs

0 56 56

0 74.3 85.6

2,298

2,333.20

a

Data from the Centers for Disease Control and Prevention (from Mead, P.S., L. Slutsker, V. Dietz, L.F. McCaig, J.S. Bresee, C. Shapiro, P.M. Griffin, and R.V. Tauxe. 1999. Food-related illness and death in the United States, Emerg. Infect. Dis. 5) except for physician visits and hospitalized case subcategories, which were estimated by ERS. bThese estimated cases and costs are for the congenital and newborn infections resulting in chronic disability or impairment. cCost calculations are based on the labor market approach for valuing the cost of premature deaths. Source: Buzby, J.C. 2000. Estimated annual costs due to foodborne listeriosis. Economic Research Service, USDA. http://www.ers.usda.gov/Briefing/FoodborneDisease/listeria/. Accessed December 23, 2004.

In 2001–2002, the New South Wales (NSW) Department of Health and the Commonwealth Department of Health and Ageing funded a National Risk Validation Project [49]. The aims of the project were to identify potentially high-risk food industry sectors, use risk assessment principles to validate the categorization of selected sectors as high risk, and include costs and benefits of implementing food safety programs in high-risk food industry sectors. The most frequently encountered hazards based on all the material reviewed were faulty temperature control of hazardous foods, poor worker hygiene, cross-contamination, and contaminated raw materials. Food businesses that ranked as high risk in order of priority were as follows: 1. 2. 3. 4. 5.

Food service for sensitive populations Producers, harvesters, processors, and vendors of RTE seafood Catering operations serving food to the general population Eating establishments Producers of manufactured and fermented meats

Other areas of concern were processed raw foods not treated listericidally by heat, and processed foods treated listericidally by heat but subject to potential recontamination during subsequent handling. Scott et al. [92] adopted a value of NZ$2 million for the cost of a fatality in New Zealand (approximately equivalent to A$2 million) but provided no details on how this figure was determined. In view of problems interpreting results when using the COI methodology for valuing a statistical life, the National Risk Validation Project chose the WTP approach. Costs were estimated for the top five high-risk businesses. For the 36 cases (33 hospitalized and 7.1 dead) resulting from catering operations supplying food with L. monocytogenes to sensitive populations, costs were estimated at A$39,098, of which a large proportion was attributed to the deaths (A$35,722) and cost per meal of A$0.1108, higher than in any other category, served to sensitive populations. For the 42 cases (39 hospitalized and 8.4 dead) resulting from contaminated fermented and manufactured meats, costs were estimated at A$46,206, of which a large proportion was also attributed to the deaths (A$42,217) and cost per meal of A$0.2310, the highest cost per meal served by any of the businesses estimated.

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In New Zealand an estimated 119,320 episodes of foodborne infectious disease occur annually [92]. The total cost of these cases is NZ$55.1 million (NZ$462 per case) made up of direct medical costs of NZ$2.1 million, direct nonmedical costs of NZ$0.2 million, indirect cost of lost productivity of NZ$48.1 million, and intangible cost of loss of life of NZ$4.7 million. Campylobacteriosis generated most of these costs. Lost productivity was the major cost component for all diseases. Infectious intestinal disease in England and Wales occurs in one of every five people each year, of whom one in six presents to a general practitioner [114]. The proportion of cases not recorded by national laboratory surveillance is large and varies widely by microorganism. Listeriosis was not included in organism-specific incidences based on cases for which a stool specimen was obtained and analyzed. The U.K. Food Standards Agency (which spends 12% of its budget on foodborne illness) is committed to reducing foodborne illness by 20% by 2006 [77]. The Food Standards Agency estimates that foodborne illness currently costs the economy £350 million a year, whereas other estimates have suggested the true cost may be as much as £1 billion per year. Another study found that the care cost to the National Health Service for 100,000 people treated for food poisoning in 1991–1994 was £83 million. These types of costs can be seen in relation to advertising costs of almost £600 million per annum in the United Kingdom, the budget of which grossly outweighs the promotion of improved diets and safety.

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77. Lang, T. and G. Raynor. 2002. Why health is the key to the future of food and farming: a report of the future of farming and food. 59 pp. http://www.gfw.co.uk/PDF/whyhealthisthekey.pdf. Accessed October 1, 2004. 78. Lindqvist, R. and A. Westöö, 2000. Quantitative risk assessment for Listeria monocytogenes in smoked or gravad salmon and rainbow trout in Sweden. Int. J. Food Microbiol. 58: 181–196. 79. Maison du Lait. Listeria. CNIEL (Centre National Interprofessionnel de l’Economie Laitière/National Interprofessional Centre of the Dairy Economy). http://www.cniel.com/site.asp?where=prodlait/ prodlait.html. Accessed December 1, 2004. 80. Marinsˇek, J. and S. Grebenc. 2002. Listeria monocytogenes in minced meat and thermally untreated meat products in Slovenia. Slov. Vet. Res. 39: 131–136. 81. Mead, P.S., L. Slutsker, V. Dietz, L.F. McCaig, J.S. Bresee, C. Shapiro, P.M. Griffin, and R.V. Tauxe. 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5. 82. Nørrung, B., J.K. Andersen, and J. Schlundt. 1999. Incidence and control of Listeria monocytogenes in foods in Denmark. Int. J. Food Microbiol. 53: 195–203. 83. Notermans, S., J. Dufrenne, P. Teunis, and T. Chackraborty. 1998. Studies on the risk assessment of Listeria monocytogenes. J. Food. Prot. 61: 244–248. 84. NSW. 2001. A New Approach to Food Safety in New South Wales: A NSW Health Information Paper, NSW Health Department. 31 pp. http://www.health.nsw.gov.au/health-public-affairs/publications/ food-safety/food-safety.pdf. Accessed October 1, 2004. 85. NSW. 2004. NSW Food Authority, an introduction. http://www.foodsafetycentre.com.au/presentations/Davey.pdf. Accessed October 1, 2004. 86. NZFSA. 2003. New Zealand Fishing Industry Agreed Implementation Standards: 003.9 Listeria Circular 1995, Amended, November, New Zealand Food Safety Authority. http://www.nzfsa.govt.nz/ animalproducts/seafood/iais/3/3-9/. Accessed October 1, 2004. 87. NZFSA. 2004. Fishing Industry Council Guidelines, Seafood — Guidelines for the Management of Listeria in Fish Packing Houses. http: //www.nzfsa.govt.nz/animalproducts/seafood/guidelines/listeria/index.htm. Accessed October 1, 2004. 88. Peeler, J.T. and V.K. Bunning. 1994. Hazard assessment of Listeria monocytogenes in the processing of bovine milk. J. Food Prot. 57: 689–697. 89. ProMED. 2002. Listeriosis, poultry — USA: recall, bacteria fears prompt largest ever U.S. meat recall. October 14, Archive Number 20021014.5550. http://www.promedmail.org/pls/askus/f?p=2400:1001: 403089::NO::F2400_P1001_BACK_PAGE,F2400_P1001_PUB_MAIL_ID:1000,19555. Accessed October 1, 2004. 90. Ross, T., Personal communication based on unpublished report of Ross, T., S. Rasmussen, J. Sumner, G. Paoli, and A. Fazil, L. monocytogenes in Australian processed meat products: risks and their management. Meat and Livestock Australia, Sydney, Australia, 2004. 91. Sanaa, M., L. Coroller, and O. Cerf. 2004. Risk assessment of listeriosis linked to the consumption of two soft cheeses made from raw milk: Camembert of Normandy and Brie of Meaux. Risk Anal. 24: 389–399. 92. Scott, W.G., H.M. Scott, R.J. Lake, and M.G. Baker. 2000. Economic cost to New Zealand of foodborne infectious disease. New Zealand Med. J. 113: 281–284. 93. Shank, F.R., E.L. Elliot, I.K. Wachsmuth, and M.E. Losikoff. 1996. U.S. position on Listeria monocytogenes in foods. Food Control. 7: 229–234. 94. Sumner, J. and J. Gallagher. The Australian seafood risk assessment. Seafood Services Australia, Hamilton, Australia, 2001, 9 pp. http: //www.asic.org.au/seafooddirections/2001/pdf/34.pdf. Accessed October 1, 2004. 95. Swaminathan, B., T.J. Barrett, S.B. Hunter, and R.V. Tauxe. 2001. PulseNet: the molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg. Infect. Dis. 7: 382–389. 96. Todd, E.C.D. 1985. Economic loss from foodborne disease outbreaks associated with foodservice establishments. J. Food Prot. 48: 169–180. 97. Todd. E.C.D. 1985. Economic loss from foodborne disease and non-illness related recalls because of mishandling by food processors. J. Food Prot. 48: 621–633. 98. Todd, E.C.D. 1989. Preliminary estimates of costs of foodborne disease in the United States. J. Food Prot. 52: 595–601.

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99. Todd, E.C.D., J.M. Farber, M.-A. Rivers, M. Smith, and W.H. Ross. 2001. Quantitative risk assessment for Listeria monocytogenes in cabbage in Canada. Health Canada Report (unpublished): 24. 100. Todd, E.C.D and T. Roberts. 1996. Approaches to estimating the benefits and costs of foodborne disease control choices. In WHO Consultation on Costs and Preharvest Treatment of Animals, June 8–10, 1995, Washington, DC. 101. Twisselmann, B. 2000. Epidemiology, treatment, and control of listeriosis. Eurosurveillance Weekly. 4(18). 102. Twisselmann, B. 2002. Cluster of listeriosis cases in France. Eurosurveillance Weekly. 6(27). 103. UK. 1995. The Dairy Products (Hygiene) Regulations, No. 1086, Schedule 6. http: //www.legislation.hmso. gov.uk/si/si1995/Uksi_19951086_en_1.htm. Accessed October 1, 2004. 104. USDA. 2000. Audit report for Netherlands, February 10 through February 28. http://www.fsis.usda. gov/OPPDE/FAR/Netherlands/Netherlands2002.pdf. Accessed October 1, 2004. 105. USDA. 2000. Federal Register, May 8, 65(89): 26563–26565. http://www.fsis.usda.gov/OPPDE/rdad/ FRPubs/00-016n.htm. Accessed on October 1, 2004. 106. USDA. 2001. Audit report for Germany July 18 through August 6, 2001. http://www.fsis.usda.gov/ OPPDE/FAR/Germany/Germany2002.pdf. Accessed October 1, 2004. 107. USDA. 2001. Audit report for Italy, May 7 through June 6, 2001. http://www.fsis.usda.gov/ OPPDE/FAR/Italy/Italy2002.pdf. Accessed October 1, 2004. 108. USDA. 2002. Audit Report For Austria, March 12 through March 21, 2002. http://www.fsis.usda. gov/OPPDE/FAR/Austria/Austria2002.pdf. Accessed October 1, 2004. 109. USDA. 2004. Control of Listeria monocytogenes in Ready-to-Eat Meat and Poultry Products. Federal Register. December 2, 69(231), Docket No. 97-013FE. 110. Van Schothorst, M. 1996. Sampling plans for Listeria monocytogenes. Food Control 7: 203–208. 111. Van Schothorst, M. 1997. Practical approaches to risk assessment. J. Food Prot. 60: 1439–1443. 112. Viscusi, W.K.1993. The value of risks to life and health. J. Econ. Lit. 31: 1912–1946. 113. Vorst, K.L., E.C.D Todd, and E.T. Ryser. 2006. Transfer of Listeria monocytogenes during mechanical slicing of turkey breast, bologna, and salami. J. Food Prot. 69: 619–626. 114. Wheeler, J.G., D. Sethi, J.M. Cowden, P.G. Wall, L.C. Rodrigues, D.S. Tompkins, M.J. Hudson, and P.J. Roderick. 1999. Study of infectious intestinal disease in England: rates in the community, presenting to general practice, and reported to National Surveillance. Br. Med. J. 318: 1046–1050. 115. WHO, Application of Risk Analysis to Food Standards Issues. Report of the Joint FAO/WHO Expert Consultation, March, 13–17, 1995, WHO, Geneva, 31.

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on Research 19 Perspectives Needs Elmer H. Marth, Robert E. Brackett, R. Bruce Tompkin, Sophia Kathariou, and Ewen C.D. Todd CONTENTS Introduction ....................................................................................................................................814 References.............................................................................................................................815 Research Needs: An FDA Perspective ..........................................................................................815 Introduction...........................................................................................................................815 Research Needs ....................................................................................................................816 Exposure of Consumers to L. monocytogenes.........................................................816 Behavior of L. monocytogenes in Foods..................................................................817 Pathogenicity ............................................................................................................817 Detection and Enumeration of L. monocytogenes ...................................................818 Elimination or Destruction of L. monocytogenes ....................................................818 References.............................................................................................................................819 Research Needs: An Industry Perspective.....................................................................................819 Significance of the FAO/WHO and FDA/FSIS Risk Assessments to the Development of Control Strategies ...........................................................................819 Outbreaks vs. Sporadic Cases ..............................................................................................820 Reducing Consumer Exposure .............................................................................................821 Preventing Contamination ........................................................................................821 Postpasteurization .....................................................................................................823 Preventing Growth....................................................................................................824 Estimating Consumer Exposure ...............................................................................826 Concluding Remarks ............................................................................................................827 Acknowledgment ..................................................................................................................827 References.............................................................................................................................827 Research Needs: An Academic Perspective ..................................................................................830 What the Organism Has and Expresses: The Genomic and Proteomic Endowment of L. monocytogenes ........................................................................................831 How, Where, and When L. monocytogenes Expresses Special Genome Components: Listerial Pathogenesis, Ecology, and Stress Responses Further Considered ......................833 Concluding Thoughts ...........................................................................................................834 References.............................................................................................................................835 Research Needs: A Food Safety Perspective.................................................................................835 Regulatory Tolerance............................................................................................................836 Control Options ....................................................................................................................837 Risks Associated with Processed Food................................................................................838

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Deli Meats ................................................................................................................838 More Focus on Retail Product .................................................................................838 Other Foods of Concern...........................................................................................839 Labeling and Consumer Education......................................................................................839 References.............................................................................................................................840

INTRODUCTION ELMER H. MARTH

The bacterium later named Listeria monocytogenes [9] was first isolated from diseased laboratory animals, characterized, and a description published in 1926 [8]. This publication did not result in an immediate groundswell of research interest in the organism. Instead, for years afterward the research consisted largely of reports of different species of animals and birds that suffered from listeriosis. Transmission of the disease to humans who were exposed to infected animals was also described. Other limited studies on the pathogen and the disease it caused were reported. It was not until the 1940s through 1960s that a sustained research effort on the organism was established by M.L. Gray and associates at Montana State College (now University). From this work we learned, among many other things, about methods to isolate the organism [5,6] and that it is widespread in the environment [4]. Sufficient research was done at Montana State College and elsewhere, so at least two conferences, with published proceedings, were organized by Gray and his co-workers. Meanwhile, H.P.R. Seeliger began to do a lifetime of research on L. monocytogenes at the University of Berlin and continued the work when he moved to the University of Würzburg. Although Seeliger’s work emphasized disease aspects, he also studied other properties of the bacterium, and in 1961 published his book, Listeriosis, in English [11]. In the late 1940s, there was some evidence in Germany of the possible role of food in human listeriosis. However, at the time no one became sufficiently concerned to do research on this potential cause of foodborne illness. This changed in the 1980s when there were three major outbreaks of listeriosis in North America attributed to contaminated food. The first outbreak involved coleslaw and prompted food microbiologists Robert Brackett and Larry Beuchat of the University of Georgia–Griffin to initiate research on behavior of L. monocytogenes in vegetable products and on methods to isolate the bacterium from various foods [1,3]. The next outbreak involved pasteurized milk. Soon after this outbreak occurred, Ralston B. Read, then of the FDA, encouraged food microbiologists Elmer H. Marth and Michael P. Doyle of the University of Wisconsin–Madison to determine the behavior of L. monocytogenes during manufacture and subsequent storage of nonfat dry milk and cottage cheese. Thus, a research program was initiated in 1984 with results reported in 1985 [2,10]. These reports were among the first, worldwide, to describe behavior of the pathogen during manufacture and subsequent storage of food products. The third outbreak occurred in 1985 and involved Mexican-style cheese made and distributed in California. This outbreak received considerable news media attention and also prompted the scientific community to organize research programs to explore various aspects of the bacterium and the disease. Thus, research efforts were undertaken by persons in such fields as food, veterinary and medical microbiology, immunology, epidemiology, cell and molecular biology, clinical medicine, pathology, and microbial taxonomy [7]. With this much effort being exerted, it was believed by the Society for Industrial Microbiology that a conference to share the new information was needed. This conference was held in October 1988 in Sonoma Valley, California. Proceedings of the conference appeared in 1990 in a book titled Foodborne Listeriosis [7]. A year later, in 1991, the first edition of the present book was published, and it was followed by the second edition in 1999. The current edition again describes results of recent and not-so-recent research.

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After about 20 years of concentrated effort by researchers, do we still need more information about the pathogen and the disease? We have all read or heard the expression, “further studies are needed.” Does this apply to L. monocytogenes and listeriosis? Four experts were invited to shed some light on this question. After you read their contributions, you, too, will agree that further studies are needed.

REFERENCES 1. Beuchat, L.R., R.E. Brackett, D.Y.-Y. Hao, and D.E. Conner. 1986. Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice. Can. J. Microbiol. 32: 791–795. 2. Doyle, M.P., L.M. Meske, and E.H. Marth. 1985. Survival of Listeria monocytogenes during the manufacture and storage of nonfat dry milk. J. Food Prot. 48: 740–742. 3. Golden, D.A., L.R. Beuchat, and R.E. Brackett. 1988. Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured and freeze-injured Listeria monocytogenes from foods. Appl. Environ. Microbiol. 54: 1451–1456. 4. Gray, M.L. 1963. Epidemiological aspects of listeriosis. Am. J. Pub. Health 53: 554–563. 5. Gray, M.L., H.J. Stafseth, and F. Thorp. 1950. The use of potassium tellurite, sodium azide and acetic acid in a selective medium for the isolation of Listeria monocytogenes. J. Bacteriol. 59: 443–444. 6. Gray, M.L., H.J. Stafseth, F. Thorp, Jr., L. B Shell, and W.F. Riley, Jr. 1948. A new technique for isolating Listerellae from the bovine brain. J. Bacteriol. 55: 471–476. 7. Miller, A.L., J.L. Smith, and G.A. Somkuti. 1990. Foodborne Listeriosis. Elsevier, Amsterdam. 8. Murray, E.G.D., R.A. Webb, and M.B.R. Swann. 1926. A disease of rabbit characterized by a large mononuclear leucocytosis, caused by a hitherto undescribed bacillus Bacterium monocytogenes (n. sp.). J. Pathol. Bacteriol. 29: 407–439. 9. Pirie, J.H.H. 1940. The genus Listerella Pirie. Science 91: 383. 10. Ryser, E.T., E.H. Marth, and M.P. Doyle. 1985. Survival of Listeria monocytogenes during manufacture and storage of cottage cheese. J. Food Prot. 48: 746–750. 11. Seeliger, H.P.R. 1961. Listeriosis. Hafner Publishing Co., New York.

RESEARCH NEEDS: AN FDA PERSPECTIVE ROBERT E. BRACKETT

INTRODUCTION Although much has been learned about the many facets of the Listeria monocytogenes problem over the past decade, it has also become clear that we still have much to learn about this organism, how to detect it in foods, and its role in foodborne disease. In 2003, the FDA and USDA published a quantitative L. monocytogenes risk assessment (LMRA) [2] to determine the public health impact from L. monocytogenes in various foods. An important benefit of conducting a risk assessment is the identification of knowledge, gaps in available data, and research needs. In the course of collecting and evaluating the data for the LMRA, it became apparent that additional data could enhance the certainty and reduce variability in the risk assessment results. As a result, new data were generated specifically for this risk assessment, and data were obtained from the published literature on levels of L. monocytogenes in food, growth in deli salads, home storage practices, and other data. These new data significantly improved the predicted risk estimates and reduced the amount of uncertainty associated with those estimates. New data and information would also facilitate development of commodity- or product-specific risk assessments. The research needs summarized in the following paragraphs were identified as important in providing data needed to continue filling existing gaps to facilitate future L. monocytogenes risk assessment work and to improve efforts to reduce listeriosis.

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RESEARCH NEEDS Research needs are driven by the type of information needed for the FDA to fulfill its public health mission. When dealing with L. monocytogenes, the type of information for which research is needed falls primarily into five general categories, each of which will be discussed: • • • • •

Exposure of consumers to L. monocytogenes Behavior of L. monocytogenes in foods Pathogenicity of L. monocytogenes Detection and enumeration of L. monocytogenes Elimination or destruction of L. monocytogenes

Exposure of Consumers to L. monocytogenes Before one can assess the potential public health impact of L. monocytogenes, it is necessary to determine the concentration of the organism to which consumers are exposed and which they ultimately consume, and the frequency of those exposures. Among the more obvious factors influencing exposure is the level of contamination in the food itself. Although a variety of studies and reports have been conducted to determine the presence of L. monocytogenes in foods, accurate and precise data on concentration of this bacterium in foods are still lacking, particularly at concentrations below 100 CFU/g. As will be discussed later in this subsection, the lack of good enumeration methods for L. monocytogenes in foods has also hindered collection of enumeration data by the scientific community. Moreover, “zero tolerance” (no detectable L. monocytogenes) regulatory policies have resulted in a situation in which there is little practical need for the food industry to enumerate L. monocytogenes in foods. Demonstration of its mere presence was sufficient information on which to judge the acceptability of the product. Consequently, additional research is needed on precisely how many viable L. monocytogenes cells are present in foods at the time of consumption. Another important factor that affects exposure is how the food is actually handled and prepared before consumption because preparation practices can affect survival of the bacterium. Estimating survival of L. monocytogenes in foods intended to be eaten in a raw state is relatively straightforward. However, the same cannot be said for all foods at all stages of preparation. Various surveys have been done to determine estimates of specific foods that are being consumed, but these surveys typically do not determine how the foods are prepared. Consequently, data on consumer food preparation and eating practices are limited. A survey on home storage times for deli meats and frankfurters was conducted [1], but additional information is needed for other food categories. Because L. monocytogenes can grow during refrigerated storage, storage time and temperature are major factors in the degree of hazard. Related factors include the time after opening the original package (particularly if it is a vacuum or modified-atmosphere package) and likely subsequent cross-contamination, such as might occur at a deli counter, or in the home refrigerator, or kitchen. More research into how consumers purchase, store, and prepare foods would enable better estimates of the numbers of viable L. monocytogenes consumed. Finally, it is important to know the actual amount of specific foods consumed if one is to estimate the potential contribution of those foods to overall listeriosis. The LMRA attempted to quantify risk and in doing so had to determine the relative quantities of various foods that consumers normally ate. Although this task on the surface appears simple and straightforward, risk assessors very quickly found that this was not true. Indeed, there is a paucity of information on consumption of specific foods in a form that lends itself for use in a microbial risk assessment. The LMRA relied primarily on two food consumption surveys to provide estimates of food consumption. However, these surveys were designed primarily for nutritional purposes rather than for a microbiological exposure assessment. They were not designed to collect information on aspects of food consumption related to food safety questions. Examples of such questions include whether a cheese was made from unpasteurized fluid milk; whether the milk or juice that was consumed

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was pasteurized or unpasteurized; whether smoked seafood is hot- or cold-smoked; whether peas put into a pasta salad were freshly cooked, frozen, or canned; and whether steamed shrimp or crabs and fried chicken were eaten freshly cooked or allowed to cool before eating. Specific dietary information was limited or lacking for many of the susceptible subpopulations. For example, data are not available to characterize consumption by elderly people living in nursing homes or other forms of assisted living out of the home. In addition, better information is needed about the health status of consumers. Future food consumption surveys that collect consumption information of the type just described would greatly enhance the utility of their results for addressing food safety as well as human nutrition issues. However, such information is also among the most difficult to obtain. Behavior of L. monocytogenes in Foods One of the most well-recognized areas of research for which additional data are needed is behavior of L. monocytogenes in different types of foods. Additional inoculated pack studies are needed on selected foods to determine survival, growth rates, and maximum growth in the presence of normal spoilage flora. Essential information from these studies should include the physical properties (such as pH, aw, or NaCl concentration) of the food studied. In addition, future studies should be conducted using a group of well-characterized L. monocytogenes strains, to allow for both direct comparisons across foods and information on the diversity of strain responses. The growth and survival characteristics of L. monocytogenes in certain foods is a related category of information that is needed for better assessment of the potential role of foods in listeriosis. The risk of contracting foodborne listeriosis is greatly reduced for foods that do not support growth of L. monocytogenes. Hence, knowing which foods do and which do not support growth of the bacterium would allow industry and government to better prioritize resources toward those of highest risk. Pathogenicity Public health and regulatory policies have typically been developed based on the assumption that all L. monocytogenes isolates are essentially equally virulent and pathogenic. However, microbiologists and clinicians have recognized for decades that this simple assumption is usually not true. Listeriosis, as most other foodborne diseases, is a function of the genotype of the bacterium, concentration of organism consumed, immunological state and age of the host, and a variety of other known and unknown factors. The immunological response of an individual is critical in determining whether an exposure to L. monocytogenes will result in clinical signs of illness. Objective measures of the immune status of symptomatic and asymptomatic individuals would help to provide a better assessment of an individual’s vulnerability to listeriosis. Likewise, information about the role of the human immune system in preventing listeriosis is also limited. Most of the information on resistance to L. monocytogenes infection has been obtained based on studies utilizing laboratory animals, primarily mice. The relevance of these studies to immune mechanisms important in human infection, particularly in pregnancy, should be investigated more thoroughly. As suggested in the previous paragraph, much is still unknown about how and why L. monocytogenes strains differ in pathogenicity. In animal models, there is at least a 5-log range in virulence among L. monocytogenes strains. The traditional serotyping system (1/2a, 4b, etc.) is often cited as being indicative but is not related to or based on specific virulence mechanisms. Development of methodologies to rapidly quantify the virulence of strains would allow more effective assessment of the public health threat of L. monocytogenes found in foods, and allow both food processors and regulatory agencies to focus on those strains of greatest pathogenicity. More also needs to be known about the effect of the food matrix and factors such as stomach acidity, achlorhydria, and use of antacids on development of listeriosis. Such information would be useful in understanding differential susceptibility in humans and would allow physicians to provide specific advice to at-risk individuals on how to minimize their odds of contracting listeriosis.

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Finally, more and better epidemiological studies are needed. A collection of data on attack rates and consumption of L. monocytogenes would aid in the development of better dose–response models. In addition, animal and biochemical tests need to be correlated to the epidemiological data to enable assessment of L. monocytogenes isolates and to establish relevant biomarkers of human susceptibility. Detection and Enumeration of L. monocytogenes Methods to detect L. monocytogenes in foods have improved greatly over the past 2 decades, with detection times decreasing from 30 days to 48 h. Newer techniques are capable of detecting very low populations of L. monocytogenes in many different types of foods. However, the relative sensitivity, selectivity, and time frame for detection are still often dependent on the specific food matrix. To be of practical use for regulatory purposes, methods must be precise and reproducible in addition to being sensitive. Consequently, not only is additional research needed to improve existing methods of achieving these characteristics, but also much more effort needs to be expended on properly validating the methods in the wide variety of food matrices for which FDA has regulatory authority. There is a growing trend internationally toward supporting the use of performance standards or regulatory limits that would allow very low populations of L. monocytogenes in certain foods. Such policies would require some use of enumeration to verify that the limits are being achieved. However, despite the improvements in detection methods for L. monocytogenes in foods, the same cannot be said about enumeration methods. The primary options for estimating populations of L. monocytogenes in foods are much the same as they were in the 1980s, that is, the direct plating and most probable number (MPN) techniques. Direct plating is relatively simple but is only capable of providing population estimates exceeding about 100 CFU/g. In contrast, MPNs are capable of providing estimates at lower concentrations, but this technique is very imprecise. Because typical contamination levels in foods are less than 100 CFU/g, neither method is acceptable for reliable and precise enumeration of the bacteria. Consequently, sensitive and precise enumeration methods still need to be developed and validated for both research and regulatory actions requiring accurate estimates of L. monocytogenes populations in foods. Sampling strategies for detection and enumeration of L. monocytogenes in various foods comprise another research need overlooked or ignored. Techniques for concentrating very low populations of L. monocytogenes cells distributed throughout large volumes of samples would improve sensitivity of methods. A related problem that needs to be addressed is how to develop proper sampling plans that would ensure detection of L. monocytogenes in foods in which distribution is nonrandom. Such research would likely require a more multidisciplinary approach than is typically seen in microbiology, and require participation by food technologists, mathematicians, statisticians, microbiologists, and professionals from even more unrelated sciences, such as physical chemists. Finally, there is a great need for research to determine appropriate surrogates and indicators for L. monocytogenes in the food processing and natural environment. A thorough knowledge of the microbial ecology of the bacterium could lead to identification of nonpathogenic indicators of contamination or presence of ecological niches in which L. monocytogenes is likely to become established. Likewise, such information would allow for better evaluation of testing programs often conducted as part of Good Manufacturing Practices and Hazard Analysis Critical Control Point programs. Elimination or Destruction of L. monocytogenes Finally, there is still a great need for techniques that are capable of destroying or eliminating L. monocytogenes on environmental harborages, food contact surfaces, and in foods. In particular, innovative techniques to destroy L. monocytogenes in ready-to-eat foods that support their growth, while still maintaining food quality, could greatly advance efforts to reduce the number of cases of listeriosis. Although it is not generally the role or within the purview of regulatory agencies to

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develop such techniques, their development in the academic or private sector would benefit regulatory efforts by allowing regulatory agencies to shift their focus to foods and environments in which L. monocytogenes would remain the highest risk. The FDA will likely continue to view listeriosis as a significant public health threat into the foreseeable future. However, successfully addressing the research needs just described would accelerate the agency’s efforts to eliminate listeriosis associated with the foods it regulates.

REFERENCES 1. American Meat Institute. 2001. Consumer Handling of RTE Meats. (Unpublished data submitted to Docket No. 99N–1168.) 2. Anonymous. 2003. Quantitative Assessment of Relative Risk to Public Health from Foodborne Listeria monocytogenes Among Selected Categories of Ready-to-Eat Foods. http://www.foodsafety.gov/~dms/ lmr2-toc.html.

RESEARCH NEEDS: AN INDUSTRY PERSPECTIVE R. BRUCE TOMPKIN

From an industry perspective, Listeria research generally falls into four categories: (1) elucidating factors that lead to foodborne listeriosis, (2) preventing contamination of ready-to-eat (RTE) foods, (3) developing lethal postpackaging treatments, and (4) preventing growth of Listeria monocytogenes on food. Through this research, control measures are developed and implemented to ensure consumer protection and regulatory compliance. Prevalence and persistence of L. monocytogenes in certain food processing environments indicate that long-term strategies are needed for consumer protection. The strategies should lead to continuous improvement in reduction of listeriosis when measured in cases per 100,000 population per year. Progress should be measured by an epidemiologic system that is consistent from year to year and sufficiently sensitive to detect trends. Other measurements can involve data from product samples collected at manufacturing plants, for example, in the Food Safety and Inspection Service (FSIS) monitoring program, and/or at retail [18,31].

SIGNIFICANCE OF THE FAO/WHO AND FDA/FSIS RISK ASSESSMENTS TO THE DEVELOPMENT OF CONTROL STRATEGIES The Food and Agriculture Organization/World Health Organization (FAO/WHO) risk assessment [19] evaluated the risk per serving and predicted the number of annual cases of listeriosis using a distribution of L. monocytogenes levels in foods. For levels less than 100 CFU/g in all servings of RTE foods eaten during an entire year in the United States, the model predicted 5.7 cases of listeriosis per year. This outcome of risk assessment is important to industry because manufacturers can strive to develop strategies to ensure that L. monocytogenes in RTE foods will not exceed a specified level, as may be stated in a performance objective or food safety objective [13,36]. The FAO/WHO and Food and Drug Administration/Food Safety and Inspection Service (FDA/FSIS) risk assessments both conclude that foods in which L. monocytogenes can multiply are of greatest concern [19,21]. Thus, frozen, acidified, or dried foods or foods containing additives to inhibit growth can be placed into a low-risk category. An important strategy for industry, then, is to conduct research on medium- and high-risk foods to shift them to a lower-risk category. Management strategies that focus on limiting the maximum concentration of L. monocytogenes in food at the consumer level should have considerable public health impact toward reducing listeriosis [15,19]. Research is needed to more clearly define the risk from specific types of foods within the FDA/FSIS categories. For example, deli meats are considered high risk, but epidemiologic data have identified only cooked noncured poultry breast products as an important source of foodborne listeriosis. Numerous other deli meats (e.g., cured poultry breast products, ham, bologna, corned

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beef, pastrami, roast beef, souse, headcheese, dry sausage, and loaf items) sold through the deli counter have not been implicated. Thus, research (e.g., challenge studies) may be necessary to better discriminate the level of risk associated with various products within these categories. Research also is needed at the retail level, with an emphasis on the deli counter, to better understand the risk factors leading to contamination of foods [18,56]. It is significant that the list of implicated foods has changed very little over the past 10 years and, thus, consumer guidance from the Centers for Disease Control and Prevention (CDC), FSIS, and FDA has changed very little. Is it possible that the major food sources have been identified? Smoked seafood has been categorized as a high-risk food [21] despite very little evidence linking this product to listeriosis. Large quantities are consumed annually throughout the world. A retail survey in two regions of the United States found the prevalence of L. monocytogenes in smoked seafood of all types to be 4.31%, with 3 of 114 positive samples (2.6%) in the range of 103 to 106 CFU/g [31]. Similar or higher prevalence rates in RTE fish products have been reported in other countries at the retail level, with some samples exceeding 103 CFU/g [17,39,40]. Why has a stronger epidemiological link not been made with this food group? Does the ecology of coldsmoked salmon operations, in particular, lead to strains that are less prone to cause illness or are there other reasons? Even more broadly, why are certain strains found in food more virulent, especially those few that have been responsible for major outbreaks in different regions of the world [56,61]? To date, very few foods containing sodium nitrite have been implicated in listeriosis. Is this because cured products normally contain a moderate amount of salt and the combination creates a less favorable environment for growth? Frankfurters are an exception, but this might reflect the greater risk of contamination because of the complexity of processes used for chilling, casing removal, sorting, packaging, and the many potential harborage sites that have been detected [61]. Industry has made considerable progress between 1990 and 2004 toward reducing the risk of contamination of frankfurters, but exceptions remain [26,66].

OUTBREAKS

VS.

SPORADIC CASES

If continuous progress is to be made in reducing the incidence of listeriosis, conditions that lead to sporadic cases must be resolved. Since 1988, CDC has conducted three case control studies to evaluate the role of food in sporadic listeriosis. The foods identified have been undercooked chicken and non-reheated frankfurters [58], soft cheeses, and foods purchased at deli counters [57] and, more recently, Camembert cheese, hummus, and sorbet [65]. Experience has verified the significance of frankfurters, certain soft cheeses, and certain foods purchased at the deli counter. Furthermore, it has been estimated that 99% of all listeriosis is foodborne [45]. However, do all cases of listeriosis result from commercially manufactured foods? Will existing regulatory policies and industry efforts be adequate to prevent sporadic cases? If industry were to succeed in consistently providing foods with ≤100 CFU per serving, would the baseline of listeriosis actually decrease to fewer than 10 cases per year, as has been estimated for the United States? Commercial facilities have large kitchens and special equipment for preparing foods on a large scale. Because the presence of L. monocytogenes in commercially prepared RTE foods is primarily caused by environmental contamination, could similar contamination occur in other locations (e.g., restaurants, retail outlets, catering, facilities for special events, and homes)? To what degree are such foods involved in listeriosis? One example of this is the 1993 outbreak of gastroenteritis in Italy, which was most likely caused by rice salad [55]. The salad of boiled rice, Swiss cheese, picked vegetables, hardboiled eggs, and mixed frozen vegetables was prepared 24 h in advance of a party and allowed to remain at ambient temperature until serving. Average daily temperatures in that region during June are 27–28°C. This dramatic event demonstrates the potential significance of food handling at the home level. A few studies have suggested home refrigerators as a source of contamination, but other potential sites also may exist in these locations (e.g., drains in sinks; seams along countertops; can openers; blenders; slicers; cloth used for cleaning dishes, countertops, and refrigerators; and

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other sites where food residue and moisture accumulate). If other food-handling locations are found to be a source, what can be done to reduce illness?

REDUCING CONSUMER EXPOSURE Preventing Contamination The goal here is to minimize or prevent contamination during the time RTE foods are exposed between a kill step and when the food is packaged for distribution. Considerable progress has been made in environmental control. This is directly the result of industry’s willingness to share best practices and treat pathogen control as a noncompetitive issue. Through workshops, publications, and other means, continued progress has been made as new interventions have been identified, shared, and implemented [1,28,35,63]. This practice should be continued to achieve further improvement. Food manufacturers must work closely with equipment manufacturers to ensure that equipment is cleanable, free of harborage sites, and performs as expected. Although there have been standards for the design of dairy and egg processing equipment, this has not been true of meat and poultry equipment since FSIS discontinued its equipment approval program. Dairy and egg processing typically involves closed systems with equipment that must meet rigid standards of hygiene design. Meat and poultry processing typically involves open systems with equipment that has lacked the level of hygiene design necessary to control L. monocytogenes. This situation has been changing through a joint effort involving equipment manufacturers and meat and poultry processors, and is leading toward improved control [2,3]. Continued research and development is needed to eliminate harborage sites in equipment as a source of L. monocytogenes [33]. Floors can be a significant source of listeriae in food manufacturing facilities unless an effective program of control has been established. New sanitizers are being developed that have improved microbial control and maintain their effectiveness over time on difficult surfaces. Less corrosive sanitizers (e.g., granulated quaternary ammonium compounds and hydrogen peroxide in a powdered form) have become available and offer two advantages. One is that floors can be maintained in a drier state, and the other is that they replace granulated citric acid, an effective but corrosive sanitizer that necessitates periodic floor resurfacing. New systems for delivering sanitizers have been developed and implemented (e.g., floor foamers at entrances of rooms and hallways). Some manufacturers strive to control L. monocytogenes by maintaining dry floors during production. Others maintain wet floors by applying a liquid sanitizer periodically during production. Some manufacturers have installed elaborate systems for washing and sanitizing footwear as employees enter certain rooms, whereas others avoid using footbaths. Some manufacturers fog a sanitizer into rooms as a final sanitization step. The decisions to maintain dry or wet floors, install permanent footbaths, apply sanitizer in the form of a fog, and adopt many other control measures are often made at the corporate level and may not reflect the uniqueness of each establishment or the food that is being produced. Data from the environmental sampling program should be evaluated and used to determine the best approach for each plant. An early observation was that the ratio of L. monocytogenes to other listeriae in the food processing environment appears to be unique to each facility and tends to be relatively stable [62]. In any particular facility, for example, the ratios seem to be relatively stable over time. Why do some facilities harbor a lower proportion of L. monocytogenes? Could it result from a decreased ability to compete with the normal flora of the facility? This has given rise to the idea of using competitive exclusion to reduce the presence of L. monocytogenes in food processing environments. Is it really possible to manage the ecology of a food processing environment and control the presence of a pathogen such as L. monocytogenes? The significance of L. monocytogenes in drains remains highly debatable. Many companies now maintain an aggressive program of cleaning and sanitizing floor drains. Some have replaced conventional drains with new stainless steel drains for improved cleanability. Others assume that L. monocytogenes will likely be present and merely clean and sanitize the basket and cover of the

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drains. Many companies use solid drain rings containing a quaternary ammonium sanitizer that slowly dissolves and is released. The significance of drains is further complicated by the FSIS assumption that drains reflect the microflora in the surrounding area and finding L. monocytogenes is evidence that the isolated strains likely came from nearby equipment and that product may have been contaminated. This interpretation has led to regulatory action against some facilities. The likelihood that a drain can be a harborage site in which L. monocytogenes can become established and multiply should be considered in any investigation. It may be established that a drain is a harborage site and the surrounding equipment is not involved or, when properly managed, the risk of contaminating the product contact surfaces is low and controllable. In each instance, the likelihood that L. monocytogenes can be dispersed from a positive drain to contaminate exposed food should be investigated and proved, not assumed. Experience has shown that equipment positioned close to a drain (e.g., the bottom of an incline conveyor) is more likely to become contaminated. Some of this may be the result of splashing and aerosols created during cleaning. Unclear, however, is whether drains “breathe” and contaminate the immediate environment during production as headspace in the drains fluctuates when large volumes of liquid flow through the drainage system of the plant. This could be evaluated by sampling the immediate environment around drains to determine if the frequency of positives increases over time and whether the frequency of positives decreases as distance from the drain increases. Although this may provide better information on the significance of drains in the environment in which RTE products are exposed, this information alone would not be sufficient to close the gap between the significance of a positive drain and the probability of product contamination. Very little research has been published on the role of drains in the overall hygiene of food operations [34]. The relevance of airborne contamination needs additional investigation. Efforts to recover L. monocytogenes from the air in food operations have yielded negative results [5,33,37,41]. This has been corroborated by unpublished industry results. Yet, regulatory action has been taken when an agency has concluded that the air of a facility is a source of product contamination even in the absence of supporting data. It is highly unlikely that a negative air pressure could account for equipment surfaces or product samples testing positive repeatedly over days or months with the same PFGE type of L. monocytogenes. Existing knowledge of food processing environments indicates that a variety of strains would more likely be detected. Additional research is needed to clarify the significance of ambient air. This question must not be confused with compressed air that can be a direct source of contamination, for example, when an air filter is moist, dirty, and has become a harborage site. Research is needed to better understand the diversity of L. monocytogenes strains found in the food processing environment and on food from different facilities throughout the food industry. One study found 10 pulsotypes were common to two or more types of food and 17 pulsotypes were detected in foods of more than one producer having no apparent association with each other. In addition, similar pulsotypes were recovered from foods in different European countries over several years [6]. Other studies have found the same PFGE types of L. monocytogenes in different facilities [39,44,54]. During an investigation of isolates from dairy farms and sporadic cases in the U.S. Pacific Northwest, some strains matched those associated with previous epidemics (e.g., outbreak in 1983 in Massachusetts, Mexican-style soft cheese outbreak in 1985 in California, pâté outbreak in 1988–1990 in the United Kingdom, sliced turkey deli meat in 2000 in the United States). It was suggested that these strains might have an on-farm reservoir [10]. This information demonstrates that certain strains of great public health significance are more widely spread geographically and in nature than was previously thought. Unfortunately, the existing regulatory climate in the United States that borders on “zero presence” for L. monocytogenes in the environment in which RTE foods are exposed during processing and packaging discourages such investigation. An untapped body of data, however, does exist within the FSIS database for L. monocytogenes isolates from meat and poultry products. This database should be used to help answer the diversity question. Because industry does not have access to the FSIS data, this evaluation must be conducted by the agency. A study of 149 isolates collected by

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the FDA from late 1999 to early 2003 found several strains in samples from different sources. The most common ribotype pattern was found in samples from Chile, Germany, Spain, and seven different states in the United States [30]. Monitoring programs by control authorities and others must consider whether the analytical method may yield biased results for the strains detected. Analysis of food and environmental samples typically involves an enrichment step before isolation. Enrichment of samples having more than one strain in a selective medium (e.g., University of Vermont medium) is biased against detection of those strains more likely to cause illness [11]. This bias can have great impact on epidemiologic studies to identify a specific food or processing plant as the source of illness. The bias also raises questions about programs such as the FSIS monitoring program for L. monocytogenes in RTE meat and poultry products. Each isolate is subjected to PFGE analysis and the results shared with public health agencies to determine if matches occur with clinical isolates. Research is needed to better understand and correct the bias in the analytical method. Direct plating may be more productive when analyzing foods in which growth may have occurred (e.g., food collected from retail or home refrigerators) [11,43]. Existing methodology for assessing control of L. monocytogenes in food operations appears to be adequate to detect a problem, if one exists. The sponge method has proved very sensitive provided sufficient samples are collected from the environment and at frequent intervals (e.g., weekly) in higher-risk environments. More rapid and/or more sensitive methods may be desirable, but this is not a high priority for L. monocytogenes research. There is a greater need for a cost-effective system that smaller manufacturers can use to assess control. In addition, when investigating the source of a contamination that is detected infrequently but is cause for concern, hundreds of samples may need to be collected over several weeks before the source is detected and corrections can be implemented. Low-cost analytical methods are necessary for producers of all sizes when a difficult, elusive problem must be resolved. Two approaches that are being used for sampling the environment deserve comment. One involves the zone concept, in which the emphasis is on detecting L. monocytogenes in zones of lower risk to product contamination. Some proponents of this strategy suggest that if L. monocytogenes can be controlled in lower-risk zones, then the likelihood that product contact surfaces and product will become contaminated is reduced. This strategy does not ensure that a harborage site will not develop in close proximity to product contact surfaces. There is a continuing need to sample product contact surfaces at a frequency that can provide confidence that the focus on lower-risk zones continues to be effective. A strategy that offers greater confidence is to routinely sample higher-risk zones (i.e., product contact surfaces) and include a small number of samples from lower-risk zones. The second approach is to create a list of sample sites associated with a processing line or area. Sites are randomly selected from the list for each sampling interval. An inherent weakness of this approach is that infrequent sampling of critical sites (i.e., product contact surfaces) may not permit an assessment of the risk of product contamination. Site selection is a very important attribute of this approach. The list of sites should consist of those that permit a short- and longterm assessment of the likelihood that the food may be contaminated. Each manufacturer of medium- and high-risk food should be able to assess on an ongoing basis whether the foods being packaged on each line are likely to have become contaminated between the lethality step and final packaging. For the present, each plant must develop the best sampling plan for its situation and for the level of risk associated with the food that is being produced. Of equal or, perhaps, greater concern is the urgency and effectiveness of the response by the manufacturer when positive sample sites are detected. Postpasteurization The goal here is to ensure the absence of viable cells of L. monocytogenes on medium- and highrisk RTE foods. With categorization of foods according to risk, food manufacturers are applying

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newer technologies to shift their products from higher- to lower-risk categories. One method is to apply a listericidal treatment after the food is in its final package. Many foods have traditionally received a listericidal treatment immediately before (i.e., aseptically packaged foods) or after packaging. Examples include canned shelf-stable foods, canned perishable hams and luncheon meat, certain fluid dairy and egg products, puddings, and juices. New equipment and packaging materials have been developed to expand the options available for delivering listericidal treatments after final packaging, while retaining quality of product in an acceptable package at a cost that is competitive. The lethality treatments generally fall into three methods: heat, high pressure, and irradiation. Heat has been used commercially to postpasteurize meat and poultry products for control of L. monocytogenes since at least 1990 and is now widely applied throughout the world. Most of the systems involve passing the packaged product through hot water or steam. Research and improvement in such systems continue [15,46,48,49], and more should be encouraged. Similarly, high hydrostatic pressure is being used commercially for a variety of foods. This technology shows great promise of broader acceptance if the cost and certain performance issues can be resolved. Additional research on the use of high hydrostatic pressure, alone or in combination with other control options, is needed [7]. Although extensive research on irradiation has been done over the past 50 years and still continues [8,15, 16, 27, 60], this is not an approved process for medium- and high-risk RTE products of concern. Application will require approval by control authorities responsible for food protection at the national level and appropriate labeling and education of consumers. Additional research and development is needed to improve upon these technologies to provide more choices, greater flexibility in processing, minimal space requirements, reliable performance, minimal cost, and optimal product quality. Research is needed to learn whether combinations (e.g., heat, high pressure, additives, changes in pH, etc.) can be more effective than single treatments. Can synergism be demonstrated for certain combinations? In addition, considerable research is necessary to validate the efficacy of these systems to ensure destruction of L. monocytogenes in a variety of foods. For RTE meat and poultry products manufactured in the United States, FSIS has not specified the log10 reduction required to validate the effectiveness of a postpasteurization process [23], but this is likely to change. Research is needed to understand the number and distribution of cells that occur when RTE foods become contaminated before final packaging. This information is critical to design of validation studies for control measures (e.g., lethality treatments and additives to prevent growth). Preventing Growth The goal here is to manage the intrinsic and extrinsic factors to prevent multiplication of L. monocytogenes in foods between final packaging and when foods are eaten. Manufacturers of medium- and high-risk foods should be informed that consumer risk is significantly reduced if L. monocytogenes growth is delayed or, ideally, prevented, so that the concentration of L. monocytogenes remains low throughout the code date on the package. This result of the Listeria risk assessments should drive research to identify effective, approved additives that reduce consumer risk and manufacturer liability. Intrinsic factors (e.g., pH, aw) of some foods prevent L. monocytogenes growth. Examples of foods in which L. monocytogenes cannot multiply include citrus juices, yogurt, hard cheeses, and dried foods. Foods that have been implicated as vehicles for listeriosis typically are high in pH (e.g., >5.5), high in free moisture, and require refrigeration because of their perishable nature. A high priority in the minds of some has been the relationship between samples from product contact surfaces that test positive and the probability of product contamination. This should be expanded to include an estimate of the numbers and distributions of L. monocytogenes that can occur when product becomes contaminated. When antimicrobial agents are added to control growth of L. monocytogenes, they should be validated for efficacy and the supporting data should be available for review by control authorities [22–25]. One means of generating realistic data is to set aside positive production lots and determine the initial level of contamination and the rate of growth

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during storage. The information also could be used to understand whether inoculated pack studies truly reflect the risk associated with the low level of contamination that likely occurs with natural contamination. To what degree is the physiology of natural contaminants the same as laboratory cultures? Are natural contaminants able to compete with the other flora that occurs on freshly packaged product? Intrinsic and extrinsic factors associated with each food further complicate this issue and may result in different responses in different foods. There is some evidence that natural contaminants of L. monocytogenes may not grow on certain foods stored under commercial conditions. This is suggested by a study on frankfurters in which the rate of positive packages did not increase during extended storage at 4 and 10°C [66]. Furthermore, when unopened packages from lots that tested negative in the FSIS monitoring program were held at 4°C for 6 weeks, only 18 of 1984 (0.91%) packages tested positive [66]. For cold-smoked salmon, many packages from contaminated lots do not show multiplication of L. monocytogenes during the normal shelf life. One reason appears to be the presence of other microorganisms (e.g., Carnobacterium spp.) that inhibit outgrowth of L. monocytogenes [12,32,51,52]. At very low inoculum levels, an increasing salt concentration can become increasingly inhibitory. Stationaryphase and sublethally injured cells have a lower probability of growth from low populations [53]. Additional research is needed to better understand conditions when growth from naturally contaminated foods will not occur. Extensive research has been conducted on a wide variety of additives (e.g., chemicals and bacteriocins), but very few (e.g., sodium and potassium lactate and sodium diacetate) have been used commercially. A variety of antimicrobial agents coupled with other hurdles (e.g., reduced pH) are being used to control pathogens such as L. monocytogenes in perishable foods. Many trade associations (e.g., manufacturers of prepared salads) have sponsored research, some of which has not been published, that is used as guidance for its members. A summary of research on the use of additives to control growth of L. monocytogenes in RTE meat and poultry products is included in the FSIS guidelines [24]. To this information should be added research on additives in cold-smoked fish [38] and other foods. Research by Seman et al. [59] as well as Legan et al. [42] has been used to generate software that enables manufacturers to formulate their RTE meat and poultry products to control growth of L. monocytogenes. The fact that this information has been made available to others in the industry is an excellent example of the commitment that has evolved within many companies to help competitors enhance the safety of their products. A variation on adding antimicrobial agents to food as an ingredient during formulation is to apply a listericidal treatment, such as heat [29] and/or an antimicrobial agent, to the surface of a food product (e.g., frankfurters and deli meat, or poultry) just before the food is packaged. Combinations of pasteurization before and after packaging also have been explored [47]. Such treatments can have the effect of killing microbial contaminants, such as L. monocytogenes, at the surface of the product and, in combination with an additive, retard or prevent growth of surviving cells during subsequent storage. Research should continue to identify additional listericidal treatments, additives, and combinations of listericidal treatments and additives that kill or prevent growth of L. monocytogenes. In addition to the research is the need to pursue regulatory approval for use of the treatments and additives in a wide variety of foods. This would enable selection of one or more options for control that provide the best fit for each specific type of food. Freezing, of course, is an option to prevent growth of L. monocytogenes in certain foods. Code-dating practices can influence consumer exposure to high concentrations of L. monocytogenes. The risk associated with perishable foods that may be exposed to contamination before packaging can be reduced by using shorter code dates. The National Advisory Committee on Microbiological Criteria for Foods has developed guidance that could be used to validate the safety of “use by” dates that are applied by industry [50]. Similar guidance has been developed to ensure that product shelf life is assessed in a standardized manner by European food producers and retailers [9]. This guidance could be used to ensure compliance with European directives that require establishment of appropriate shelf life dates for food.

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Numerous issues must be resolved through research to arrive at an acceptable, standardized method of validation. Among the issues is whether a laboratory-prepared culture of L. monocytogenes will behave in the same manner as a natural contaminant of L. monocytogenes. The source of the strains (e.g., food or clinical isolates), methods of preparing the inoculum (e.g., 24-h broth culture), and method of inoculation (e.g., on the surface of the product) should be defined. What is an appropriate level of inoculum? What levels (i.e., prevalence and concentration) actually occur when a food is contaminated in a processing plant? Can strains having little or no virulence be used to minimize risk among laboratory personnel? What constitutes a sufficient change in a food, process, or package that would necessitate revalidation? What temperatures should be used to represent the temperatures occurring throughout the food chain? What maximum concentration of L. monocytogenes is acceptable for code-date validation, i.e., what is the food safety objective for L. monocytogenes in RTE foods? Some of these questions are being investigated [64]. Estimating Consumer Exposure Research is needed to better estimate consumer exposure to contaminated foods and, ideally, estimate the number of cells consumed. As foods are identified as the source of listeriosis, a statistically based sampling plan should be used to examine representative lots to develop estimates of prevalence, concentration, and distributions. This is seldom possible, because the food is often no longer available or too few samples remain. Occasionally, however, opportunities occur, such as the 1998 multistate outbreak when approximately 100 illnesses were recognized. The recall of all frankfurters and other cooked meat and poultry products with “sell-by” dates before February 1999 involved about 35 million lb. Because only 5.9 million lb were recovered, it can be assumed the remainder had been consumed. Although the quantity of frankfurters was not specified, it is reasonable to assume that the amount of potentially contaminated frankfurters consumed exceeded 5 million pounds. The plant reportedly produced 250,000 lb of frankfurters per day [4] and, if true, the quantity of frankfurters produced between July 4, 1998, and February 1999 should have been far greater than 5 million lb. During 1998 antimicrobial agents were not added to frankfurters and many companies in the United States used sell-by or use-by dates of 60 days or greater from the date of production. Growth to levels exceeding 103 CFU/frankfurter (i.e., serving) would have been expected in some portion of the packages. The potential for product contamination existed over an extended period (≥6 months). During the 6 weeks before the July 4th weekend, about 25% of the plant’s environmental samples tested positive for Listeria spp. After the July 4th weekend, when a cooling unit was removed and replaced, the rate of positive samples reportedly increased during the following 6 weeks to 92% and remained at nearly 70% over the subsequent 9 weeks of testing [4]. The contamination was attributed to construction dust; however, during removal of the refrigeration unit, the room temperature would have significantly increased, allowing for a substantial increase in the microbial population. Subsequent cleaning and sanitizing may not have been adequate to cope with the increased population. The possibility of harborage sites from which a persistent strain of L. monocytogenes could be dispersed over a period of weeks or months was not recognized at the time by public health officials. Retail frankfurters are normally manufactured to yield 8 or 10 per pound. It can be assumed that more than one individual would have eaten frankfurters from many of the packages and, in many instances, the frankfurters would have been eaten on multiple occasions until none in the package remained. The recall notice does not provide production dates or number of production lots. The available information suggests that consumer exposure was frequent and would have involved packages with high numbers of cells. It is surprising that only approximately 100 instances of illness were recognized. Why were so few cases recognized, given the probable extent of exposure? If a more complete analysis had been made of the product and information made available to the agencies, an assessment, for example, of the relative virulence of the unique strain may have been possible.

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Although the foregoing description includes many assumptions, the information is provided to illustrate the opportunity for expanding our knowledge of consumer exposure and the risk of illness. More complete investigation and information gathering during such events could enhance our knowledge of dose–response and exposure assessment for L. monocytogenes in RTE foods.

CONCLUDING REMARKS The road to controlling L. monocytogenes since the early 1980s has been long and difficult. The food industry had no previous experience with controlling a human pathogen that was capable of multiplying in refrigerated rooms in which perishable RTE foods may be exposed to contamination. For many foods, research at the processing plant level and in the laboratory was necessary to, first, understand the issues involved, second, develop strategies for control, and third, implement effective controls appropriate for the foods of concern. Enormous changes have occurred throughout the food industry since the year 2000, particularly in the U.S. meat and poultry industry, the source of several major outbreaks. Additional improvements toward control of L. monocytogenes continue to be developed through research, and it is anticipated that future risk assessments will have to consider the many changes that have occurred within the medium- and high-risk food categories. A conclusion of the FAO/WHO risk assessment is that compliance by industry to control contamination and ensure low numbers of cells when foods are consumed is one of the keys to reducing the incidence of listeriosis. This represents an important challenge to the food industry, but it also clarifies the goal that must be achieved. Another outcome was the importance of temperature throughout the life of perishable RTE foods. This will require an effective educational program to achieve the desired results. Validation of the educational program’s effectiveness will be as important as validation of other control measures applied throughout the food chain. Additional research and application of the results are needed to achieve the desired level of control with optimal, costeffective systems.

ACKNOWLEDGMENT Comments from Randy Huffman, American Meat Institute, and Jenny Scott, Food Products Association, are gratefully appreciated and acknowledged by the author.

REFERENCES 1. AMI (American Meat Institute). 2003. Listeria Control Manual, A comprehensive Step by Step Guide for Processors. Meat Marketing Technol., December Supplement. 2. AMI (American Meat Institute) 2004. Eleven Principles of Sanitary Facility Design. http://www.meatami. com/Template.cfm?Section=Sanitation&CONTENTID=2765&TEMPLATE=/ContentManagement/ Content Display.cfm. 3. AMI (American Meat Institute). 2004. Sanitary Equipment Design Checklist. http://www.meatami.com/ Template.cfm?Section=Sanitation&CONTENTID=2278&TEMPLATE=/ContentManagement/ContentDisplay.cfm. 4. Anonymous. 1999. CDC report links Bil Mar outbreak to construction. Food Chem. News. June 14: 1, 25–26. 5. Autio, T., S. Hielm, M. Miettinen, et al. 1999. Sources of Listeria monocytogenes contamination in a cold-smoked rainbow trout processing plant detected by pulsed-field gel electrophoresis typing. Appl. Environ. Microbiol. 65:150–155. 6. Autio, T., J. Lundén, M. Fredriksson-Ahomaa, et al. 2002. Similar Listeria monocytogenes pulsotypes detected in several foods originating from different sources. Int. J. Food Microbiol. 77: 83–90. 7. Aymerich, T., A. Jofré, M. Garriga, and M. Hugas. 2005. Inhibition of Listeria monocytogenes and Salmonella by natural antimicrobials and high hydrostatic pressure in sliced cooked ham. J. Food Prot. 68: 173–177.

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8. Bari, M.L., M. Nakauma, S. Todoriki, et al. 2005. Effectiveness of irradiation treatments in inactivating Listeria monocytogenes on fresh vegetables at refrigeration temperature. J. Food Prot. 68: 318–323. 9. Betts, R.P. 2005. European perspectives on shelf life dating. Food Prot. Trends 25: 57–58. 10. Borucki, M.K., J. Reynolds, C.C. Gay, et al., 2004. Dairy farm reservoir of Listeria monocytogenes sporadic and epidemic strains. J. Food Prot. 11: 2496–2499. 11. Bruhn, J.B., B. Fonnesbech Vogel, and L. Gram. 2005. Bias in the Listeria monocytogenes enrichment procedure: lineage 1 strains in University of Vermont selective enrichment. Appl. Environ. Microbiol. 71:961–967. 12. Budde, B.B., T. Hornbaek, T. Jacobson, et al. 2003. Leuconostoc carnosum 4010 has the potential for use as a protective culture for vacuum-packed meats: culture isolation, bacteriocin identification and meat application experiments. Int. J. Food Microbiol. 83: 171–184. 13. CCFH (Codex Committee on Food Hygiene). 2005. Proposed draft guidelines on the application of general principles of food hygiene to the control of Listeria monocytogenes in ready-to-eat foods. CX/FH 05/37/5, December 2004, revised. Available at ftp://ftp.fao.org/codex/ccfh37/fh37_05e.pdf. 14. Chen, C.-M., J.G. Sebranek, J.S. Dickson, and A.F. Mendonca. 2004. Combining pediocin (ALTA 2341) with postpackaging irradiation for control of Listeria monocytogenes on frankfurters. J. Food Prot. 67: 1866–1875. 15. Chen, Y., W.H. Ross, V.N. Scott, and D.E. Gombas. 2003. Listeria monocytogenes: Low levels equal low risk. J. Food Prot. 66: 570–577. 16. Clardy, S., D.M. Foley, F. Caporaso, et al. 2002. Effect of gamma irradiation on Listeria monocytogenes in frozen, artificially contaminated sandwiches. J. Food Prot. 65: 1740–1744. 17. Coillie, E. van, H. Werbrouck, Heyndrickx, et al. 2004. Prevalence and typing of Listeria monocytogenes in ready-to-eat food products on the Belgian market. J. Food Prot. 67: 2480–2487. 18. Elson, R., F. Burgess, C.L. Little, et al. 2004. Microbiological examination of ready-to-eat cold sliced meats and pâte from catering and retail premises in the U.K. J. Appl. Microbiol. 96: 499–509. 19. FAO/WHO (Food and Agriculture Organization of the United Nations/World Health Organization). 2004. Risk Assessment of Listeria monocytogenes in Ready-to-Eat Foods. Interpretive Summary and Technical Report. Microbiological Risk Assessment Series. Food and Agriculture Organization of the United Nations, Rome. Available at http://www.fao.org/es/esn/food/risk_mra_riskassessment_listeria_en.stm. 20. FDA (Food and Drug Administration). 2001. Processing parameters needed to control pathogens in cold smoked fish. Available at http://www.cfsan.fda.gov/~comm/ift2-toc.html. 21. FDA-FSIS (Food and Drug Administration-Food Safety and Inspection Service). 2003. Quantitative assessment of relative risk to public health from foodborne Listeria monocytogenes among selected categories of ready-to-eat foods. Center for Food Safety and Applied Nutrition, Food and Drug Administration, U.S. Department of Health and Human Services and the Food Safety and Inspection Service, U.S. Department of Agriculture, Washington, D.C. Available at http://www.foodsafety.gov/ ~dms/lmr2-toc.html. 22. FSIS (Food Safety and Inspection Service). 2003. Control of Listeria monocytogenes in ready-to-eat meat and poultry products; final rule. Fed. Regist. 68: 34208–34254. 23. FSIS (Food Safety and Inspection Service). 2004. Questions and Answers. Resource 3. Available at: http://www.fsis.usda.gov/oa/haccp/lmworkshop.htm. 24. FSIS (Food Safety and Inspection Service). 2004. Compliance guidelines to control Listeria monocytogenes in post-lethality exposed ready-to-eat meat and poultry products. Available at http://www. fsis.usda.gov/oa/haccp/lmworkshop.htm. 25. FSIS (Food Safety and Inspection Service). 2004. Assessing the effectiveness of the “Listeria monocytogenes” interim final rule. Available at http://www.fsis.usda.gov/Oppde/rdad/frpubs/97-013F/LM_ Assessment_Report_2004.pdf. 26. FSIS (Food Safety and Inspection Service). 2004. Microbiological testing programs for ready-to-eat meat and poultry products. Available at http://www.fsis.usda.gov/ophs/rtetest/index.htm. 27. Fu, A.-H., J.G. Sebranek, and E.A. Murano. 1995. Survival of Listeria monocytogenes and Salmonella typhimurium and quality attributes of cooked pork chops and cured ham after irradiation. J. Food Sci. 60: 1001–1005, 1008. 28. Gall, K., V.N. Scott, R. Collette, et al. 2004. Implementing targeted good manufacturing practices and sanitation procedures to minimize Listeria contamination of smoked seafood products. Food Prot. Trends 24: 302–315.

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29. Gande, N. and P. Muriana. 2003. Prepackage surface pasteurization of ready-to-eat meats with a radiant heat oven for reduction of Listeria monocytogenes. J. Food Prot. 66: 1623–1630. 30. Gendel, S.M. 2004. Riboprint analysis of Listeria monocytogenes isolates obtained by FDA from 1999 to 2003. Food Microbiol. 21: 187–191. 31. Gombas, D.E., Y. Chen, R.S. Clavero, and V.N. Scott. 2003. Survey of Listeria monocytogenes in ready-to-eat foods. J. Food Prot. 66: 559–569. 32. Gram, L. 2001. Potential hazards in cold-smoked fish: Listeria monocytogenes. J. Food Sci. S-1072–1081, Supplement to 66(7). 33. Gudbjörnsdóttir, B., M-L. Suihko, P. Gustavsson, et al. 2004. The incidence of Listeria monocytogenes in meat, poultry and seafood plants in Nordic countries. Food Microbiol. 21: 217–225. 34. Heldman, D.R., T.I. Hedrick, and C.W. Hall. 1965. Sources of air-borne microorganisms in food processing areas—drains. J. Milk Food Technol. 28: 41–45. 35. Hicks, D., M. Wiedmann, V.N. Scott, et al. 2004. Minimizing Listeria contamination in smoked seafood: training plant personnel. Food Prot. Trends 24: 953–960. 36. ICMSF (International Commission on Microbiological Specifications for Foods). 2002. Microorganisms in Foods. 7. Microbiological Testing in Food Safety Management. Kluwer Academic/Plenum Publishers, New York. 37. Jacquet, C., J. Rocourt, and A. Reynaud. 1993. Study of Listeria monocytogenes contamination in a dairy plant and characterization of the strains isolated. Int. J. Food Microbiol. 20: 13–22. 38. Jahncke, M.L., R. Collette, D. Hicks, et al. 2004. Treatment options to eliminate or control growth of Listeria monocytogenes on raw material and on finished product for the smoked fish industry. Food Prot. Trends 24: 612–619. 39. Johansson, T., L. Rantala, L. Palmu, and T. Honkanen-Buzalski. 1999. Occurrence and typing of Listeria monocytogenes strains in retail vacuum-packed fish products and in a production plant. Int. J. Food Microbiol. 47: 111–119. 40. Jorgensen, L.V. and H.H. Huss. 1998. Prevalence and growth of Listeria monocytogenes in naturally contaminated seafood. Int. J. Food Microbiol. 42: 127–131. 41. Lawrence, L.M. and A. Gilmour. 1994. Incidence of Listeria spp. and Listeria monocytogenes in a poultry processing environment and in poultry products and their rapid confirmation by multiplex PCR. Appl. Environ. Microbiol. 60: 4600–4604. 42. Legan, J.D., D.L. Seman, A.L. Milkowski, et al. 2004. Modeling the growth boundary of Listeria monocytogenes in ready-to-eat cooked meat products as a function of the product salt, moisture, potassium lactate, and sodium diacetate concentrations. J. Food Prot. 67: 2195–2204. 43. Loncarevic, S., W. Tham, and M.L. Danielsson-Tham. 1996. The clones of Listeria monocytogenes detected in food depend on the method used. Lett. Appl. Microbiol. 22: 381–384. 44. Lundén, J.M., T.J. Autio, A.-M. Sjöberg, and H.J. Korkeala. 2003. Persistent and nonpersistent Listeria monocytogenes contamination in meat and poultry processing plants. J. Food Prot. 66: 2062–2069. 45. Mead, P.S., L. Slutsker, V. Dietz, et al. 1999. Food-related illness and death in the United States. Emerging Infect. Dis. 5: 607–625. 46. Muriana, P.M., W. Quimby, C.A. Davidson, and J. Grooms. 2002. Postpackage pasteurization of ready-toeat deli meats by submersion heating for reduction of Listeria monocytogenes. J. Food Prot. 65: 963–969. 47. Muriana, P.M., N. Gande, W. Robertson, et al. 2004. Effect of prepackage and postpackage pasteurization on postprocess elimination of Listeria monocytogenes on deli turkey products. J. Food Prot. 67: 2472–2479. 48. Murphy, R.Y., K.H. Driscoll, M.E. Arnold, et al. 2003. Lethality of Listeria monocytogenes in fully cooked and vacuum packaged chicken leg quarters during steam pasteurization. J. Food Sci. 68: 2780–2783. 49. Murphy, R.Y., L.K. Duncan, K.H. Driscoll, et al. 2003. Determination of thermal lethality of Listeria monocytogenes in fully cooked chicken breast fillets and strips during postcook in-package pasteurization. J. Food Prot. 66: 578–583. 50. NACMCF (National Advisory Committee on Microbiological Criteria for Foods). 2004. Considerations for establishing safety-based consume-by date labels for refrigerated ready-to-eat foods. Adopted August 27. Washington, DC. Available at http//www.fsis.usda.gov/ophs/nacmcf/2004/ NACMCF_Safety-based_Date_Labels_082704.pdf.

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51. Nilsson, L., J. Christiansen, B. Jorgensen, et al. 2004. The contribution of bacteriocin to inhibition of Listeria monocytogenes by Carnobacterium piscicola strains in cold smoked salmon systems. J. Appl. Microbiol. 96: 133–134. 52. Nilsson, L., T.B. Hansen, P. Garrido, et al. 2005. Growth inhibition of Listeria monocytogenes by a nonbacteriocinogenic Carnobacterium piscicola. J. Appl. Microbiol. 98: 172–183. 53. Pascual, C., T.P. Robinson, M.J. Ocio, et al. 2001. The effect of inoculum size and sublethal injury on the ability of Listeria monocytogenes to initiate growth under suboptimal conditions. Lett. Appl. Microbiol. 33: 357–361. 54. Peccio, A., T. Autio, H. Korkeala, et al. 2003. Listeria monocytogenes occurrence and characterization in meat-producing plants. Lett. Appl. Microbiol. 37: 234–238. 55. Salamina, G., E.D. Donne, A. Niccolini, et al. 1996. A foodborne outbreak of gastroenteritis involving Listeria monocytogenes. Epidemiol. Infect. 117: 429–436. 56. Sauders, B.D., K. Mangione, C. Vincent, et al. 2004. Distribution of Listeria monocytogenes molecular subtypes among human and food isolates from New York State shows persistence of human diseaseassociated Listeria monocytogenes strains in retail environments. J. Food Prot. 67: 1417–1428. 57. Schuchat, A., K.A. Deaver, J.D. Wenger, et al. 1992. Role of foods in sporadic listeriosis. I. Casecontrol study of dietary risk factors. JAMA 267: 2041–2045. 58. Schwartz, B., C.A. Ciesielski, C.V. Broome, et al. 1988. Association of sporadic listeriosis with consumption of uncooked hot dogs and undercooked chicken. Lancet 2: 779–782. 59. Seman, D.L., A.C. Borger, J.D. Meyer, et al. 2002. Modeling the growth of Listeria monocytogenes in cured ready-to-eat processed meat products by manipulation of sodium chloride, sodium diacetate, potassium lactate and product moisture content. J. Food Prot. 65: 651–658. 60. Sommers, C.H. and D.W. Thayer. 2000. Survival of surface-inoculated Listeria monocytogenes on commercially available frankfurters following gamma irradiation. J. Food Saf. 20: 127–137. 61. Tompkin, R.B. 2002. Control of Listeria monocytogenes in the food-processing environment. J. Food Prot. 65: 709–725. 62. Tompkin, R.B., L.N. Christiansen, A.B. Shaparis, et al. 1992 Control of Listeria monocytogenes in processed meats. Food Aust. 44: 370–376. 63. Tompkin, R.B., V.N. Scott, D.T. Bernard, et al. 1999. Guidelines to prevent post-processing contamination from Listeria monocytogenes. Dairy Food Environ. Sanit. 19: 551–562. 64. Uyttendaele, M., A. Rajkovic, G. Benos, et al. 2004. Evaluation of a challenge testing protocol to assess the stability of ready-to-eat cooked meat products against growth of Listeria monocytogenes. Int. J. Food Microbiol. 90: 219–236. 65. Varma, J.K., M.C. Samuel, R. Marcus, et al. 2004. Dietary and medical risk factors for sporadic Listeria monocytogenes infection: A FoodNet case-control study: United States, 2000–2003. Int. Conf. Emerg. Infect. Dis., Program and Abstracts, Atlanta, p. 101. 66. Wallace, F.M., J.E., Call, A.C.S. Porto, G.J. Cocoma, The ERRC Special Projects Team, and J.B. Luchansky. 2003. Recovery rate of Listeria monocytogenes from commercially prepared frankfurters during extended refrigerated storage. J. Food Prot. 66: 584–591.

RESEARCH NEEDS: AN ACADEMIC PERSPECTIVE SOPHIA KATHARIOU

After an intense and close scrutiny of its physiology, genomic composition, and its interactions with mammalian model systems during the past two decades, Listeria monocytogenes remains both intriguing and interesting, and yet threatening. It is intriguing to those who see it as a multilayered system with complex, sophisticated adaptations for impressively diverse environments, be it mammalian host cells, food processing plants, or the organism’s other habitats in nature; interesting to those who see it as a paradigm for foodborne pathogens constantly responding to shifts in their available habitats, often made possible through human, social, and economic trends that impact the way food is processed, distributed, and handled; and threatening to those humans and animals for whom it can cause frequently devastating illness. It also remains threatening in a related sense to the food industry, the economic well-being of which can be seriously compromised when contaminated ready-to-eat products are recalled or implicated in outbreaks of listeriosis.

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In the next series of research studies to come, I expect that we, as students of the organism, will continue to pursue further understanding of the organism’s attributes that render it, in the aforementioned sense, intriguing, interesting, and threatening. New generations of scientists, equipped with an increasingly rich collection of the tools of genomics, proteomics, and biomathematics, will carry the frontiers of Listeria research further, posing new questions. Interesting and novel answers will be pursued to address fundamental problems of the sort that have occupied the Listeria community heretofore; these can be summarized as questions of what virulence and other features the organism has and expresses, as well as how, where, and when it does so. Following are thoughts on some projected trends in studies along these lines, from the point of view of one researcher involved in some of these issues, and what these trends might look like during the next several years.

WHAT THE ORGANISM HAS AND EXPRESSES: THE GENOMIC ENDOWMENT OF L. MONOCYTOGENES

AND

PROTEOMIC

A milestone in the study of the organism was the first complete genome sequence of L. monocytogenes (strain EGD-e), along with the sequence of the genome of L. innocua, a nonpathogenic species closely related to L. monocytogenes [3]. L. monocytogenes was found to have a remarkably conserved genomic backbone compared to its nonpathogenic relative. Underlying the identification and analysis of genomic segments found in L. monocytogenes, but absent from L. innocua, was the realization that some of these genomic segments would be specific to the strain or serotype of the strain that was chosen for genome sequencing, strain EGD, of serotype 1/2a. Earlier genomic surveys had suggested that indeed strains of other serotypes harbor genomic fragments unique to them and absent from the genome of serotype 1/2a strains [5,6]. The complete genome sequencing of three additional strains (one of serotype 1/2a and two of serotype 4b) confirmed and extended these findings [8]. The clonal population structure of L. monocytogenes suggests that detailed genomic analysis of strains of diverse serotypes and clonal groups would be valuable, and indeed necessary, for an adequate understanding of the genomic makeup of the species. Genomic initiatives that will reveal the complete genomic sequence of several additional strains are now anticipated during the next few years. How will the choice be made of which new strains will have their complete genome sequenced? This is an issue of considerable importance, because complete genome sequencing, even though it currently can be accomplished in a relatively short period of time, remains a large, and expensive, undertaking and should be designed to maximize use of its findings by scientists of as many diverse research interests, backgrounds, and program objectives as may be possible. Ideally, funding agencies would make such choices, following open discussion and evaluation of feedback by the Listeria research community as a whole. Leading in priority would be strains implicated in outbreaks. At this time, genome sequence information has been reported for only three strains implicated in outbreaks of human listeriosis [8]. A number of additional outbreak strains may be chosen for complete genome sequencing, including (a) strain Scott A (serotype 4b) used in many food microbiology studies, (b) a strain of serotype 3a implicated in a butter-associated outbreak, (c) and strains implicated in febrile gastroenteritis outbreaks. Even though outbreaks receive most of our attention, the public health burden of clinical listeriosis primarily involves sporadic cases. None of the four strains, the genomes of which have been already sequenced, was from sporadic cases of human illness. The genome sequence of selected strains from sporadic cases is now eagerly awaited. Choosing among isolates from sporadic cases will be demanding and, again, should ideally be done in consultation with the Listeria community. Special efforts should be made to utilize feedback from sources that can evaluate temporal and geographical trends in sporadic illness incidence of isolates with specific genomic

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fingerprints, such as the Centers for Disease Control and Prevention, the PulseNet database, and equivalent agencies or databases in other countries. It would be valuable to include strains with genomic fingerprints common among isolates from sporadic cases, as well as selected strains with fingerprints that are unique, or rarely encountered. It would also be valuable to include representatives of the prevalent clinical serotypes (1/2a, 1/2b, and 4b) as well as of those serotypes rarely encountered in human illness (1/2c, 3a, 3b, and 3c). Comparative genomic analysis of such isolates would yield a wealth of information that may be critical to our understanding of how (and whether) human virulence may be reflected at the genomic level of the microbe. In addition, genome sequencing of selected isolates from a poorly understood group (Lineage III), primarily consisting of strains of serotype 4a and 4c, would be valuable for furthering our understanding of the evolution of L. monocytogenes. Lineage III strains appear to be significantly divergent from other members of the species and were reported to be significantly more frequent among animal listeriosis isolates than among those implicated in human illness [9]. In addition to strains chosen on the basis of their epidemiological significance, there is still a keen need to know the genomic composition of isolates frequently implicated in colonization of food processing plants and consequently in contamination of food products. Of interest would be recently identified isolates from foods that harbor the genetic fragments typical of epidemicassociated strains [10] as well as isolates that are frequently encountered in foods and processing environments but are relatively rare in illness, for example, isolates of serotype 1/2c. An additional major contribution in Listeria genomics would be the genome sequence of isolates from the organism’s potential reservoirs in nature (animals, soil, vegetation, and others as yet unidentified). These genome sequencing projects are expected to provide further information of the genomic attributes that characterize different serotypes and clonal groups and may be unique to isolates with special epidemiological or ecological (plant colonization, reservoir persistence) features. They would ideally identify targets of special relevance for subsequent functional analysis. Such analysis will further our understanding of the mechanisms that contribute to differential prevalence of certain strains of the organism in illness, environmental (including processing plant) contamination, or persistence in selected reservoirs. In addition, they have the potential to provide us with the molecular tools for subsequent design of sensitive detection and monitoring systems not just for L. monocytogenes as a species but of specific subpopulations within the species. Current trends in technology suggest that design for such systems would likely employ arraytype formats that incorporate entire genomic sequences or selected signature sequences of diverse serotypes and clonal groups. In addition to detection and monitoring of known clonal groups, these systems would facilitate detection of emergent strain types, with genomic fingerprints that deviate from those previously encountered. Clearly, successful application of such systems would require construction and careful maintenance of a molecular signature database, for which successful prototypes such as PulseNet already exist. Molecular subtyping with such high-resolution systems would be greatly aided by suitable equipment for automated processing of the arrays. Proteomic analysis will continue to be invaluable for characterization of bacterial responses to different conditions and identification of likely targets for subsequent functional analysis. Although proteomics-based tools can also be used for high-resolution subtyping of the organism, stringent conformity would be required in conditions for growth of the bacterium, because the proteomic profile (unlike the genomic content) would be highly condition dependent. Reaching an agreement on conditions most suitable for proteomic analysis would therefore be crucial. Because regulation of expression of a certain gene may differ among strains, and may be sensitive to even slight changes in environmental conditions, conclusions that are related to strain-specific proteins would need to be quite carefully formulated. Furthermore, regulatory changes within the genome that alter the expression of specific genes may be selected in the process of growth and serial passage in the laboratory, potentially creating further sources of artifacts and difficulties in use of proteomic signatures for molecular epidemiology purposes.

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Agreed-upon criteria on what differences actually are allowed for two isolates to be considered “different” will need to be carefully thought through by the Listeria community for all softwaredependent applications that might involve large collections of data at the genomic or proteomic level. Hopefully, development of such criteria would only occur following careful studies that examine results obtained from a carefully selected library of strains. Such a library would include, among others, strains that represent lineages that may differ in genomic stability, those that are epidemiologically related isolates (multiple patient and food isolates from a confirmed commonsource outbreak), and finally, those isolates that are obtained following repeated transfers in the laboratory. For proteomic analysis, it would be crucial to determine intra- and interlaboratory reproducibility of proteomic profiles, and the possible perturbations of proteomic signatures by unavoidable or subtle changes in growth conditions, such as those resulting from different lots of the same growth medium.

HOW, WHERE, AND WHEN L. MONOCYTOGENES EXPRESSES SPECIAL GENOME COMPONENTS: LISTERIAL PATHOGENESIS, ECOLOGY, AND STRESS RESPONSES FURTHER CONSIDERED A tremendous amount of information has been obtained to date on the mechanisms by which L. monocytogenes interacts with mammalian host cells [2]. The next few years, nonetheless, promise to further expand the frontiers and to help us understand how pathogenesis actually evolved in the history of this organism. Does L. monocytogenes interact with nonmammalian animal systems in ways that may have set the stage for its evolving as a pathogen of mammals, including humans? The terrestrial ecosystem which Listeria often inhabits in its “nonpathogenic” mode abounds in many animal forms that may interact with the bacteria; protozoa, nematodes, insects and their larvae; all these may serve as amplification reservoirs, and they may themselves be actually infected by the pathogen. In this context, recent reports of Drosophila melanogaster as a potential model system for listerial pathogenesis [1,7] remind us of the hidden surprises that the pathogenesis-related study of this organism holds. Future studies will address whether virulence of Listeria to mammals is perhaps increased following the microorganism’s passage through invertebrate hosts, as is known for other pathogens [4]. If virulence is indeed enhanced following such passages, a subsequent question is whether this contributes to the observed differences in the prevalence of certain serotypes, or clonal groups, in human illness. These are questions that this author anticipates will be productively explored in the next few years and, similar to many others, eagerly awaits the findings. Further studies along these lines also hold potential for development of a new generation of models for pathogenesis of the organism. Potential hosts with highly characterized genomes and with available libraries of mutants of key developmental genes, such as Drosophila and the nematode Caenorhabditis elegans, hold further promise of understanding the genetic give-and-take operating in the evolution of pathogenesis, through implementation of a molecular version of Koch’s postulates that encompasses both host and pathogen. How the genome is expressed under selected circumstances, such as during infection (or colonization) of animals, or as the pathogen becomes established and persists in the processing plant, will undoubtedly continue to be explored with heavy reliance on the accumulating genomic and proteomic knowledge. In addition to currently pursued lines of research such as the expression arrays, new tools will undoubtedly be developed to get glimpses of gene expression of the organism in situ. There is little doubt that the currently developing appreciation of Listeria’s existence as a component of biofilms will be strengthened and that gene and protein expression profiles of the organism will be investigated in the actual surface associations that are of relevance both to its ecology and its pathogenesis, and to the food industry. Currently our understanding along these lines remains primitive.

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In this context, a key component of interest is the associations of Listeria with other microbes. Characterization of the microbial communities with which Listeria is associated in nature, and in the processing plants and foods, will occupy the Listeria community in the next few years, with hopefully intriguing results. Community-based perspectives will also reach out to other areas of keen interest, such as the study of stress response. The stress response of the microorganism has been extensively investigated in selected strains, but in the admittedly artificial, isolated system of pure monocultures. Although such studies will still be needed to provide the database of how individual strains respond to various stresses, the biological and ecological relevance of the findings would be greatly complemented by investigations of how the organism responds and copes with the stresses encountered in the actual microbial communities of which it is a denizen. This necessarily includes further understanding of not only how Listeria may interact with other bacterial species, but also how different strains of L. monocytogenes may interact with each other. In ecosystems commonly serving as harborage sites in food processing plants, such as floor drains, this author’s laboratory and others have found that multiple strains of L. monocytogenes typically do coexist, although their population dynamics, physical associations, and interstrain interactions remain completely unknown at this time. The establishment and augmentation of a database of microbial consortia or communities that might harbor the organism ought to be accompanied by bio-mathematical modeling of the complexity and dynamics of community establishment and compositional shifts. Such modeling would need to be developed to take into adequate account the distinct variables (community composition and diversity, environmental conditions) that shape the quite different ecosystems that are of relevance to the organism. Clearly, modeling of community shifts and strain composition, and prevalence in the food processing plant may involve variables very different from those in ecosystems such as soil and vegetation, or the gastrointestinal tract of animals.

CONCLUDING THOUGHTS An expansion in the genomic and proteomic characterization of L. monocytogenes will continue to occupy scientists in the next few years, and will also provide tools for targeted functional analysis of the organism. Expected deliverable outcomes will include a new generation of detection, monitoring, and subtyping strategies, not only with unprecedented potential but also with significant challenges at the experimental and interpretational levels. These challenges would need to be carefully addressed if the new tools and findings are to fully realize their potential for public health, epidemiology, and the food industry. Other challenges are of nonscientific nature, and they involve costs and accessibility of the technology. Genomics- and proteomics-based tools are expensive, and their costs will likely remain high, especially if the technology becomes proprietary and relies on automated equipment and standardized reagents. It will be the task of an enlightened Listeria community to develop programs and venues to ensure that high-resolution tools of the expected high potential will be accessible to all members of the community for the purposes of further understanding the organism in the natural environment, in the processing plant, and as a human and animal pathogen. At the basic science level, the projected shifts from contained laboratory monocultures to communities and surface-associated systems to be analyzed in situ will constitute novel venues for the study of the organism and will result in findings that are currently nonexistent, enriching our views of how the organism survives in relevant ecosystems and responds to environmental stresses. Projected shifts from pathogenesis studies focusing on mammalian models (animals and cell lines) not only may result in potentially novel models for pathogenesis studies, but will also elucidate aspects of Listeria’s lifestyle that have remained largely hidden, including a whole spectrum of the organism’s interactions with nonmammalian biota in its various habitats. This author has little doubt that, with the proper direction of resources and encouragement of young scientists, the next few years will bring to fruition a new generation of studies that will

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continue to justify this organism’s reputation as both intriguing and interesting, and to clarify its evolution as a pathogen of concern to public health, as well as to the food industry. In so many ways, for students of Listeria, the best is still to come.

REFERENCES 1. Cheng, L.W. and D.A. Portnoy. 2003. Drosophila S2 cells: an alternative infection model for Listeria monocytogenes. Cell. Microbiol. 5: 875–885. 2. Dussurget, O., J. Pizarro-Cerda, and P. Cossart. 2004. Molecular determinants of Listeria monocytogenes virulence. Annu. Rev. Microbiol. 58: 587–610. 3. Glaser, P., L. Frangeul, C. Buchrieser, C. Rusniok, A. Amend, et al. 2001. Comparative genomics of Listeria species. Science 294: 849–852. 4. Harb, O. S., L.Y. Gao, and Y. Abu Kwaik. 2000. From protozoa to mammalian cells: a new paradigm in the life cycle of intracellular bacterial pathogens. Environ. Microbiol. 2: 251–265. 5. Herd, M. and C. Kocks. 2001. Gene fragments distinguishing an epidemic-associated strain from a virulent prototype strain of Listeria monocytogenes belong to a distinct functional subset of genes and partially cross-hybridize with other Listeria species. Infect. Immun. 69: 3972–3979. 6. Lei, X. H., F. Fiedler, Z. Lan, and S. Kathariou. 2001. A novel serotype-specific gene cassette (gltAgltB) is required for expression of teichoic acid-associated surface antigens in Listeria monocytogenes of serotype 4b. J. Bacteriol. 183: 1133–1139. 7. Mansfield, B.E., M.S. Dionne, D.S. Schneider, and N.E. Freitag. 2003. Exploration of host-pathogen interactions using Listeria monocytogenes and Drosophila melanogaster. Cell. Microbiol. 5: 901–911. 8. Nelson, K.E., D.E. Fouts, E.F. Mongodin, J. Ravel, et al. 2004. Whole genome comparisons of serotype 4b and 1/2a strains of the food-borne pathogen Listeria monocytogenes reveal new insights into the core genome components of this species. Nucl. Acids Res. 32: 2386–2395. 9. Wiedmann, M., J.L. Bruce, C. Keating, A.E. Johnson, P.L. McDonough, and C.A. Batt. 1997. Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential. Infect. Immun. 65: 2707–2716. 10. Yildirim, S., W. Lin, A.D. Hitchins, L.-A. Jaykus, E. Altermann, T. R. Klaenhammer, and S. Kathariou. 2004. Epidemic clone I-specific genetic markers in strains of Listeria monocytogenes serotype 4b from routine surveys of foods. Appl. Environ. Microbiol. 70: 4158–4164.

RESEARCH NEEDS: A FOOD SAFETY PERSPECTIVE EWEN C.D. TODD

In the United States, data for the passive foodborne outbreak reporting system at the state level submitted to CDC may be unrepresentative of the real illness situation, and the information gathered is typically incomplete. However, outbreaks that are investigated provide the best process to identify the food vehicles associated with illnesses. Obtaining better data depends on the commitment by the different public health authorities from local to national levels to provide the necessary resources for extensive case interviews, along with the use of molecular typing methods such as PFGE to match cases with implicated food products. This will allow policy making and inspection services to better monitor the effectiveness of regulations and other programs to reduce listeriosis. At present, it is unclear how much of a correlation exists between outbreak reports, FoodNet data on listeriosis, and prevalence and concentration of L. monocytogenes in ready-to-eat (RTE) foods. For instance, FSIS regulatory testing for L. monocytogenes showed a 25% reduction of positive samples collected in 2003 over 2002, recalls dropped from 40 in 2002 to 14 in 2003, and there were no large outbreaks involving meat and poultry products since 2003. The 2003 CDC FoodNet preliminary data showed a slight increase in listeriosis incidence after a 4-year downward trend [2]; there were 3.3 cases of listeriosis for every 1 million people in 2003, compared with 2.7 cases per million in 2002. This translates into 951 cases in the United States, but this is different from the 629 cases of listeriosis provisionally reported by the CDC Electronic Foodborne Outbreak Reporting System (EFORS)

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[13] for the same year. The assumption is that the active reporting system (FoodNet) is more accurate than the passive system (EFORS), and thus, while it would appear that L. monocytogenes contamination is down, cases are probably up slightly. How does a regulatory agency interpret this collection of information? Although it is recognized that disease patterns have to be measured over several years in order to see long-term trends, these types of challenges make policy setting difficult for food control. Regulators will view the contamination reduction positively, but maybe some key foods are not being tested. For instance, they may be sampling commercially packaged products but not those products that are supplied from “under the table.” This issue has been raised for noncommercial Latino soft cheeses and some kinds of ethnic sausages that have been linked to illness. Options for federally inspected establishments in preventing product contamination and outgrowth in retail operations appear to be limited. Regulatory strategies that attempt to project the responsibility onto inspected processing establishments may not be effective in significantly reducing the likelihood of foodborne listeriosis from the deli counter and other retail-produced items. As the quality of EFORS data improves, with the use of PFGE to match cases and food products and with the use of an extensive case interview form for all lab-confirmed cases, regulatory agencies like FSIS will be better able to correlate changes in regulations with changes in illness. In time, it is hoped that active and passive systems will be more coordinated, and data therefore better correlated. In summary, long-term trends in the incidence of listeriosis reflect changes in regulation, changing industry practices, consumption patterns, educational approaches, demographics, diagnostic methods, and health-care-seeking behavior. Consequently, the relative impact of regulations will be difficult to measure precisely.

REGULATORY TOLERANCE The United States and some other countries do not have any regulatory tolerance for L. monocytogenes in RTE foods. This can be interpreted as the most restrictive approach for limiting L. monocytogenes contamination in products available for consumption. Yet, outbreaks have occurred, and it is assumed that many sporadic cases have arisen since this rule was established. In fact, based on disease surveillance data, rates do not appear substantially different in countries with or without such a policy, even allowing for variability with different national surveillance systems (0.3–7.8 cases per million population). A petition by industry groups is now with the FDA seeking change in its policy to 100 CFU/g, similar to that in other countries. One argument presented by USDA is that it is not in a position to set a regulatory tolerance partly because it cannot identify the end users of the products (presumably some would be high-risk individuals with compromised immune systems). However, the FAO/WHO risk assessment of L. monocytogenes in RTE foods [4] indicates that nearly all cases of listeriosis result from the consumption of pathogen levels exceeding 100 CFU/g by several orders of magnitude, even in immunocompromised subpopulations. The FDA/FSIS/CDC [8] risk assessment demonstrates that the probability of mortality at the 5th percentile for the neonatal population (worst-case scenario) is 1 in 1.7 × 108. The model also predicts that consumption of low numbers of L. monocytogenes has a low probability of causing illness; for example, when the level of L. monocytogenes was assumed not to exceed 1000 CFU/g, the number of cases in the United States would be less than 26 per year versus the estimated 2500 [4]. The report states that the vast majority of listeriosis cases are associated with foods that do not meet current standards for L. monocytogenes in foods, whether the standard is “zero tolerance” (e.g., <0.04 CFU/g) or 100 CFU/g. In other words, dealing with compliance is just as important as setting a standard. For instance, the FAO/WHO model predicts that if a limit of 0.04 CFU/g with a 0.018% defect rate (2133 cases) was replaced by a 100 CFU/g limit and a 0.001% defect rate (124 cases), the predicted result based on the scenario would be an approximate 95% reduction in foodborne listeriosis. Thus, risk assessments give government potential tools to alter policies so that the public would be at even lower risk.

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It can be argued that the zero-tolerance approach to dealing with L. monocytogenes was a cautious enforcement policy based on the state of the science during the 1980s—a time when there was no understanding of the ubiquity of the microorganism, nor effective methods for finding it in foods and food processing environments. Since then, knowledge has increased sufficiently to be able to set a regulatory limit. Regulatory agencies and the industry will then be able to focus their resources in proportion to the level of public health significance on measures to provide a strong incentive for development of products that do not support growth of L. monocytogenes, encourage aggressive sampling programs, and facilitate collection of better quantitative data on L. monocytogenes.

CONTROL OPTIONS Because problems still exist with RTE meats and poultry, FSIS has proposed three possible options for industry to adopt to reduce the risk of L. monocytogenes contamination in RTE meat and poultry products [10–12]. These include the use of (1) a postlethality treatment and addition of antimicrobial agents, (2) a postlethality treatment or addition of antimicrobial agent, and (3) neither of these with increased sanitation used as the only Listeria control strategy. Coupled with these alternatives are differing degrees of verification testing, with alternative (3) requiring the most. Some details of the testing were revealed in the January 2005 FSIS RTE001 program for sampling (postlethalityexposed RTE meat and poultry products only) [13]. A survey of 2900 establishments found that 27% have started using an antimicrobial agent, 17% have started using postlethality treatments, and 59% have started enhanced testing for L. monocytogenes or Listeria-like organisms. If done properly, postlethality treatments will be effective in destroying the pathogen, and antimicrobial agents will eliminate or at least prevent growth of the organism (presumably low numbers from sporadic environmental contamination) to potentially hazardous levels. However, the concentrations of these antimicrobials will likely show wide variability, which would compromise their effectiveness. Establishments that produce certain high-risk products may be able to use levels of lactate that are acceptable for incorporation into their product, but likely ineffective. Attempting to inhibit L. monocytogenes outgrowth with greater lactate/diacetate combinations, and at higher levels, alters the palatability and marketability of the product. However, some low-risk deli products containing antimicrobial agent formulations may also represent a significant hazard in retail deli operations if refrigeration is not properly controlled, especially where these are sliced, handled, and packaged. These agents become increasingly less effective in inhibiting outgrowth as the storage temperature rises. The only truly effective way to verify and validate the effectiveness of an antimicrobial agent in a product is to conduct challenge tests using inoculated product held throughout the shelf life. Even if these controls are followed, problems may remain. Any L. monocytogenes-positive products produced under alternative (2) (use of antimicrobials to prevent growth) would still be subject to recall. The degree of reduction of Listeria contamination is uncertain because the new testing procedures will be the only control measure under alternative (3), which may represent over 50% of all RTE operations. Alternative (3) is statistically based and depends on development and implementation of a regular and adequate testing regimen for food contact surfaces in the particular facility. However, unlike the use of postlethality treatments and antimicrobial agents, verification testing is very much dependent on worker expertise and commitment. The noncompliance (NC) records are well documented from very small plants (56%), and most establishments with NC records had chosen alternative (3) (the least protective alternative) to control L. monocytogenes. In addition, most L. monocytogenes-related NCs concern fully cooked, perishable products. Managers and employees in very small plants will most likely have the least knowledge and expertise of all producers of RTE meat and poultry products. Because companies typically look for Listeria spp. rather than L. monocytogenes, due to the cost of testing and the adverse consequences of finding a positive test result, a minimum number of samples are likely to be tested with a possible cover up of any undesirable findings. In other words, the human element is much more vulnerable to lack of compliance than a measurable heat treatment or addition of a specified amount of an

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antimicrobial agent, both of which would be carefully monitored at critical control points in the HACCP plan. Retail delicatessens present a potential public health concern but are not currently under the active purview of a federal agency. Most states have adopted the Food Code as their regulatory food safety framework. Unfortunately, retail delis complied with all Food Code controls only some of the time [7].

RISKS ASSOCIATED

WITH

PROCESSED FOOD

Deli Meats The FDA/FSIS/CDC [8] risk assessment indicated that deli meats were the products associated with the highest number of cases. Outbreaks involving deli meats have been reported over several years and recalls occur on a regular basis. In one outbreak in 2002, 54 people were diagnosed with listeriosis associated with consumption of sliced meat, which resulted in over 27 million pounds of product at risk. Although much of the product had been consumed or discarded, the company experienced more than $100 million in lost sales. Scenarios to evaluate the impact of changing the maximum storage time were run for deli meats, with the simulation end point being the predicted annual mortality for the elderly subpopulation. The simulations showed that reducing storage time by half (28-day baseline to 14 days) reduced the median number of listeriosis cases in the elderly population from 228 to 197 (13.6%), whereas shortening storage time to 10 days further reduced the cases to 154 (32.5%). The exercise concluded that achieving a 50% reduction in listeriosis cases from consumption of deli meats would require eliminating storage above approximately 8°C or all storage times longer than 8 days. The interaction of modifying both storage time and temperature was also simulated. An example of a combination that would reduce cases of listeriosis by 50% was 10°C for no more than 11 days. This assessment was based on generic deli meat that supported Listeria growth. The ILSI report on Achieving Continuous Improvement in Reductions in Foodborne Listeriosis— A Risk-Based Approach [9] outlines the five most effective strategies to control L. monocytogenes in high-risk foods: 1. Good manufacturing practices, standard sanitation operating procedures and HACCP programs to minimize environmental L. monocytogenes contamination and to prevent cross-contamination in processing plants and at retail 2. An intensive environmental sampling program in processing plants along with an effective corrective action plan to reduce the likelihood of contamination of high-risk foods 3. Time and temperature controls throughout the entire distribution and storage period including establishing acceptable storage times for foods that support growth of L. monocytogenes to high numbers 4. Reformulating foods to prevent or retard L. monocytogenes growth, and using postpackaging treatments to destroy L. monocytogenes on products 5. Using postpackaging treatments to destroy L. monocytogenes on products FSIS is incorporating four of these approaches into the Interim Rule (2004) [9], but much less emphasis is placed on the third strategy to have effective time and temperature controls. More Focus on Retail Product The entire Interim Rule [12] has focused on minimizing Listeria contamination of RTE meat and poultry products at the producer level. Only in the FSIS December 2004 Docket are potential problems at retail, such as Listeria transfer and cross-contamination during slicing and packaging, mentioned. Although some research is being done to estimate the contribution of these

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operations to the overall caseload, more work is clearly needed. This is important because the risk of illness from delicatessen-sliced meats is approximately seven times higher than prepackaged sliced products coming directly from the manufacturer. In the survey by Gombas et al. [6], 0.4% of prepackaged, compared to 2.7% of delicatessen-sliced luncheon meats, were contaminated with L. monocytogenes. Even more problematic is the fact that four of every five consumers purchase deli-sliced meats as opposed to products sliced and packaged at the manufacturing facility. Given an estimated 320 fatal listeriosis cases each year from RTE meat products, 242 of these deaths could be considered associated with the consumption of delicatessen-sliced luncheon meats alone. Problems with retail delis have been documented. Only 73% of them complied with all Food Code controls [7]. Proper holding time and temperature were observed only 43.3% of the time, and opportunity for equipment to become contaminated was seen over 79.4% of the time. Both of these are critical controls for RTE foods. The Docket [12] indicates that small and very small establishments need better guidance, and a similar strategy will be needed for retail establishments. At present, the incidence of L. monocytogenes on food contact surfaces within delicatessen environments remains unknown along with the most likely routes of contamination. These two areas, along with more vigilant training of delicatessen workers, are in need of further study. Other Foods of Concern Deli meats and pasteurized milk are widely consumed by the population at large. However, there is some risk for RTE foods less frequently eaten by the general population being contaminated and causing listeriosis. These include smoked fish, crustaceans, pâtés, and some cheeses. Certain aficionados, who are often in the higher-risk category such as the aged, may consume these frequently. Cheeses of concern are soft unripened varieties (usually Hispanic or Latino in North America) and fresh soft cheeses made from unpasteurized milk. Smoked fish and raw milk cheeses may be sold by small family operations “under the counter” or at the farm, and are not identified during routine retail store inspections. These products are sought after by gourmands or those with strong cultural links to the product, and their purchase is difficult to monitor, let alone control. In countries or states where dairy regulations prevent consumers from buying raw milk in stores or directly from farmers, some individuals are entering into cow share or farm share agreements with the farmer. In a cow share agreement, consumers pay a farmer a fee corresponding to the share owned for boarding, caring for, and milking the cow [5]. The cow shareowner then is given milk from the cow without any formal purchase being conducted. This arrangement is similar to arrangements of owning a share in a racehorse or a bull. Where the regulations specifically forbid cow share agreements, consumers and farmers may set up corporations in which consumers hold nonvoting shares, although these are more difficult and expensive to set up than a cow share program. The same approach can be made for any products made from raw milk. Other would-be consumers tell the farmer that they are using the milk for their pets, but they drink most of it themselves. Arguments given for seeking out raw milk products are that milk from the cow is a highly health-promoting fresh food, and pasteurization damages its quality (by destroying enzymes, vitamins, and beneficial bacteria, denaturing milk proteins, and promoting pathogens through lack of competition by the beneficial lactic acid organisms) [1]. There is also a cultural element in the consumption of raw milk products and other traditionally produced RTE foods for centuries without any large-scale concern. Thus, eliminating the small proportion of any population consuming such RTE foods with increased risks of listeriosis and other enteric diseases will be very difficult.

LABELING

AND

CONSUMER EDUCATION

Labeling to advise consumers that products have received some type of treatment to reduce contamination (such as postlethality treatment or added antimicrobial agents) needs to be considered along with safety-based dates and advice on proper storage conditions. The interim final rule states that establishments can declare any processing methodology on their label that they use to address L. monocytogenes.

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However, at present, no company is using such incentive labeling on a voluntary basis. Although such information could be perceived by industries as an advantageous marketing strategy, it is not certain how the public would respond. At present, it is probable that industry perceives that labeling statements would be too confusing or misinterpreted by consumers. More research is needed on labeling as an effective means of educating consumers about proper food storage and handling practices, including the use of focus groups, to help develop labeling statements. However, growth studies are also required to determine how long and under what conditions RTE foods can be stored before L. monocytogenes will reach unacceptable levels at the time of consumption. The FSIS Interim Rule [12] suggests no more than a 2-log increase, which could be interpreted as 100–1000 CFU/g, assuming that most processed packages contain no, or very low levels of, L. monocytogenes, e.g., < 0.04 CFU/g. However, any “best consumed by” date will depend on the product, storage temperature and length of storage. Different labels also may be required for different types of consumers (high vs. low risk). Sciencebased education and risk communication strategies aimed at susceptible populations and focused on high-risk foods should be delivered through health-care providers or other credible sources. Exquisitely sensitive consumers may become ill when exposed to low numbers of L. monocytogenes or other opportunistic pathogens. So reducing the risk to this population could be achieved by maintaining them on restricted low microbial diets during those periods when they are most severely immunocompromised. The ILSI report [9] indicates the critical importance of education and communication. High-risk individuals, i.e., the elderly, pregnant women, and most immunocompromised individuals, should receive guidance on healthy eating habits, including specific information on high-risk foods that should be avoided, and strategies to reduce their risk, such as thorough cooking, avoidance of cross-contamination, and short-term refrigerated storage of cooked perishable foods. Those at low risk for listeriosis should receive information on safe food handling practices, preferably starting at a preschool age. L. monocytogenes labeling statements should be further developed by conducting focus group research studies to produce statements that would provide flexibility to the industry while still remaining truthful and not misleading. Consumer education messages can also be delivered using multimedia channels, e.g., radio and television networks for Hispanic or Arab-American communities, Web sites, and mobile exhibits and community health fairs. Apart from consumer education, there are education and training needs for regulatory agencies, especially for inspecting personnel when new approaches are being taken for risk-based policies.

REFERENCES 1. A Campaign for Real Milk. 2005. Cow Share and Management Agreement. http://www.realmilk.com/cowshare-austr.doc. Accessed March 21, 2005. 2. CDC. 2004. Centers for Disease Control and Prevention. Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food—selected sites, United States, 2003. Morb. Mort. Wkly. Rep. 53: 338–343. 3. CDC. 2004. Centers for Disease Control and Prevention. Food Outbreak Response and Surveillance Unit Electronic Foodborne Outbreak Investigation and Reporting System (EFORS) http: //www.cdc.gov/foodborneoutbreaks/reporting_outbreak.htm. Accessed March 21, 2005. 4. FAO/WHO, Buchanan, R., R. Lindqvist, T. Ross, M. Smith, E. Todd, and R. Whiting. 2004. Risk Assessment of Listeria monocytogenes in Ready-to-Eat Foods—Technical report. Microbiological Risk Assessment Series 5, 304 pp. 5. Fausett, M.R. and K.C. Dhuyvetter. 1995. Beef Cow Leasing Arrangements MF-2163. Kansas State University Agricultural Experiment Station and Cooperative Extension Service. http: //www.oznet.ksu.edu/ library/agec2/mf2163.pdf. Accessed March 21, 2005. 6. Gombas, D.E., Y. Chen, R.S. Claver, and V.N. Scott. 2003. Survey of Listeria monocytogenes in readyto-eat foods. J. Food Prot. 66: 559–569. 7. FDA. 2000. Report of the FDA Retail Food Program Database of Foodborne Illness Risk Factors, prepared by the FDA Retail Food Program Steering Committee. http://www.cfsan.fda.gov/acrobat/ retrsk.pdf. Accessed August 7, 2006.

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8. FDA/FSIS/CDC. 2003. Center for Food Safety and Applied Nutrition, Food and Drug Administration/Food Safety and Inspection Service, USDA and Centers for Disease Control and Prevention. Quantitative assessment of the relative risk to public health from foodborne Listeria monocytogenes among selected categories of ready-to-eat foods. http://www.foodsafety.gov/~dms/lmr2-toc.html. Accessed October 1, 2004. 9. ILSI. 2004. Achieving continuous improvement in reductions in foodborne listeriosis. reduction in foodborne listeriosis, draft report by an expert panel. International Life Sciences Institute, Washington, DC. http: //www.ilsi.org/publications/pubslist.cfm?publicationid=541. Accessed March 21, 2005. 10. USDA. 2003. Control of Listeria monocytogenes in Ready-to-Eat Meat and Poultry Products; Final Rule, 9 CFR Part 430. Federal Register 68 (109): 34207–34254. http://a257.g.akamaitech.net/7/257/2422/ 14mar20010800/edocket.access.gpo.gov/2003/pdf/03–14173.pdf. Accessed March 21, 2005. 11. USDA. 2003. Verification Procedures for the Listeria monocytogenes Regulation and Microbial Sampling of Ready-to-Eat (RTE) Products for the FSIS Verification Testing Program. FSIS Directive 10,240.4, October 2, pp. 1–25. http://www.fsis.usda.gov/oppde/rdad/FSISDirectives/10240-4.pdf. Accessed March 21, 2005. 12. USDA. 2004. Control of Listeria monocytogenes in Ready-to-Eat Meat and Poultry Products. 9 CFR Part 430. Federal Register 69(231): 70051. http://www.fsis.usda.gov/oppde/rdad/frpubs/97-013FE.pdf. Accessed March 21, 2005. 13. USDA. 2004. FSIS Notice Listeria monocytogenes Risk-Based Verification Testing Program–Phase 1: Introduction of a New Sampling Project—RTE001 61-04. December 23, pp. 1–5.

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Index A Aarnisalo studies, 291 Abachin studies, 118 Abdalla studies, 467 Abdel-Gawad studies, 443 Abou-Donia and Al-Medhagi studies, 467 Abou-Eleinin studies, 366 Academic perspective food safety perspective, 835–840 fundamentals, 830–831, 834–835 gene expression, 833–834 genomic endowment, 831–833 proteomic endowment, 831–833 Accessory virulence factors catalase, 131 fundamentals, 130 iron uptake systems, 132 protein p60, 130–131 stress response mediators, 131–132 superoxide dismutase, 131 Acidity, 169, 169–171 Acid sanitizers, 194 Acriflavine, 221–222 Active packaging, 196–197 Adak studies, 98 Addison, Farber and, studies, 292–293 Adesiyun studies, 522 AFLP, see Amplified fragment length polymorphism (AFLP) Agents, selective, 219–223, 220 Aguado studies, 661, 745 Ahmed studies, 467 Al-Azawi, Al-Ghazali and, studies, 30 Al-Ghazali and al-Azawi studies, 30 Al-Medhagi, Abou-Donia and, studies, 467 Alpas and Bozoglu studies, 673 Al-Sheddy and Richter studies, 579 Alternative processing technologies fundamentals, 187 high-intensity pulsed light, 188–189 high-pressure processing, 189, 189 ionizing radiations, 187–188, 188 irradiation, 187–189 pulsed electric field processing, 189–190, 190 ultraviolet radiation, 188–189 Amelang and Doores studies, 391 Amezquita and Brashears studies, 558 Amoril and Bhunia studies, 516 Amplified fragment length polymorphism (AFLP), 294–295 Amtsberg studies, 528 Animal feeds, 32–34

Animal listeriosis animals, transmission to, 57–72 cattle, 62–66 crustaceans, 71–72 ecology, 40–42 fish, 71–72 fowl, 67–69 fundamentals, 55 goats, 60–62 humans, transmission to, 57 incidence, 56 minor species, 69–71 predisposing factors, 56–57 sheep, 57–60 swine, 66–67 transmission, 40–42, 57–72 treatment, 72 Anjaria, Brahmbhatt and, studies, 522 Anjou, France, 37 Anne (Queen of England), 2 Antelope, 70 Antibody-based methods, 264–267 Antimicrobial agents and additives, 589, 598–599 Antimicrobial components, foods antioxidants, 180–181 benzoic acid derivatives, 175, 178 extracts, plant, 181–182, 182 fatty acids, 178–180 food processing importance, 174–184 free fatty acids, 178–179, 179 herbs, 181–182, 182 hydrogen peroxide, 183 lactate, 175, 176 lactoferrin, 184 lactoperoxidase system, 183–184 lysozyme, 182–183 monoesters, 179–180 organic acids, 175, 175–178 parabens, 175, 178 plant extracts, 181–182, 182 potassium sorbate, 175, 177–178 salt, 174–175 smoke, 181 sodium benzoate, 175, 178 sodium diacetate, 175, 176–177 sodium nitrite, 180 sodium propionate, 175, 177 spices, 181–182, 182 Antimicrobial susceptibility testing, 286 Antioxidants, 180–181 Arias studies, 364 Arimi studies, 36, 39 ARS-MMLA media, 229–230

843

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844 Arumugaswamy studies, 662 Ashenafi studies, 446 Ashton, Wesley and, studies, 37 Asia, 662 Asperger studies, 452 Aspic, 328–329 Asymptomatic carriage, 91, 92–93, 93 Asymptomatic carriers, 67 Attachment to surfaces, 190–192, 191 Aureli, Gianfranceschi and, studies, 666 Australia foodborne infection, 332 food processing, facilities, 747–748, 748 listeriosis outbreak, 332 risk assessment, 773–774 standards and criteria, 791–793 Austria, 796 Autio studies, 631, 638, 744 Autoclaved milk, 380–387 Automation, 259 Avery studies, 548 Ayre studies, 444 Aytac and Gorris studies, 667

B Babic studies, 670 Bachmann and Spahr studies, 456 Bacillus relationship, 4 Back studies, 480 Bacteria identification, Listeria fundamentals biochemical characteristics, 10 culture, 9 genus characteristics, 9–10 metabolism, 10 morphology, 9 nutritional requirements, 10 species identification, 10–12 Bacterial surface-ripened cheeses, 454–456 Bacteriocins biocontrol, 185–186 meat products, 557–559 Bacteriophage typing, 285–286 Baigent, Donnelly and, studies, 225–226 Bailey studies, 68, 229, 232, 577 Bakery and pastry items, 177, 243 Ball studies, 605–606 Baloga and Harlander studies, 288, 297 Banks studies, 452, 461 Bannerman and Bille studies, 218, 223–224, 431 Bannerman studies, 286 Barbalho studies, 586 Barbosa studies, 532 Barrier establishment, 715 Bats, 41 Batt, Norton and, studies, 262 Baxter studies, 34 Bearns and Girard studies, 227, 229 Beckers studies, 227, 363, 428 Beef, see also Cattle; Cow’s milk cooked roast beef, 535 growth and survival, 530–533

Listeria, Listeriosis, and Food Safety modified-atmosphere packaging, 548–549, 549 transmission pathways, 43 Beerens and Tahon-Castel studies, 221 Behavior, FDA perspective, 817 Behavior, fermented products bacterial surface-ripened cheeses, 454–456 blue cheese, 453, 454 brick cheese, 455–456 brine solutions, 483–486, 484–486 Bulgarian white-pickled cheese, 467 Camembert cheese, 449–453, 450–451 cheddar cheese, 459, 460, 461–462 Chihuahua cheese, 465 coagulants, 447–448, 448 colby cheese, 458–459, 459 cold-pack cheese food, 476–477, 476–477 coloring agents, 448–449 composition of cheese, 478–481, 479–480 cottage cheese, 471–474, 472 cream cheese, 474 Domiati cheese, 467 ewe’s milk cheese, 468–470 feta cheese, 465–467, 466 fundamentals, 446 goat’s milk cheese, 468–470 Gouda cheese, 458 hard cheeses, 457–463 hard Italian-type cheese, 463 Hispanic cheeses, 464–465 Kachkaval cheese, 468–469 Maasdam cheese, 458 Manchego cheese, 464–465, 469 Mexican Manchego cheese, 464–465 mold-ripened cheeses, 449–453 mozzarella cheese, 457 Parmesan cheese, 462–463, 463 pasteurized process cheese, 477 pickled cheeses, 465–468 Queso blanco cheese, 464 Queso de los Ibores cheese, 464 raw milk, 481 semisoft cheeses, 457–463 soft Italian cheese, 457 soft unripened cheese, 471–474 starter distillates, 448–449 Sudanese white-pickled cheese, 467–468 Swiss cheese, 462 Taleggio cheese, 456 Tilsiter cheese, 456 Trappist cheese, 456 Turkish white-brined cheese, 467 whey, 482, 482–483 whey cheeses, 474–477 Yugoslavian white-pickled cheese, 468 Behavior, fish and seafood products fundamentals, 636 growth, 638–640 inactivation, 643–646, 644 inhibition, 640–643, 641 survival, 638–640 transmission modes, 636–638

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Index Behavior, meat products bacteriocins, 557–559 beef, 530–533, 535, 548–549, 549 cooked products, 534–538 cooked roast beef, 535 cooked smoked sausage, 540–542, 542 cured ham, 534–535 domestic livestock, 528 dry fermented sausage, 545–547 fermented sausage, 544, 558–559 fresh sausage, 538–540, 539–540 fundamentals, 527–528 growth, 530–533 ham, cured, 534–535 lamb, 533–534, 549–550, 550 luncheon meats, 535–538, 536–537 modified-atmosphere packaging, 547–557 pork, 533–534, 550–552, 551 raw ground meat, 557–558 raw meat, 530–533 ready-to-eat products, 534–538 roast beef, cooked, 535 sausage, 538–547, 549 semidry fermented sausage, 544–545 smoked sausage, 540–543, 542 specialty items, 543–544 survival, 530–533 thermal inactivation, 552–557, 553–556 tissue localization, 528–530 uncooked smoked sausage, 543 unfermented sausage, 538–547 Behavior, plant origin products chemical sanitizers, 668–669 fruits and fruit juices, 672–673, 674 fundamentals, 663, 672–676, 674 growth, 663–666, 664 heat, 667–668 inactivation, 667–671 modified atmosphere storage, 666–667 plant components, 669–670 processing techniques, 671, 671 survival, 663–666 Behavior, poultry products chicken, raw, 587–590, 588 cooked products, 590–592, 591–593 fundamentals, 587 growth, 587–592, 588, 591–593 ready-to-eat products, 590–592, 591–593 thermal inactivation, 593–597, 594–596 Behavior, unfermented dairy products autoclaved milk, 380–387 chocolate milk, 380–387 cream, 380–387 evaporated milk, 387 fundamentals, 376 intensively pasteurized milk, 379–380 mixed cultures, 388–389 other products, 367–376 pasteurized milk, 367–376, 379–380 raw milk, 377–379, 378–380

845 sweetened condensed milk, 387 ultrafiltered milk, 388 Behrsing studies, 673 Belgium, 31 Bemrah studies, 775–776 Benagozzi studies, 32 Bendig and Strangeways studies, 661 Benech studies, 461 Benkerroum and Sandine studies, 474 Benzoic acid derivatives, 175, 178 Berang studies, 391 Berche studies, 60 Berrang studies, 666, 731 Berry studies, 558 Beuchat, Brackett and, studies, 600, 604, 639 Beuchat, Holliday and, studies, 393 Beuchat, Hwang and, studies, 589 Beuchat, Taormina and, studies, 556 Beuchat and Brackett studies, 665, 667–668, 670, 675 Beuchat and Doyle studies, 670 Beuchat studies, 663, 667, 670, 814 Bhunia, Amoril and, studies, 516 Bierne and Cossart studies, 116 Biester and Schwarte studies, 66, 217 Bile salt hydrolase, 129–130 Bille, Bannerman and, studies, 218, 223–224, 431 Bille and Rocourt studies, 284, 297 Biochemical characteristics, 10 Biocontrol, 185–186 Biofilms factory and equipment design, 696–697, 697–704, 699, 702–704 formation, 190–192, 191 fundamentals, 685–692, 686–692 maintenance and repair practices, 693, 693–697 Biosensors, rapid method detection, 269–271, 270 Blanco studies, 234 Blenden studies, 67 Blood donations, 100 Blood-free plating media, 229 Blue-mold or hard cheese, 314 Blue mussels, 32 Blue-veined cheese, 453–454 Bockemühl studies, 221 Boerlin studies, 287, 293, 637 Bohner and Bradley studies, 708–709 Bojsen-Møller studies, 217–218 Bolton and Frank studies, 481 Borucki studies, 296 Boston (United States), 37 Botzler studies, 30 Bourry studies, 64 Bovine, see Cattle Bower, Betsy, 647 Boyle studies, 552, 755 Bozoglu, Alpas and, studies, 673 Brackett, Beuchat and, studies, 665, 667–668, 670, 675 Brackett, Cassiday and, studies, 218 Brackett, Hao and, studies, 669 Brackett and Beuchat studies, 600, 604, 639 Brackett studies, 71, 655–676, 814–819

DK3089_C020.fm Page 846 Tuesday, February 20, 2007 7:13 PM

846 Braden studies, 305–349 Bradley, Bohner and, studies, 708–709 Brahmbhatt and Anjaria studies, 522 Brashears, Amezquita and, studies, 558 Bréand studies, 555 Breer and Schöpfer studies, 518 Breer studies, 431, 483, 584 Brehm-Stecher studies, 257–274 Brehm studies, 136 Bremer and Osborne studies, 644 Brick cheese cold enrichment, 217 fermented dairy products, 455–456 Brie cheese, 231 Brie de Meaux, 97, 313–314 Briggs, Donnelly and, studies, 383 Brine solutions, 483–486, 484–486, see also specific solution Brosch studies, 291–292 Buazzi studies, 457, 462 Buchanan and Klawitter studies, 557 Buchanan studies, 220, 229–230, 603, 659, 777 Buchrieser studies, 1–12, 291 Budu-Amoako studies, 643 Buffalo, 41 Bulgaria, 56 Bulgarian white-pickled cheese, 467 Buni studies, 527 Bunning, Peeler and, studies, 778 Bunning studies, 167 Burden estimates, foodborne infection, 348–349 Busani studies, 599 Busch and Donnelly studies, 245 Busse, Sulzer and, studies, 452 Butter, see also Dairy products foodborne infection, 318 human outbreaks, 37 potassium sorbate, 177 unfermented dairy products, 392–393 Buyong studies, 462

C Cabbage antioxidants, 181 direct plating media, 231 epidemic listeriosis, 94 fecal and sewage contamination, 31 soil contamination, 30 CAC, see Codex Alimentarius Commission (CAC) Cagri studies, 599 Cai studies, 296 Calcinated calcium solution, 196 California (United States) listeriosis outbreak, 329 Mexican-style cheese, 315 transmission to animals, 63, 71–72 Camembert cheese cold enrichment, 217 fatty acid monoesters, 178 fermented dairy products, 449–453, 450–451 shrimp, 337

Listeria, Listeriosis, and Food Safety CAMP tests, 10–11 Canada animal feeds, 33 foodborne infection, 314–315 monitoring programs, 419–421, 420, 508–514 plant origin products, 660 risk assessment, 775 seafood products, 333 standards and criteria, 794–796, 795 surveillance programs, 419–421, 420 transmission to animals, 64–65, 67 vegetables, 335 Canaries, 41 Canillac and Mourney studies, 747 Cantoni, Comi and, studies, 584 Cantoni studies, 430, 518 Cantor, Schwartz and, studies, 291 Capita studies, 599 Carlier studies, 552 Carlin and Nguyen-the studies, 665 Carlin studies, 665 Carminati, Ribeiro and, studies, 444 Carpenter, Harrison and, studies, 597 Carpenter and Harrison studies, 594–595 Carpentier, Midelet and, studies, 538 Carvacrol, 196 Casolari studies, 526 Cassiday and Brackett studies, 218 Cassiday studies, 231 Cassin studies, 778 Catalase, 131 Cats, 41 Cattle, see also Beef; Cow’s milk animal feeds, 32–33 animal listeriosis, 62–66 fecal material, 41 predisposing factors, 56–57 soil contamination, 30 Caugant studies, 287 Cauliflower, 337 Ceftazidime, 223 Cell separation and concentration, 266–267 Cellular adhesion, 118 Cereals, 177 Channel catfish, 71 Characteristics, food processing importance acid damage and tolerance, 170–171 acidity, 169–171 acid sanitizers, 194 active packaging, 196–197 alternative processing technologies, 187–190 antimicrobial components, 174–184 antioxidants, 180–181 attachment to surfaces, 190–192, 191 bacteriocins, 185–186 benzoic acid derivatives, 175, 178 biocontrol, 185–186 biofilm formation, 190–192, 191 chlorine and chlorinated compounds, 193–194 cold tolerance, 161–163, 162 elevated sublethal temperature, 163

DK3089_C020.fm Page 847 Tuesday, February 20, 2007 7:13 PM

Index enhanced acid tolerance, 171 extrinsic factors, heat resistance, 167 fatty acid monoesters, 179–180 fatty acids and related compounds, 178–180 free fatty acids, 178–179 freezing temperature, 163–164 fundamentals, 158–159 growth, water activity, 171–172, 172 growth kinetics, 160–161, 161 growth temperature, 159–163 heat resistance, 167–168 herbs, 181–182, 182 high-intensity pulsed light, 188–189 high-pressure processing, 189, 189 high salt concentration, 174 hydrogen peroxide, 183 inactivation, low pH, 169–170 intrinsic factors, heat resistance, 167–168 ionizing radiations, 187–188, 188 irradiation, 187–189 kinetics, thermal inactivation, 165–167 lactate, 175, 176 lactoferrin, 184 lactoperoxidase system, 183–184 lethal temperature, 164–168 live fermentate, 185 low pH, 169–170 lysozyme, 182–183 miscellaneous sanitizing agents, 195–196 modified atmosphere, 186–187 multiple antimicrobial treatments, 197, 197–198 optimum temperature, 159–160 organic acids and salts, 175, 175–178 osmotolerance factors, 172–173, 172–173 ozone, 194–195, 195 parabens, 175, 178 physiology, high salt concentration, 174–175 plant extracts, 181–182, 182 potassium sorbate, 175, 177–178 pulsed electric field processing, 189–190, 190 quaternary ammonium compounds, 194 range, temperature, 159–160 salt concentration, 174 salts, 174–178 sanitizers, 192–196 smoke, 181 sodium benzoate, 175, 178 sodium diacetate, 175, 176–177 sodium nitrite, 180 sodium propionate, 175, 177 spices, 181–182, 182 stress adaptation, 163 surrogate microorganisms, 168 survival, 171–172, 172, 174 temperature, 159–168 thermal inactivation, 164–167 tolerance, acid, 170–171 ultraviolet radiation, 188–189 virulence variations, 163 water activity, 171–173 Charpentier and Courvalin studies, 286

847 Chasseignaux studies, 522, 743 Cheddar cheese cold enrichment, 217 fermented dairy products, 459–460, 461–462 Cheese and fermented products, see also specific type bacterial surface-ripened cheeses, 454–456 behavior, 446–486 blue cheese, 453, 454 brick cheese, 455–456 brine solutions, 483–486, 484–486 Bulgarian white-pickled cheese, 467 Camembert cheese, 449–453, 450–451 Canadian monitoring programs, 419–421, 420 cheddar cheese, 459, 460, 461–462 Chihuahua cheese, 465 coagulants, 447–448, 448 colby cheese, 458–459, 459 cold-pack cheese food, 476–477, 476–477 coloring agents, 448–449 composition of cheese, 478–481, 479–480 cottage cheese, 471–474, 472 cream, 436–445 cream cheese, 474 cultured buttermilk, 439–441, 440 cultured cream, 441 cultured milks, 436–445 domestic cheese recalls, 407–414 Domiati cheese, 467 ergo, 445–446 European countries, 418–419, 421–435 ewe’s milk cheese, 468–470 febrile gastroenteritis, 342–343 fermented milks, 436–446 feta cheese, 465–467, 466 France, 414–418, 421 fraser broth, 227 fundamentals, 313–317, 342–343, 406–407 Germany monitoring programs, 428–430, 429 goat’s milk cheese, 468–470 Gouda cheese, 458 hard cheeses, 457–463 hard Italian-type cheese, 463 Hispanic cheeses, 464–465 hydrogen peroxide, 183 imported cheese, 414–419 Italy monitoring programs, 430 Kachkaval cheese, 468–469 kefir, 445 labneh, 445–446 Maasdam cheese, 458 Manchego cheese, 464–465, 469 mesophilic starter cultures, 436–439, 437–438 Mexican Manchego cheese, 464–465 mold-ripened cheeses, 449–453 monitoring programs, 421 mozzarella cheese, 457 non-European countries, 435 Parmesan cheese, 462–463, 463 pasteurized process cheese, 477 pickled cheeses, 465–468 potassium sorbate, 177

DK3089_C020.fm Page 848 Tuesday, February 20, 2007 7:13 PM

848 Queso blanco cheese, 464 Queso de los Ibores cheese, 464 raw milk, 481 recalls, 407–419 semisoft cheeses, 457–463 soft Italian cheese, 457 soft unripened cheese, 471–474 starter cultures, 436–445 starter distillates, 448–449 Sudanese white-pickled cheese, 467–468 surveillance programs, 407–435 Swiss cheese, 462 Switzerland monitoring programs, 430–432, 431 Taleggio cheese, 456 thermophilic starter cultures, 441–442, 441–443 Tilsiter cheese, 456 Trappist cheese, 456 Turkish white-brined cheese, 467 U.S. surveillance programs, 407–419 whey, 482, 482–483 whey cheeses, 474–477 yogurt, 443–444 Yugoslavian white-pickled cheese, 468 Cheese production facilities, 743 Chemical sanitizers, 668–669 Chemotaxonomy, 3–4 Chen and Hotchkiss studies, 474 Chicken, raw, 577–579, 587–590, 588 Chicken products, 332–333, see also Poultry products Chickens, 41, 67 Chihuahua cheese, 465 China, 775 Chinchillas, 41 Chip-based microanalytical systems, 271, 272 Chitosan, 196 Chlorine and chlorinated compounds, 193–194 Chlorohexidine, 196 Chocolate milk, see also Milk; Raw milk febrile gastroenteritis, 338–339 gastrointestinal illness, 90 unfermented dairy products, 380–387 Chocolate production facilities England, 742 Europe, 741–742 Choi studies, 439, 443–444, 523 Chromosomal DNA restriction endonuclease analysis, 287–288 Chung studies, 529 Cirigliano and Keller studies, 393 Cirigliano studies, 544 Claire studies, 604 Clarifiers, 750 Cleaning, control method, 707–710 Clinical manifestations, humans, 87–88 Coagulants, 447–448, 448 Codex Alimentarius Commission (CAC), 783–790 Colburn studies, 23, 32 Colby cheese, 458–459 Cold enrichment, 217–219

Listeria, Listeriosis, and Food Safety Cold-pack cheese food fermented dairy products, 476–477, 476–477 sodium propionate, 177 Cold-smoked fish products direct plating media, 230 foodborne infection, 334–335 smoking, 181 Cold-smoked rainbow trout, 340–341 Cold tolerance, 161–163, 162 Coleslaw fecal and sewage contamination, 31 human outbreaks, 37 soil contamination, 30 transmission, 36 Collins studies, 4 Collins-Thompson, Slade and, studies, 222, 224–225, 362 Coloring agents, 448–449 Comi and Cantoni studies, 584 Comi and Valenti studies, 463 Commercial availability, 258–259 Comparison of methods, 297, 298 Composition of cheese, 478–481, 479–480 Connecticut (United States) deli meat and frankfurters, 321 foodborne infection, 328 Conner studies, 663 Consequences, enhanced tolerance, 170–171 Consumer education, 839–840 Consumer exposure FDA perspective, research needs, 816–817 industry perspective, research needs, 821–827 specific measures for reducing, 102 Consumption, risk assessment, 784 Contamination prevention, 821–823 Continental Europe, 582–583, 584–585, see also Europe Control, febrile gastroenteritis, 343–344 Control guidelines, poultry products, 597–599 Control options, 837–838 Conventional methods, detection and isolation acriflavine, 221–222 agents, selective, 219–223, 220 ARS-MMLA media, 229–230 blood-free plating media, 229 ceftazidime, 223 cold enrichment, 217–219 direct plating media comparisons, 230–232 Food and Drug Administration method, 234–238, 236–237 Fraser broth, 226–227 fundamentals, 216–217, 248 gum base nalidixic acid, 229 β-hemolysis, 233–234 incubation conditions, 232 injured Listeria recovery, 244–248 International Dairy Federation method, 238–239, 239 isolation media, 227–232 lithium chloride, 220–221 lithium chloride-phenylethanol-moxalactam agar, 228 McBride Listeria agar, 227–228 modified Oxford agar, 228 moxalactam, 223 nalidixic acid, 221

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Index Netherlands Government Food Inspection Service, 243 oblique illumination, 232–234, 233 official methods, 234–243 Oxford agar, 228 PALCAM agar, 228–229 phenylethanol, 220–221 plating, 219–223 polymyxin B, 222–223 potassium tellurite, 219–220 potassium thiocyanate, 222 recovering injured Listeria, 244–248 research advances, 244 selective enrichment, 219–223 selective media, 223–243 thallous acetate, 222 trypaflavine, 221–222 USDA-FSIS method, 239–243, 240 UVM broth, 225–226 Conventional methods, subtyping, 284–286 Cooked products meat products, 505, 506–508, 507–508, 513, 534–538 poultry products, 586–587, 590–592, 591–593 Cooked roast beef, 535 Cooked smoked sausage, 540–542, 542 Corn and tuna salad, 340 Corned beef, 755 Corrective actions, HACCP, 718 Corry, Lewis and, studies, 218 Cortesi studies, 640 Cossart, Bierne and, studies, 116 Cossart, Johansson and, studies, 135 Cossart studies, 116 Costa Rica neonatal disease, 88 transmission pathways, 43 Costs, 801–807, 802–803, 805–806 Cottage cheese cold enrichment, 217 fermented dairy products, 471–474, 472 Cottin studies, 474, 528 Courvalin, Charpentier and, studies, 286 Cows, water contamination, 32 Cow’s milk, 358–366, see also Beef; Cattle Cox studies, 27, 578, 739, 741, 745, 748 Crabmeat, see also Fish and seafood products fatty acid monoesters, 178 human outbreaks, 37 Cream fermented milks, 436–445 unfermented dairy products, 380–387, 381–382, 384–386 Cream cheese, 474 Criteria and standards, 790–807 Critical control point, 717 Critical limits, 717 Cross-connections, 752 Crustaceans animal listeriosis, 71–72 fecal material, 41 fish and seafood products, 626, 630 Culture, 9

849 Cultured buttermilk, 439–441, 440 Cultured cream, 441 Cultured milks, 436–445 Cured ham, 534–535 Curtis and Mitchell studies, 286 Curtis studies, 228 Cutter and Siragusa studies, 559 Cytoplasm growth, see Host cell cytoplasm growth

D Dairy processing facilities control methods, 750–754 Europe, 739–741, 740–741 Sweden, 746–747 United States, 720–723, 721–724 Dairy products, see also Fermented milks and dairy products; Unfermented dairy products foodborne infection, 310–320, 338–339, 342–343 isolation methods for, 234 McBride Listeria agar, 228 USDA-FSIS method, 243 Daley, Farber and, studies, 537 Dalgliesh, Stephanie, 647 Dalu and Feresu studies, 445 Damage mechanisms, 170–171 Dancz studies, 122 DaSilva studies, 435 Dauphin studies, 743 Davidson studies, 362 Davies studies, 475, 550 Dean and Zottola studies, 391 Debevere, El-Ziny and, studies, 473–474 Debevere, Zeitoun and, studies, 588 De Boer and van Netten studies, 543 Dedie studies, 377 Deer, 41 Degnan studies, 642 Deibel, Griffith and, studies, 444 Delaquis studies, 668 Deli meats, see also Luncheon meats; Ready-to-eat meat and poultry products epidemic listeriosis, 96–97 foodborne infection, 321–327 food safety perspective, 838 Dendritic cells uptake, 118–119 Denmark asymptomatic carriage, 91, 93 foodborne infection, 314 listeriosis outbreak, 329 sporadic incidence, 98 standards and criteria, 796–797 transmission to animals, 63, 65, 69 De Roin studies, 690 Desmarchelier studies, 601 Despierres studies, 222 Destro studies, 32, 292, 733 Destruction and detection, FDA perspective, 818–819 Detection, rapid methods advances, 265–266 antibody-based methods, 264–267

DK3089_C020.fm Page 850 Tuesday, February 20, 2007 7:13 PM

850 automation, 259 biosensors, 269–271, 270 cell separation and concentration, 266–267 chip-based microanalytical systems, 271, 272 commercial availability, 258–259 diagnostic targets, 259 DNA microarrays, 262–263 flow cytometry, 267–269, 268–269 fluorescence in situ hybridization, 263–264 fundamentals, 257–259, 274 generic species vs. specific species, 259 ideal detection, 258 nucleic-acid-based methods, 259–264 polymerase chain reaction, 260–262 potential pitfalls, 265 recombinant bateriophage, 264 spectroscopic methods, 271–274, 273 traditional methods, 258 Detection and isolation, conventional methods acriflavine, 221–222 agents, selective, 219–223, 220 ARS-MMLA media, 229–230 blood-free plating media, 229 ceftazidime, 223 cold enrichment, 217–219 direct plating media comparisons, 230–232 Food and Drug Administration method, 234–238, 236–237 Fraser broth, 226–227 fundamentals, 216–217, 248 gum base nalidixic acid, 229 β-hemolysis, 233–234 incubation conditions, 232 injured Listeria recovery, 244–248 International Dairy Federation method, 238–239, 239 isolation media, 227–232 lithium chloride, 220–221 lithium chloride-phenylethanol-moxalactam agar, 228 McBride Listeria agar, 227–228 modified Oxford agar, 228 moxalactam, 223 nalidixic acid, 221 Netherlands Government Food Inspection Service, 243 oblique illumination, 232–234, 233 official methods, 234–243 Oxford agar, 228 PALCAM agar, 228–229 phenylethanol, 220–221 plating, 219–223 polymyxin B, 222–223 potassium tellurite, 219–220 potassium thiocyanate, 222 recovering injured Listeria, 244–248 research advances, 244 selective enrichment, 219–223 selective media, 223–243 thallous acetate, 222 trypaflavine, 221–222 USDA-FSIS method, 239–243, 240 UVM broth, 225–226

Listeria, Listeriosis, and Food Safety Devlieghere studies, 551 de Vries and Strikwerda studies, 63 Diagnosis, humans, 100–101 Diagnostic targets, 259 Dickson, Niebuhr and, studies, 530 Dickson studies, 529 Dietary risk factors, 98–100 Dieuleveux and Gueguen studies, 470 Dijkstra studies, 31, 33, 41, 377 Direct plating media comparisons, 230–232 DNA macrorestriction analysis, 291–292, 293 microarrays, 262–263 relatedness, 6–7 sequence-based strategies, 25, 295–297 Documentation procedures, 718–719 Dogan and Erkmen studies, 673 Dogs, 41, 70–71 Doherty studies, 556 Domestic cheeses, 407–414, see also Fermented products and cheese Domestic livestock, see also specific type incidence, 56 meat products, 528 Domestic seafood, 618–626, see also Fish and seafood products Domiati cheese, 467 Dominguez-Rodriguez studies, 64 Dominguez studies, 469 Donachie studies, 37 Donald studies, 33 Donker-Voet, Welshimer and, studies, 28 Donker-Voet and Seeliger studies, 6 Donnelly, Busch and, studies, 245 Donnelly, Klausner and, studies, 722 Donnelly, Ngutter and, studies, 244 Donnelly, Roth and, studies, 248 Donnelly, Sallam and, studies, 245 Donnelly and Baigent studies, 225–226 Donnelly and Briggs studies, 383 Donnelly studies, 215–248, 359, 364 Doores, Amelang and, studies, 391 Dorsa studies, 555, 644 Dose-response assessment, 784–785 Doyle, Beuchat and, studies, 670 Doyle, Glass and, studies, 534–546, 558, 604 Doyle, Lammerding and, studies, 243 Doyle and Schoeni studies, 223–224, 226, 229, 238 Doyle studies, 167, 238, 369, 378, 394, 556, 814 Drevets studies, 119 Dry areas, cleaning, 707 Dry fermented sausage, 545–547 Dry milk, see Nonfat dry milk Duarte studies, 230 Ducks, see also Poultry products asymptomatic carriers, 67 fecal material, 41 thermal inactivation, 597 Duffy studies, 534 Durst studies, 163 D-value, 165–167

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Index

E Eagles, 41 Early onset, neonatal disease, 87–88 Ecology animal feeds, 32–34 animal listeriosis, 40–42 enumeration methods, 23–24 environments, various, 27 food-processing environment, 36 fundamentals, 21–22 gene expression regulation, 34–35 globals pathways and reservoirs, 42–44 human listeriosis, 35–40 isolation method, 22–23 limitations, current methods, 24–25 molecular detection, 25–27, 26 multiplication, stress conditions, 27–35 natural environment, 35–42 outbreaks, 37–38 pitfalls, current methods, 24–25 sewage, 30–31 soil, 28, 29, 30 sporadic cases, 38–40 stress conditions, 27–35 study methods, 22–27 subtyping methods, 25–27, 26 survival, stress conditions, 27–35 traditional enrichment method, 22–23 transmission dynamics, 35–44 various environments, 27 vegetation, 28, 29, 30 water, 31–32 Economic impact, see Risk assessment, regulatory control, and economic impact Egg processing facilities control methods, 757 United States, 732 Eggs and egg products, see also Poultry products fundamentals, 599–600 growth, 601–603, 601–604 incidence, 600–601 isolation methods for, 234 thermal inactivation, 165, 604–606 USDA-FSIS method, 241 Eilertz studies, 469 Eklund studies, 617–646, 733 Elevated sublethal temperature, 163 El-Gazzar and Marth studies, 447–449, 675 El-Gazzar studies, 388, 439 Elimination, FDA perspective, 818–819 El-Khateib studies, 559 Elliot and Kvenberg studies, 634 El-Shenawy and Marth studies, 471 El-Ziny and Debevere studies, 473–474 Embarek studies, 622, 642 Encephalitis, animal feeds, 34 England epidemic listeriosis, 97 foodborne infection, 327 food processing, facilities, 742–743 listeriosis outbreak, 327, 330

851 neonatal disease, 88 predisposing factors, 56 sporadic incidence, 98 transmission to animals, 59, 67 Engulfment, 167 Enhanced acid tolerance, 171 Enrichment method, 22–23 Enumeration, 23–24, 234–235, 818 Environmental signals affect, 136 Environments, 27 Epidemic listeriosis, humans, 94, 95, 96–97 Epidemiological patterns, 94–100, 95 Equines, see Horses Equipment design, 697–704 Ergo, 445–446 Erickson and Jenkins studies, 604 Ericsson studies, 294 Erkmen, Dogan and, studies, 673 Erkmen studies, 467 EU, see European Union (EU) Europe cheese and fermented products, 418–419, 421–435 epidemic listeriosis, 96 food processing, facilities, 739–742 meat products, 523–527, 524–525 plant origin products, 660–661 poultry products, 580–587 standards and criteria, 796–799 transmission to animals, 67 European Union (EU), 799–800, 800 Evans studies, 742 Evaporated milk, 387 Evolutionary aspects, 138 Ewe’s milk, see also Lambs; Sheep cheese and fermented dairy products, 468–470 unfermented dairy products, 366, 366–367 Exposure assessment, 635 Extracts, plant, 181–182, 182 Extrinsic factors, 167

F Factors, food processing facilities, 710–711 Factory design, 697–704 Fairchild and Foegeding studies, 168 Fanelli, Zaika and, studies, 160 FAO/WHO risk assessment significance, 819–820 Farber, Sontakke and, studies, 291 Farber, Zhang and, studies, 668 Farber and Addison studies, 292–293 Farber and Daley studies, 537 Farber studies Camembert cheese, 503–559 Canadian cheeses sold, 453 cheeses exported to Canada, 420 fermented sausages, 523, 546 generation times, 383 growth and survival, 639 incidence, 600 nalidixic acid, 221 raw cow’s milk isolation, 362 risk assessment, 775–777

DK3089_C020.fm Page 852 Tuesday, February 20, 2007 7:13 PM

852 selective enrichment media, 224 thermal inactivation, 552, 595 vegetable incidence, 660 Farm environment, 750 Farrag and Marth studies, 388–389 Farrag studies, 387 Fatty acid monoesters, 179–180 Fatty acids, 178–180 FDA/FSIS risk assessment significance, 819–820 FDA perspective, research needs behavior in foods, 817 consumer exposure, 816–817 destruction, 818–819 detection, 818 elimination, 818–819 enumeration, 818 FAO/WHO risk assessment significance, 819–820 FDA/FSIS risk assessment significance, 819–820 fundamentals, 815 pathogenicity, 817–818 risk assessment significance, 819–820 FDA surveys, 618–626, 619–625 Febrile gastroenteritis cheese, 342–343 chocolate milk, 338–339 cold-smoked rainbow trout, 340–341 control, 343–344 corn and tuna salad, 340 fresh, raw-milk cheese, 342–343 fundamentals, 336–337, 337, 343–344 ready-to-eat meat and poultry products, 341–342 rice salad, 338 shrimp, 337–338 Fecal carriage, see Asymptomatic carriage Fecal materials, transmission, 41 Fedio studies, 474 Fenlon, David, 44 Fenlon studies, 28, 30, 33, 41, 637 Feresu, Dalu and, studies, 445 Feresu and Jones studies, 3, 10 Ferguson and Shelef studies, 675 Fermented milks and dairy products, see also Dairy products control methods, 754 cream, 436–445 cultured buttermilk, 439–441, 440 cultured cream, 441 cultured milks, 436–445 ergo, 445–446 food processing, facilities, 754 fundamentals, 436 kefir, 445 labneh, 445–446 mesophilic starter cultures, 436–439, 437–438 polymyxin B, 223 starter cultures, 436–445 thermophilic starter cultures, 441–442, 441–443 traditional products, 445–446 yogurt, 443–444 Fermented products and cheese bacterial surface-ripened cheeses, 454–456 behavior, 446–486

Listeria, Listeriosis, and Food Safety blue cheese, 453, 454 brick cheese, 455–456 brine solutions, 483–486, 484–486 Bulgarian white-pickled cheese, 467 Camembert cheese, 449–453, 450–451 Canadian monitoring programs, 419–421, 420 cheddar cheese, 459, 460, 461–462 Chihuahua cheese, 465 coagulants, 447–448, 448 colby cheese, 458–459, 459 cold-pack cheese food, 476–477, 476–477 coloring agents, 448–449 composition of cheese, 478–481, 479–480 cottage cheese, 471–474, 472 cream, 436–445 cream cheese, 474 cultured buttermilk, 439–441, 440 cultured cream, 441 cultured milks, 436–445 domestic cheese recalls, 407–414 Domiati cheese, 467 ergo, 445–446 European countries, 418–419, 421–435 ewe’s milk cheese, 468–470 fermented milks, 436–446 feta cheese, 465–467, 466 France, 414–418, 421 fundamentals, 313–317, 342–343, 406–407 Germany monitoring programs, 428–430, 429 goat’s milk cheese, 468–470 Gouda cheese, 458 hard cheeses, 457–463 hard Italian-type cheese, 463 Hispanic cheeses, 464–465 imported cheese, 414–419 Italy monitoring programs, 430 Kachkaval cheese, 468–469 kefir, 445 labneh, 445–446 Maasdam cheese, 458 Manchego cheese, 464–465, 469 mesophilic starter cultures, 436–439, 437–438 Mexican Manchego cheese, 464–465 mold-ripened cheeses, 449–453 monitoring programs, 421 mozzarella cheese, 457 non-European countries, 435 Parmesan cheese, 462–463, 463 pasteurized process cheese, 477 pickled cheeses, 465–468 Queso blanco cheese, 464 Queso de los Ibores cheese, 464 raw milk, 481 recalls, 407–419 semisoft cheeses, 457–463 soft Italian cheese, 457 soft unripened cheese, 471–474 starter cultures, 436–445 starter distillates, 448–449 Sudanese white-pickled cheese, 467–468 surveillance programs, 407–435

DK3089_C020.fm Page 853 Tuesday, February 20, 2007 7:13 PM

Index Swiss cheese, 462 Switzerland monitoring programs, 430–432, 431 Taleggio cheese, 456 thermophilic starter cultures, 441–442, 441–443 Tilsiter cheese, 456 Trappist cheese, 456 Turkish white-brined cheese, 467 U.S. surveillance programs, 407–419 whey, 482, 482–483 whey cheeses, 474–477 yogurt, 443–444 Yugoslavian white-pickled cheese, 468 Fermented sausage, 544, 558–559, 560 Ferreira and Lund studies, 474 Ferron and Michard studies, 243 Feta cheese, 465–467 Fett, Ukuku and, studies, 673 Filling, control methods, 752 Finfish, 630 Finland foodborne infection, 340–341 food processing, facilities, 744 human outbreaks, 37 Fish and seafood processing facilities control methods, 757–758 Europe, 741 Finland, 744 United States, 732–738, 733, 735–737 Fish and seafood products animal listeriosis, 71–72 behavior, 636–646 crustaceans, 626, 630 domestic seafood, 618–626 exposure assessment, 635 FDA surveys, 618–626, 619–625 fecal material, 41 finfish, 630 foodborne infection, 333–335, 337–338, 340–341 fundamentals, 617–618, 646 growth, 638–640 hazard identification and characterization, 635 human listeriosis, 632–633 imported seafood, 618–626 incidence, 627–629 inhibition, 640–643, 641 isolation methods for, 234 lightly processed fish products, 631–632 McBride Listeria agar, 228 regulatory aspects, 634–635 risk assessment, 635–636 shellfish, 630 smoked fish products, 630–631 surveys, 618–632 survival, 638–640 transmission modes, 636–638 USDA-FSIS method, 242 Fisher studies, 668 Flanders studies, 245 Fleming, Kyung and, studies, 670 Flies, fecal material, 41 Flow cytometry, 267–269, 268–269

853 Fluid milk, 317–318, see also Milk Fluorescence in situ hybridization, 263–264 Foegeding, Fairchild and, studies, 168 Foegeding, Leasor and, studies, 600 Foegeding and Leasor studies, 603, 605 Foegeding and Stanley studies, 606 Foegeding studies, 558 Food and Drug Administration method, 234–238, 236–237 Foodborne infection aspic, 328–329 Australia, 332 blue-mold or hard cheese, 314 Brie de Meaux, 313–314 burden estimates, 348–349 butter, 318 Canada, 314–315 cheese, 310–317, 342–343 chicken products, 332–333 chocolate milk, 338–339 cold-smoked fish products, 334–335 cold-smoked rainbow trout, 340–341 Connecticut (United States), 328 dairy products, 310–320, 338–339, 342–343 deli meat, 321–327 Denmark, 314 England, 327 febrile gastroenteritis, 336–344, 337, 343–344 Finland, 340–341 fluid milk, 317–318 France, 313–314, 330–332 frankfurters, 321–327 fresh, raw-milk cheese, 342–343 fundamentals, 306, 307 identification as, 308, 308–309 illicitly produced/distributed cheese, 315–317 Ireland, 327 Italy, 338 Japan, 343 Los Angeles County, 310–312, 311 Maryland (United States), 328 meat products, 320–333 Mexican-style cheese, 310–312 molecular subtyping role, 309–310 mussels, smoked, 334 New York (United States), 328 New Zealand, 341–342 outbreak detection and investigation, 309–310, 310 paté, 327–328 pork tongue, 328–329 poultry products, 320–333 Quebec, Canada, 314–315 raw-milk fresh, unripened cheese, 315–317 raw-milk soft cheese, 314–315 ready-to-eat meat and poultry products, 321–327, 341–342 reduction, 333 rice salad, 338 rillettes, 330–331 seafood products, 333–335, 337–338, 340–341 semihard soft cheese, 314–315 shrimp, 337–338

DK3089_C020.fm Page 854 Tuesday, February 20, 2007 7:13 PM

854 smoked mussels, 334 soft, unripened Mexican-style cheese, 310–312 sporadic listeriosis, 344–348 surveillance, 344–349, 346–347 Sweden, 342–343 Switzerland, 312–313 United States, 321–328, 337–339, 341–342, 344–349 Vacherin Mont d’Or, 312–313 vegetables, 335–336 Wales, 327 Food composition, 167 FoodNet clusters of listeriosis, 345 historical development, 98 surveillance, 347–348 Food processing, facilities Australia, 747–748, 748 barrier establishment, 715 biofilms, 685–704 cheese production facilities, 743 chocolate production facilities, 741–742 clarifiers, 750 cleaning, control method, 707–710 corned beef, 755 corrective actions, 718 critical control point, 717 critical limits, 717 cross-connections, 752 dairy processing facilities, 720–723, 721–724, 739–741, 740–741, 746–747, 750–754 documentation procedures, 718–719 dry areas, cleaning, 707 egg processing facilities, 732, 757 England, 742–743 equipment design, 697–704 Europe, 739–742 factors to consider, 710–711 factory design, 697–704 farm environment, 750 fermented dairy products, 754 filling, 752 Finland, 744 France, 743 frankfurters, 755–756 frozen dairy products, 753 fruit processing facilities, 738, 758–759 fundamentals, 683–684, 683–685 growth, 685–704 hazard analysis critical control point, 711–713, 712, 716–719 household kitchens, 748–749 hygiene, 38 incidence, 719–748 industry-specific control approaches, 749–759 Italy, 744 kitchens, household, 748–749 link-type products, 755–756 luncheon meats, 756 maintenance practices, 693, 693–697 meat processing facilities, 724–730, 725–726, 728–730, 741, 744, 754–756

Listeria, Listeriosis, and Food Safety miscellaneous production facilities, 742–743, 745–746 monitoring, 711, 717 Netherlands, 745–746 packaging, 752 pasteurization, 750–752, 751 pipeline connections, 752 poultry processing facilities, 730–731, 731, 741–742, 742, 743, 756–757 rebagged products, 755 reclaimed and reworked products, 753 record-keeping procedures, 718–719 repair practices, 693, 693–697 roast beef, 755 sampling plans, 713–715, 714 sanitation, control method, 707–710 seafood processing facilities, 732–738, 733, 735–737, 741, 744, 757–758 separators, 750 smoked-salmon processing facilities, 743 Spain, 745 Sweden, 746–747 Switzerland, 747 traditional control approaches, 704–710 traffic patterns, 710 United Kingdom, 742–743 United States, 719–738 vegetable processing facilities, 738, 745, 758–759 verification procedures, 718 wet processing areas, cleaning, 707–709 Food processing, importance of characteristics acid damage and tolerance, 170–171 acidity, 169–171 acid sanitizers, 194 active packaging, 196–197 alternative processing technologies, 187–190 antimicrobial components, 174–184 antimicrobial components, foods, 174–184 antioxidants, 180–181 attachment to surfaces, 190–192, 191 bacteriocins, 185–186 benzoic acid derivatives, 175, 178 biocontrol, 185–186 biofilm formation, 190–192, 191 chlorine and chlorinated compounds, 193–194 cold tolerance, 161–163, 162 elevated sublethal temperature, 163 enhanced acid tolerance, 171 extrinsic factors, heat resistance, 167 fatty acid monoesters, 179–180 fatty acids and related compounds, 178–180 free fatty acids, 178–179 freezing temperature, 163–164 fundamentals, 158–159 growth, water activity, 171–172, 172 growth kinetics, 160–161, 161 growth temperature, 159–163 heat resistance, 167–168 herbs, 181–182, 182 high-intensity pulsed light, 188–189 high-pressure processing, 189, 189 high salt concentration, 174

DK3089_C020.fm Page 855 Tuesday, February 20, 2007 7:13 PM

Index hydrogen peroxide, 183 inactivation, low pH, 169–170 intrinsic factors, heat resistance, 167–168 ionizing radiations, 187–188, 188 irradiation, 187–189 kinetics, thermal inactivation, 165–167 lactate, 175, 176 lactoferrin, 184 lactoperoxidase system, 183–184 lethal temperature, 164–168 live fermentate, 185 low pH, 169–170 lysozyme, 182–183 miscellaneous sanitizing agents, 195–196 modified atmosphere, 186–187 multiple antimicrobial treatments, 197, 197–198 optimum temperature, 159–160 organic acids and salts, 175, 175–178 osmotolerance factors, 172–173, 172–173 ozone, 194–195, 195 parabens, 175, 178 physiology, high salt concentration, 174–175 plant extracts, 181–182, 182 potassium sorbate, 175, 177–178 pulsed electric field processing, 189–190, 190 quaternary ammonium compounds, 194 range, temperature, 159–160 salts, 174–178 sanitizers, 192–196 smoke, 181 sodium benzoate, 175, 178 sodium diacetate, 175, 176–177 sodium nitrite, 180 sodium propionate, 175, 177 spices, 181–182, 182 stress adaptation, 163 surrogate microorganisms, 168 survival, 171–172, 172, 174 temperature, 159–168 thermal inactivation, 164–167 tolerance, acid, 170–171 ultraviolet radiation, 188–189 virulence variations, 163 water activity, 171–173 Food-processing environments, 36, see also Food processing, facilities Food safety perspective consumer education, 839–840 control options, 837–838 deli meats, 838 foods of concern, 839 labeling, 839–840 processed food risks, 838–839 regulatory tolerance, 836–837 retail product focus, 838–839 Foods of concern, 839 Fowl, 67–69 Foxes, 41 France, see also Rillettes cheese and fermented products, 414–418 epidemic listeriosis, 97

855 fluid milk, 317 foodborne infection, 313–314, 330–332 food processing, facilities, 743 human outbreaks, 37 listeriosis outbreak, 330, 332 predisposing factors, 56 risk assessment, 775–776 sporadic incidence, 98 standards and criteria, 797 surveillance programs, 414–418, 421, 428 transmission to animals, 67 USDA-FSIS method, 243 Franco studies, 585 Frank, Bolton and, studies, 481 Frankfurters, see also Hot dogs foodborne infection, 321–327 food processing, facilities, 755–756 organic acids and salts, 176–177 potassium sorbate, 177–178 sodium benzoate, 178 Fraser broth, 226–227 Free fatty acids, 178–179, 179 Freezing, temperature, 163–164 Fresh, raw-milk cheese, 342–343 Fresh sausage, 538–540, 539–540, see also Sausages Frogs, 41 Frozen dairy products, 753 Fruit processing facilities control methods, 758–759 United States, 738 Fruits and fruit juices, 671–674, 672 Fuad studies, 636 Fuchs and Surendran studies, 630 Fuerst, Hugenholtz and, studies, 704 Fundamentals bacteria identification, 9–12 biochemical characteristics, 10 chemotaxonomy, 3–4 culture, 9 fundamentals, 4–5, 12 genus characteristics, 9–10 historical background, 1–2 metabolism, 10 morphology, 9 numerical taxonomy, 3 nutritional requirements, 10 phylogenetic position, 3–5 rRNA sequencing, 4 species identification, 10–12 taxonomy, 5–9 whole genome sequencing, 4, 5 Future issues, 139 Fuzi and Pillis studies, 222

G Gabis, Silliker and, studies, 721 Gabis studies, 483, 723 Gahan studies, 461 Galindo-Cuspinera studies, 449 Gallagher, Sumner and, studies, 773

DK3089_C020.fm Page 856 Tuesday, February 20, 2007 7:13 PM

856 Gamma radiation, 589–590 Garayzabal and Genigeorgis studies, 232 Garayzabal studies, 374 Garcia-Gimeno studies, 667 Garland, Soonthoranant and, studies, 32 Garrec studies, 31 Gastrointestinal illness, 90–93 Gaya studies, 61 Gazelles, 70 Geese, 67 Geisen studies, 451 Gendel studies, 291 Gene expression academic perspective, 833–834 molecular virulence determinants, 132–137 regulation in stress conditions, 34–35 Generic species vs. specific species, 259 Genigeorgis, Garayzabal and, studies, 232 Genigeorgis studies, 478, 577–579, 742 Genome sequencing fundamentals, 4, 5, 27–28 groups, 6 molecular virulence determinants, 137–138 Genomic endowment, 831–833 Genotypic markers, 11–12 Genus characteristics, 9–10 Georgia (United States) deli meat and frankfurters, 325 Mexican-style cheese, 316 Germany asymptomatic carriage, 91 fluid milk, 317 monitoring programs, 428–430, 429 predisposing factors, 56 risk assessment, 777 standards and criteria, 797 surveillance programs, 429 Gerner-Smidt, Norrung and, studies, 287–288, 297 Gerner-Smidt studies, 288 Gianfranceschi and Aureli studies, 666 Gibbons studies, 2 Gilbert, McLauchlin and, studies, 526 Gilbert, Pini and, studies, 433, 581, 583 Gilbert studies, 516 Gill and Reichel studies, 548 Gillespie studies, 526 Gill studies, 2 Gilmour, Harvey and, studies, 36, 363, 660, 747 Gilmour, Kells and, studies, 230 Gilmour, Lawrence and, studies, 36, 586 Giovanacci studies, 522 Giraffes, 41 Girard, Bearns and, studies, 227, 229 Girard, McBride and, studies, 220, 227 Gitter studies, 33, 581 Glass and Doyle studies, 534–546, 558, 604 Glass studies, 464, 477, 539 Global pathways and reservoirs, 42–44 Globals pathways and reservoirs, 42–44 Glutaraldehyde, 196 Goats and goat’s milk

Listeria, Listeriosis, and Food Safety animal feeds, 34 animal listeriosis, 60–62 cheese and fermented dairy products, 468–470 unfermented dairy products, 366, 366–367 water contamination, 32 Godreuil studies, 286 Goebel studies, 111–139 Goetz studies, 125 Gohil studies, 522, 662 Golan, Kuchler and, studies, 803 Golden studies, 231 Goldfine and Wadsworth studies, 119 Goldfine studies, 119 Gombas studies, 413, 804 Gómez studies, 271 Gorris, Aytac and, studies, 667 Gouda cheese, 458 Gouet studies, 530–531 Goulet studies, 98 Graham, Kerlin and, studies, 66 Grant’s gazelle, 70 Grau and Vanderlinde studies, 527, 532, 535 Graves studies, 283–297, 324 Gray, M.L., 5 Gray and Killinger studies, 41, 218 Gray studies, 9, 32, 216–219, 258, 814 Great Britain animal feeds, 33 neonatal disease, 88 transmission to animals, 59, 62 Greece, 31 Gregorio studies, 221 Griffith and Deibel studies, 444 Grif studies, 39, 91 Grøntsol studies, 33 Ground meat, raw, see also specific type fraser broth, 226–227 meat products, 557–558 Groves and Welshimer studies, 6 Grow studies, 274 Growth egg products, 601–603, 601–604 enrichment and isolation methods, 22 factors, 10 fish and seafood products, 638–640 food processing, facilities, 685–704 kinetics, 160–161, 161 meat products, 530–533 nutritional requirements, 10 plant origin products, 663–666, 664 poultry products, 587–592 prevention, 824–826 risk assessment, regulatory control, and economic impact, 784 temperature, 159–163 water activity, 171–172, 172 Gudbjornsdottir studies, 741 Gueguen, Dieuleveux and, studies, 470 Guerra studies, 294 Guinea pigs, 41 Gulf Coast, United States, 32, 72

DK3089_C020.fm Page 857 Tuesday, February 20, 2007 7:13 PM

Index Gulls, 41 Gulmez and Guven studies, 445 Gum base nalidixic acid, 229 Gurtler studies, 681–759 Guven, Gulmez and, studies, 445 Guyer and Jemmi studies, 639

H Halle, Germany, 317 Ham direct plating media, 231–232 meat products, 534–535 thermal inactivation, 165 Hamon and Peron studies, 286 Hao and Brackett studies, 669 Hao studies, 226, 231, 663, 669 Hard cheeses, 457–463 Hard Italian-type cheese, 463 Hard salami, 172 Harlander, Baloga and, studies, 288, 297 Harrison, Carpenter and, studies, 594–595 Harrison, Shineman and, studies, 639 Harrison and Carpenter studies, 597 Harrison and Huang studies, 643 Harrison studies, 639 Hart studies, 587 Harvey and Gilmour studies, 36, 363, 660, 747 Haydon studies, 43 Hayes studies, 64, 223, 225, 243, 358 Hazard analysis critical control point (HACCP) corrective actions, 718 critical control points, 717 critical limits, 717 fundamentals, 711–713, 712 hazard analysis, 716–717 monitoring procedures, 717 need for methods, 216 plan development, 331 record-keeping and validation, 718–719 required by USDA-FSIS, 333 team, 716 verification procedures, 718 Hazard identification and characterization, fish, 635 Heat food processing importance, 167–168 plant origin products, 667–668 temperature, 167 Heinitz, Maxine, 647 Heisick studies, 659–660, 662 Helloin studies, 452 Hemolysin, 110 β-hemolysis, 233–234 Henderson-Hasselbalch equation, 176 Henry studies, 232–233 Herald and Zottola studies, 529 Herbs, 181–182, 182 Heredia studies, 516 Herman, Rijpens and, studies, 230 Hernandez-Sanchez, Solano-Lopez and, studies, 464–465 Hessen studies, 66

857 Hewitt studies, 804 Hicks and Lund studies, 473 Higgins, Parish and, studies, 672 Higgins and Robinson studies, 10 High-intensity pulsed light, 188–189 High-pressure processing, 189, 189 Hill, Nancy, 647 Hill, Walter E., 647 Hispanic cheeses, see Mexican-style cheese Historical background, 1–2 Hitchins studies, 778 Hobson studies, 527 Hofer studies, 221 Hoffman and Wiedmann studies, 242 Hoffman studies, 631, 638 Höhne studies, 528 Holliday and Beuchat studies, 393 Holt studies, 273 Hong Kong, 775 Hooper-Kinder studies, 515 Horses fecal material, 41 transmission to, 69–70 Host cell cytoplasm growth, 125–126 Hotchkiss, Chen and, studies, 474 Hot dogs, see also Frankfurters dietary risk factors, 99–100 epidemic listeriosis, 96 human outbreaks, 37 Household kitchens, 748–749 Huang, Harrison and, studies, 643 Huang studies, 592 Hudson and Mead studies, 742 Hudson and Mott studies, 640 Huffman, Randy, 827 Hugenholtz and Fuerst studies, 704 Hughey studies, 453, 482, 486, 538, 669 Huhtanen studies, 589 Hulphers studies, 1 Humans, listeriosis asymptomatic carriage, 91, 92–93, 93 blood donations, 100 clinical manifestations, 87–88 diagnosis, 100–101 dietary risk factors, 98–100 early onset, neonatal disease, 87–88 epidemic listeriosis, 94, 95, 96–97 epidemiological patterns, 94–100, 95 fish and seafood products, 632–633 fundamentals, 85–86, 86 gastrointestinal illness, 90–93 incidence, 97–98, 99 infection, 87–88 invasive disease, 88–90 late onset, neonatal disease, 88 neonatal disease, 87–88 noninvasive disease, 90–93 nonpregnant adults, 88–90 nosocomial transmission, 100 outbreaks, 37–38 pregnancy, 87

DK3089_C020.fm Page 858 Tuesday, February 20, 2007 7:13 PM

858 prevention, 101–102 sexual transmission, 100 sporadic disease, 97–100, 99 transmission dynamics, 35–40 treatment, 101 water contamination, 32 Humans, transmission to, 57 Humboldt-Arcata Bay, 72 Hungary, 56 Hungerford, Jim, 647 Hunter studies, 283–297 Husu studies, 41, 578 Hwang and Beuchat studies, 589 Hydrogen peroxide, 183, 196 Hyraxes, 41

I Ice cream, see also Dairy products direct plating media, 231 unfermented dairy products, 391–392 Ideal detection, 258 Identification, 308, 308–309 Ikonomov and Todorov studies, 467–468 Illicitly produced/distributed cheese, 315–317, see also Mexican-style cheese Imitation crabmeat, see also Fish and seafood products fatty acid monoesters, 178 human outbreaks, 37 Imported cheese, 414–419, see also specific type Imported seafood, 618–626, see also Fish and seafood products Inactivation low pH, 169–170 plant origin products, 667–673 Incidence animal listeriosis, 56 egg products, 600–601 fish and seafood products, 627–629 food processing, facilities, 719–748 fruits, 671–672 humans, listeriosis in, 97–98, 99 raw vegetables, 657–663 Incidence, meat products Canadian monitoring program results, 508–514, 514–518 cooked meat, 505 cooked products, 505, 506–508, 507–508, 513 Europe, 519–521, 523–527, 524–525 non-European countries, 527 non-North American countries, 516, 518–523, 519–521 North America, 515–516, 517–518, 523 raw meat, 505, 514–523 ready-to-eat products, 508, 513, 514–515, 523–527 recalls and regulatory actions, 507–508, 509–513, 514, 516 sausage, 523–527 USDA-FSIS monitoring program, 504–508, 506–508 Incidence, unfermented dairy products fundamentals, 358 incidence, 358–376

Listeria, Listeriosis, and Food Safety other products, 367–376 pasteurized milk, 367–376 raw cow’s milk, 358–366 raw ewe’s milk, 366, 366–367 raw goats milk, 366, 366–367 Incubation conditions, 232 Indiana (United States), 65 Industry perspective, research needs, 819–827 consumer exposure, 821–827 contamination prevention, 821–823 FAO/WHO risk assessment significance, 819–820 FDA/FSIS risk assessment significance, 819–820 fundamentals, 819, 827 growth prevention, 824–826 industry perspective, 819–827 outbreaks vs. sporadic cases, 820–821 postpasteurization, 823–824 risk assessment significance, 819–820 Industry-specific control approaches, 749–759 Infection, humans, 87–88, see also Humans, listeriosis Ingham studies, 590 Inhibition, fish and seafood products, 640–643, 641 Initial contamination, 783–784 Injured Listeria recovery, 244–248 Intensively pasteurized milk, 379–380, see also Pasteurized milk International, risk assessment, 781–790, 800–801 International Dairy Federation method, 238–239, 239 Intracellular motility, 126–129, 127 Intrinsic factors, 167–168 Invasive disease, 88–90, see also Humans, listeriosis Iodophors, 195–196 Ionizing radiations, 187–188, 188 Iowa (United States), 62, 66–67 Ireland, 97, 327, 330 Iron uptake systems, 132 Irradiation, 187–189 Isaac, Kwantes and, studies, 580–581 Islam studies, 599 Isolation and detection, conventional methods acriflavine, 221–222 agents, selective, 219–223, 220 ARS-MMLA media, 229–230 blood-free plating media, 229 ceftazidime, 223 cold enrichment, 217–219 direct plating media comparisons, 230–232 Food and Drug Administration method, 234–238, 236–237 Fraser broth, 226–227 fundamentals, 216–217, 248 gum base nalidixic acid, 229 β-hemolysis, 233–234 incubation conditions, 232 injured Listeria recovery, 244–248 International Dairy Federation method, 238–239, 239 isolation media, 227–232 lithium chloride, 220–221 lithium chloride-phenylethanol-moxalactam agar, 228 McBride Listeria agar, 227–228 modified Oxford agar, 228

DK3089_C020.fm Page 859 Tuesday, February 20, 2007 7:13 PM

Index moxalactam, 223 nalidixic acid, 221 Netherlands Government Food Inspection Service, 243 oblique illumination, 232–234, 233 official methods, 234–243 Oxford agar, 228 PALCAM agar, 228–229 phenylethanol, 220–221 plating, 219–223 polymyxin B, 222–223 potassium tellurite, 219–220 potassium thiocyanate, 222 recovering injured Listeria, 244–248 research advances, 244 selective enrichment, 219–223 selective media, 223–243 thallous acetate, 222 trypaflavine, 221–222 USDA-FSIS method, 239–243, 240 UVM broth, 225–226 Isolation media, 227–232 Isolation method, 22–23 Issa and Ryser studies, 446 Italy foodborne infection, 338 food processing, facilities, 744 standards and criteria, 798 surveillance programs, 430 Ivanov studies, 5–6

J Japan foodborne infection, 343 sporadic incidence, 98 Jayaro studies, 64 Jeffers studies, 38 Jemmi, Guyer and, studies, 639 Jemmi, Trüssel and, studies, 543, 545 Jemmi and Keusch studies, 646 Jemmi studies, 639 Jenkins, Erickson and, studies, 604 Jinneman studies, 617–646 Johannessen studies, 661 Johannson and Cossart studies, 135 Johannson studies, 135 Johansson studies, 230 Johnson, Jan, 647 Johnson, Lungu and, studies, 599 Johnson, Siragusa and, studies, 223 Johnson studies, 257–274, 528, 530–531, 533, 545–546 Jones, Feresu and, studies, 3, 10 Jones, Kramer and, studies, 222 Jones, Wilkinson and, studies, 3 Jonesia denitrificans, 8 Jones studies, 3 Juneja, Klein and, studies, 262 Juneja studies, 535 Junttila studies, 547

859

K Kabuki studies, 723 Kachkaval cheese, 468–469 Kallipolitis studies, 137 Kamat studies, 671 Kampelmacher studies, 64, 93 Kaneko studies, 662 Kanuganti studies, 534 Karaioannoglou and Xenos studies, 552 Katic studies, 468 Kaya and Schmidt studies, 531 Kaymaz, Sarumehmetoglu and, studies, 467 Kaysner studies, 639 Kefir, 445 Keller, Cirigliano and, studies, 393 Kells and Gilmour studies, 230 Kentucky (United States), 65 Kerlin and Graham studies, 66 Kerr studies, 196 Keusch, Jemmi and, studies, 646 Khan studies, 222, 533–534 Khattab studies, 444 Killinger, Gray and, studies, 41, 218 Kim studies, 457, 669 Kinetics growth, temperature, 160–161, 161 thermal inactivation, food processing importance, 165–167 Kitchens, household, 748–749 Kitts, Wong and, studies, 600 Klausner and Donnelly studies, 722 Klawitter, Buchanan and, studies, 557 Klein and Juneja studies, 262 Knabel studies, 245 Kornacki studies, 240, 681–759 Kovácsné Domjan studies, 527 Kovincic studies, 456 Kramer and Jones studies, 222 Kreft, J., 139 Kreft and Vazquez-Boland studies, 136 Kuchler and Golan studies, 803 Kuhn studies, 111–139 Kvenberg, Elliot and, studies, 634 Kvenburg studies, 645 Kwantes and Isaac studies, 580–581 Kyung and Fleming studies, 670

L Labeling, 839–840 Labneh, 445–446 Lachica studies, 232 Lactate, 175, 176 Lactoferrin, 184 Lactoperoxidase system, 183–184 Lado studies, 157–198 Lahti studies, 559 Lambs, 533–534, 549–550, 550, see also Ewe’s milk; Sheep Lammerding and Doyle studies, 243

DK3089_C020.fm Page 860 Tuesday, February 20, 2007 7:13 PM

860 Lamont and Postlethwaite studies, 93 Lanciotti studies, 393 Landefeld and Seskin studies, 803 Lappi studies, 734 Larsen studies, 216, 485 Larson studies, 483 Late onset, neonatal disease, 88 Lawrence and Gilmour studies, 36, 586 Lawrence studies, 292, 743 Leasor, Foegeding and, studies, 603, 605 Leasor and Foegeding studies, 600 Lecuit studies, 117 Lee, McClain and, studies, 226, 228 Lee and McClain studies, 223, 228, 239 Lee studies, 532, 666 Lefier studies, 273 Legan studies, 825 Lehmann and Schönberg studies, 285 Leighton studies, 222 Lemaitre studies, 285 Lemmings, 41 Leningham studies, 2 Lennon studies, 71 Lessons learned, 137–138 Lethal temperature, 164–168 Lettuce, 30 Lety studies, 122 Leuchner studies, 379 Levre studies, 526 Lewis and Corry studies, 218 Lieval studies, 587 Liewen, Peters and, studies, 385 Lightly processed fish products, 631–632, see also Fish and seafood products Limitations, ecology study methods, 24–25 Lindqvist and Westöö studies, 777–778 Link-type products, 755–756, see also specific type Lin studies, 274, 659 Lister, Arthur, 2 Lister, Joseph Jackson, 2 Listeria denitrificans, 8 Listeria fundamentals bacteria identification, 9–12 biochemical characteristics, 10 chemotaxonomy, 3–4 culture, 9 fundamentals, 4–5, 12 genus characteristics, 9–10 historical background, 1–2 metabolism, 10 morphology, 9 numerical taxonomy, 3 nutritional requirements, 10 phylogenetic position, 3–5 rRNA sequencing, 4 species identification, 10–12 taxonomy, 5–9 whole genome, 4, 5 Listeria grayi, 7–8 Listeria ivanovii, 6–7 Listeria monocytogenes, 6–7

Listeria, Listeriosis, and Food Safety Listeria murrayi, 7–8 Listeria seeligeri, 6–7 Listeria welshimeri, 6–7 Listeriophages, 196 Li studies, 665 Lithium chloride, 220–221 Lithium chloride-phenylethanol-moxalactam agar, 228 Live fermentate, 185 Livestock, domestic, see also specific type incidence, 56 meat products, 528 Llamas, 70 Localization in tissues, 528–530 Loessner studies, 232 Lorber, Ooi and, studies, 336 Los Angeles County asymptomatic carriage, 93 epidemic listeriosis, 96 foodborne infection, 310–312, 311 human outbreaks, 37 sporadic incidence, 98, 344–345 surveillance, 347 Lovett studies, 228, 234, 534, 638 Low pH, 169, 169–170, see also Acidity Low studies, 34, 41 Lozniewski studies, 31 Ludwig studies, 4 Lukasova studies, 442 Luncheon meats, see also Deli meats; Ready-to-eat meat and poultry products food processing, facilities, 756 meat products, 535–538, 536–537 Lund, Ferreira and, studies, 474 Lund, Hicks and, studies, 473 Lund, Nguyen-the and, studies, 670 Lunden studies, 744 Lund studies, 243 Lungu and Johnson studies, 599 Ly and Müller studies, 42 Lysozyme antimicrobial components, foods, 182–183 food processing importance, 182–183

M MA, see Modified atmosphere (MA) Maasdam cheese, 458 MacGowan studies, 28, 516 Mackey studies, 556 Macrophages uptake, 118–119 Madden, Moore and, studies, 600 Madden studies, 634 Maini studies, 518 Maintenance practices, 693, 693–697 Maisner-Patin studies, 452 Mammalian cells cellular adhesion, 118 dendritic cells uptake, 118–119 fundamentals, 113 macrophages uptake, 118–119 nonprofessional phagocytic cells, 114, 115, 116–118

DK3089_C020.fm Page 861 Tuesday, February 20, 2007 7:13 PM

Index Manchego cheese, 464–465, 469 Manu-Tawiah studies, 550 Marchisio studies, 456 Marine environments, 32 Marquet-van der Mee studies, 285 Marshall and Schmidt studies, 383, 389 Marth, El-Gazzar and, studies, 447–449, 675 Marth, El-Shenawy and, studies, 471 Marth, Farrag and, studies, 388–389 Marth, Papageorgiou and, studies, 453, 465, 484–485 Marth, Pearson and, studies, 182, 385–387 Marth, Rosenow and, studies, 381, 383–385, 392 Marth, Ryser and, studies brick cheese, 455–456 brine solutions, 484–485 Camembert cheese, 449–451 cheddar cheese, 459, 461 cold enrichment, 217–218 cold-pack cheese food, 476 International Dairy Federation method, 238 lithium chloride/phenylethanol, 221 selective enrichment and plating, 219 survival in cheddar cheese, 413 whey, 482–483 Marth, Schaack and, studies, 436–439, 441–443, 446, 471 Marth, Wenzel and, studies, 377 Marth, Yousef and, studies, 221, 458, 462, 481 Marth studies, 814–815 Martin studies, 229, 232 Maryland (United States), 328 Mascola studies, 93 Massachusetts (United States) epidemic listeriosis, 94 fluid milk, 317 human outbreaks, 37 listeriosis outbreak, 330 transmission to animals, 65 vegetables, 336 Massa studies, 430, 433 Mathew and Ryser studies, 437 Mathew studies, 380 Matthews studies, 62 Mazurier studies, 292 McAuliffe studies, 473 McBride and Girard studies, 220, 227 McBride Listeria agar, 227–228 McClain, Lee and, studies, 223, 228, 239 McClain and Lee studies, 226, 228 McKellar studies, 541, 591 McLauchlin and Gilbert studies, 526 McLauchlin and Nichols studies, 38 McLauchlin studies, 38, 285, 334 McNab, Truscott and, studies, 227 Mead, Hudson and, studies, 742 Mead studies, 98, 348 Measurement, thermal inactivation kinetics, 165 Meat processing facilities control methods, 754–756 Europe, 741 Finland, 744

861 Italy, 744 United States, 724–730, 725–726, 728–730 Meats and meat products, see also Ready-to-eat meat and poultry products bacteriocins, 557–559 beef, 530–533, 535, 548–549, 549 behavior, 527–559 Canadian monitoring program results, 508–514 cooked products, 505, 506–508, 507–508, 513, 534–538 cooked roast beef, 535 cooked smoked sausage, 540–542, 542 cured ham, 534–535 domestic livestock, 528 dry fermented sausage, 545–547 Europe, 523–527, 524–525 fermented sausage, 544, 558–559, 560 foodborne infection, 320–333 fresh sausage, 538–540, 539–540 fundamentals, 504 growth, 530–533 ham, cured, 534–535 incidence, 504–527 isolation methods for, 234 lamb, 533–534, 549–550, 550 luncheon meats, 535–538, 536–537 modified-atmosphere packaging, 547–557 non-European countries, 527 non-North American countries, 516, 518–523, 519–521 North America, 515–516, 517–518, 523 pork, 533–534, 550–552, 551 potassium sorbate, 177 raw ground meat, 557–558 raw meat, 505, 514–523, 530–533 ready-to-eat products, 508, 513, 514–515, 523–527, 534–538 recalls and regulatory actions, 507–508, 509–513, 514, 516 roast beef, cooked, 535 sausage, 523–527, 538–547, 549 semidry fermented sausage, 544–545 smoked sausage, 540–543, 542 specialty items, 543–544 survival, 530–533 thermal inactivation, 552–557, 553–556 tissue localization, 528–530 uncooked smoked sausage, 543 unfermented sausage, 538–547 USDA-FSIS monitoring program results, 504–508 Mechanisms acid damage and tolerance, 170–171 food processing importance, 164–165 positive regulatory factor A, 134–136 temperature, 164–165 thermal inactivation, 164–165 Medicated soap, 196 Mehta and Tatini studies, 461 Mesophilic starter cultures, 436–439, 437–438 Metabolism, 10 Metaxopoulos, Samelis and, studies, 535

DK3089_C020.fm Page 862 Tuesday, February 20, 2007 7:13 PM

862 Methods comparison of subtyping, 297, 298 conventional subtyping methods, 284–286 ecology, 22–27 enrichment method, 22–23 enumeration methods, 23–24 isolation method, 22–23 limitations, ecology study methods, 24–25 molecular detection methods, 25–27, 26, 287–297 pitfalls, ecology study methods, 24–25 subtyping, 25–27, 26, 284–297, 298 traditional enrichment method, 22–23 Mexican Manchego cheese, 464–465 Mexican-style cheese epidemic listeriosis, 96 foodborne infection, 310–312 human outbreaks, 37 illicitly produced/distributed cheese, 315–317 Meyer-Broseta studies, 363 Mice animal feeds, 32 fecal material, 41 human outbreaks, 39 Michard, Ferron and, studies, 243 Michigan (United States), 325 Microarrays, DNA, 262–263 Middle East, 662–663 Midelet and Carpentier studies, 538 Miles, Wilson and, studies, 2 Milk, see also Chocolate milk; Raw milk human outbreaks, 37 lactoferrin, 184 sanitizing agents, 195 Miller studies, 556 Mink, 41 Minor species, animal listeriosis, 69–71 Miscellaneous production facilities Netherlands, 745–746 United Kingdom, 742–743 Miscellaneous sanitizing agents, 195–196 Missouri (United States), 61–62, 67 Mitchell, Curtis and, studies, 286 Mixed cultures, 388–390 Modified atmosphere (MA) fundamentals, 186–187 storage, plant origin products, 666–667 Modified-atmosphere (MA) packaging beef, 548–549, 549 fundamentals, 547–548 lamb, 549–550, 550 pork, 550–552, 551 sausages, 549 thermal inactivation, meats, 552–557, 553–556 Modified Oxford agar, 228 Moir studies, 473 Mold-ripened cheeses fermented dairy products, 449–453 PALCAM agar, 229 Molecular methods detection, 25–27, 26 subtyping, 287–297

Listeria, Listeriosis, and Food Safety Molecular subtyping role, 309–310 Molecular virulence determinants accessory virulence factors, 130–132 bile salt hydrolase, 129–130 catalase, 131 cellular adhesion, 118 dendritic cells uptake, 118–119 environmentals signals affect, 136 evolutionary aspects, 138 fundamentals, 111–113, 112–113 future issues, 139 gene expression, 132–137 genome sequence, 137–138 host cell cytoplasm growth, 125–126 intracellular motility, 126–129, 127 iron uptake systems, 132 lessons learned, 137–138 macrophages uptake, 118–119 mammalian cells, 113–119 mechanisms, 134–136 nonprofessional phagocytic cells, 114, 115, 116–118 phagocytic vacuole escape, 120–125, 121, 123 positive regulatory factor A, 132–137, 133 promoters, 134–136 protein p60, 130–131 regulation, 137 stress response mediators, 131–132 superoxide dismutase, 131 transcripts, 134–136 two-component systems, 137 Molin, Ternstrom and, studies, 584 Monitoring programs, see also Surveillance Canada, 419–421, 420, 508–514 cheese and fermented products, 419–421, 420, 428–432, 429, 431 food processing, facilities, 711, 717 France, 421 Germany, 428–430, 429 Italy, 430 meats and meat products, 504–514 Switzerland, 430–432, 431 USDA-FSIS monitoring program results, 504–508 Monoesters, 179–180 Monolaurin, 196 Moore and Madden studies, 600 Morgan studies, 469 Morgen, Skovgaard and, studies, 226, 584 Morphology, 9 Morris and Ribeiro studies, 543, 586 Mosupye and von Holy studies, 662 Motes studies, 32 Mott, Hudson and, studies, 640 Mounier studies, 126 Mourney, Canillac and, studies, 747 Moxalactam, 223 Mozzarella cheese fermented dairy products, 457 free fatty acids, 178 Müller, Ly and, studies, 42 Multilocus enzyme electrophoresis, 287 Multiple antimicrobial treatments, 197, 197–198

DK3089_C020.fm Page 863 Tuesday, February 20, 2007 7:13 PM

Index Multiplication, stress conditions, 27–35 Muraoka studies, 362 Muriana, Wang and, studies, 523 Muriana studies, 556, 598, 605 Murphy studies, 590, 597–598 Murray, E.G.D., 5 Murray, Webb and Swann studies, 1 Murray and Pirie studies, 2 Murray studies, 216–217, 306 Mushrooms, 227 Mussels, smoked, 334

N Nalidixic acid, 221 Natural environment, 35–42 Neonatal disease, 87–88 Netherlands food processing, facilities, 745–746 predisposing factors, 56 risk assessment, 777 standards and criteria, 798 transmission to animals, 59, 67 Netherlands Government Food Inspection Service, 22, 243 Newman and Weiner studies, 135 New South Wales, 60, 806 New York (United States) deli meat and frankfurters, 321, 325 foodborne infection, 328 transmission to animals, 65 New Zealand costs, 807 foodborne infection, 341–342 smoked mussels, 334 standards and criteria, 791–793 Ngutter and Donnelly studies, 244 Nguyen-the, Carlin and, studies, 665 Nguyen-the and Lund studies, 670 Nichols, McLauchlin and, studies, 38 Niebuhr and Dickson studies, 530 Nilsson studies, 642 Nisin, 185–187, 196 Noah studies, 243 Nocera studies, 288, 297 Nogva studies, 262 Nohuman primates, 71 Non-European countries cheese and fermented products, 435 meat products, 527 Nonfat dry milk, 393–394 Nonfluid dairy products butter, 392–393 fundamentals, 391 ice cream, 391–392 nonfat dry milk, 393–394 Noninvasive disease, 90–93 Non-North American countries, 516, 518–523, 519–521 Nonpregnant adults, 88–90 Nonprofessional phagocytic cells, 114, 115, 116–118, see also Phagocytic vacuole escape Non-United States products, 574–576, 580–587 Non-United States programs, 419–435

863 Norrung and Gerner-Smidt studies, 287–288, 297 Norrung studies, 521 North America epidemic listeriosis, 96 meat products, 515–516, 517–518, 523 standards and criteria, 793–796 transmission to animals, 62 North Carolina (United States), 315–316 Northolt studies, 376–377, 379, 384, 458, 482 Norton and Batt studies, 262 Norton studies, 36–39, 305–349, 637 Nosocomial transmission, 100 Notermans studies, 604, 777, 783 Nova Scotia epidemic listeriosis, 94 human outbreaks, 37 transmission, 36 Nucleic-acid-based methods DNA microarrays, 262–263 fluorescence in situ hybridization, 263–264 fundamentals, 259–260 polymerase chain reaction, 260–262 recombinant bateriophage, 264 Numerical taxonomy, 3 Nutritional requirements, 10 Nyachuba studies, 215–248 Nyfeldt, Schmidt and, studies, 63 Nyfeldt studies, 2 Nykanen studies, 642

O Oblique illumination, 232–234, 233 O’Donoghue studies, 293 Official methods, detection and isolation, 234–243 Ohio (United States), 65, 321 Ojeniyi studies, 584 Oklahoma, 98 Olafson studies, 32 Olsen studies, 392 Olson studies, 219 Omary studies, 667 Ontario, Canada, 65 Ooi and Lorber studies, 336 Optimum temperature, 159–160 Organic acids, 175, 175–178 Ortel studies, 222 Osborne, Bremer and, studies, 644 Osmotolerance factors, 172–173, 172–173 Outbreaks detection and investigation, 309–310, 310 ecology, 37–38 ten or more cases, 307 vs. sporadic cases, 820–821 Oxford agar, 228 Oysters, 231–232 Ozone, 194–195, 195

P Pace studies, 643 Pacific Northwest, 65

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864 Pacific oysters, 32 Packaging, control methods, 752 Pagan studies, 198 Painter studies, 85–102 PALCAM agar, 228–229 Palumbo and Williams studies, 577 Paoli studies, 265 Papageorgiou and Marth studies, 453, 465, 484–485 Papageorgiou studies, 466, 475 Parabens, 175, 178 Parish and Higgins studies, 672 Park studies, 600 Parmesan cheese, 462–463 Parrots, 41 Partridges, 41 Pass-through phenomenon, 42 Pasteurization, control methods, 750–752, 751 Pasteurized cheese spreads, 185–186 Pasteurized milk direct plating media, 231 epidemic listeriosis, 94, 96 unfermented dairy products, 367–376, 379–380 Pasteurized process cheese, 477 Pastry and bakery items baked goods, 243 potassium sorbate, 177 USDA-FSIS method, 243 Pâté epidemic listeriosis, 97, 317 foodborne infection, 327–330 human outbreaks, 37 Paterson studies, 6, 601 Pathogenicity, 817–818 Patterson studies, 589 PCR, see Polymerase chain reaction (PCR) Pearson and Marth studies, 182, 385–387 Peccio studies, 744 Pediocin, 186 Peeler and Bunning studies, 778 PEF, see Pulsed electric field (PEF) processing Pelroy studies, 640–641 Peng and Sheref studies, 244 Pennsylvania (United States), 319–320, 326 Peptone water, 196 Peron, Hamon and, studies, 286 Peters and Liewen studies, 385 Peterson studies, 640 Petran studies, 658 PFGE, subtyping, 291–292, 293 Phage typing (bateriophage typing), 285–286 Phagocytic vacuole escape, 120–125, 121, 123, see also Nonprofessional phagocytic cells Pheasants, 41, 67 Phenotypic markers, 11 Phenylethanol, 220–221 Philadelphia, Pennsylvania, 319–320 Phylogenetic position, 3–5 Physiology high salt concentration, 174–175 resistance to heat, 168 Pickett, Sword and, studies, 286

Listeria, Listeriosis, and Food Safety Pickled cheeses, 465–468 Pigeons, 41 Pigs, 41, see also Swine Pillis, Fuzi and, studies, 222 Pine studies, 380 Pingulkar studies, 662, 665 Pini and Gilbert studies, 433, 581, 583 Pinkerton, Pat, 647 Pipeline connections, 752 Pirie, Murray and, studies, 2 Pirie studies, 2 Pitfalls ecology study methods, 24–25 rapid method detection, 265 Pitt studies, 437, 442 Plant components, 669–670 Plant extracts, 181–182, 182 Plant origin products Asia, 662 behavior, 663–676, 674 Canada, 660 chemical sanitizers, 668–669 Europe, 660–661 fruits and fruit juices, 671–674, 672 fundamentals, 655–656 growth, 663–666, 664 heat, 667–668 inactivation, 667–673 incidence, 657–663, 671–672 Middle East, 662–663 modified atmosphere storage, 666–667 plant components, 669–670 processing techniques, 671, 671 raw vegetables, 657–663, 658 risk from, 656–657, 657 survival, 663–666, 664 United States, 658–660 Plant processing facilities Australia, 747–748, 748 barrier establishment, 715 biofilms, 685–704 cheese production facilities, 743 chocolate production facilities, 741–742 clarifiers, 750 cleaning, control method, 707–710 corned beef, 755 corrective actions, 718 critical control point, 717 critical limits, 717 cross-connections, 752 dairy processing facilities, 720–723, 721–724, 739–741, 740–741, 746–747, 750–754 documentation procedures, 718–719 dry areas, cleaning, 707 egg processing facilities, 732, 757 England, 742–743 equipment design, 697–704 Europe, 739–742 factors to consider, 710–711 factory design, 697–704 farm environment, 750

DK3089_C020.fm Page 865 Tuesday, February 20, 2007 7:13 PM

Index fermented dairy products, 754 filling, 752 Finland, 744 France, 743 frankfurters, 755–756 frozen dairy products, 753 fruit processing facilities, 738, 758–759 fundamentals, 683–684, 683–685 growth, 685–704 hazard analysis critical control point, 711–713, 712, 716–719 household kitchens, 748–749 hygiene, 38 incidence, 719–748 industry-specific control approaches, 749–759 Italy, 744 kitchens, household, 748–749 link-type products, 755–756 luncheon meats, 756 maintenance practices, 693, 693–697 meat processing facilities, 724–730, 725–726, 728–730, 741, 744, 754–756 miscellaneous production facilities, 742–743, 745–746 monitoring, 711, 717 Netherlands, 745–746 packaging, 752 pasteurization, 750–752, 751 pipeline connections, 752 poultry processing facilities, 730–731, 731, 741–742, 742, 743, 756–757 rebagged products, 755 reclaimed and reworked products, 753 record-keeping procedures, 718–719 repair practices, 693, 693–697 roast beef, 755 sampling plans, 713–715, 714 sanitation, control method, 707–710 seafood processing facilities, 732–738, 733, 735–737, 741, 744, 757–758 separators, 750 smoked-salmon processing facilities, 743 Spain, 745 Sweden, 746–747 Switzerland, 747 traditional control approaches, 704–710 traffic patterns, 710 United Kingdom, 742–743 United States, 719–738 vegetable processing facilities, 738, 745, 758–759 verification procedures, 718 wet processing areas, cleaning, 707–709 Plating, 219–223 Polymerase chain reaction (PCR), 260–262 Polymyxin B, 222–223 Pork, see also Swine foodborne infection, 328–329 human outbreaks, 37 meat products, 533–534, 550–552, 551 plant extracts, 181–182 processing facilities, 743 Porritt, Sutherland and, studies, 748

865 Portnoy, Tilney and, studies, 126 Portnoy studies, 122, 131 Positive regulatory factor A environmental signals, 136 fundamentals, 132–134, 133 mechanism, 134–136 promoters, 134–136 regulation, 136 transcripts, 134–136 two-component systems, 136 Postlethwaite, Lamont and, studies, 93 Postpackaging pasteurization, 598 Postpasteurization, 823–824 Potassium sorbate, 175, 177–178 Potassium tellurite, 219–220 Potassium thiocyanate, 222 Potel studies, 2, 306 Pothuri studies, 642 Poultry processing facilities control methods, 756–757 England, 742, 742 Europe, 741 France, 743 United States, 730–731, 731 Poultry products, see also Raw chicken; Raw turkey; Ready-to-eat meat and poultry products antimicrobial agents and additives, 589, 598–599 behavior in, 587–597 chicken, raw, 577–579, 587–590 continental Europe, 582–583, 584–585 control guidelines, 597–599 cooked products, 586–587, 590–592, 591–593 deli meat and frankfurters, 321–327 European products, 580–587 frankfurters and deli meat, 321–327 fraser broth, 226 fundamentals, 320–321, 332–333, 572 gamma radiation, 589–590 growth, 587–592 isolation methods for, 234 moxalactam, 223 non-United States products, 574–576, 580–587 PALCAM agar, 229 postpackaging pasteurization, 598 raw chicken, 587–590, 588 raw poultry, 580–586 raw turkey, 578–580, 579 ready-to-eat products, 586–587, 590–592, 597–599 recalls, 574, 575–576 reduction of listeriosis, 333 thermal inactivation, 593–597, 594–596 transmission pathways, 43 turkey, raw, 578–580, 579 United Kingdom, 580–584, 582–583 United States products, 577–579 USDA control guidelines, 597–599 USDA-FSIS, 241, 572–574, 574 Poysky studies, 646 Predisposing factors, 56–57 Pregnancy, 87 Prevention, 101–102

DK3089_C020.fm Page 866 Tuesday, February 20, 2007 7:13 PM

866 Pritchard studies, 245, 721 Process, risk assessment, 771–773 Processed food risks, 838–839 Processed meat, 241 Processing techniques, 671, 671 Promoters, 134–136 Protein p60, 130–131 Proteomic endowment, 831–833 Pucci studies, 473 Pulsed electric field (PEF) processing, 189–190, 190 Pulsed-field gel electrophoresis (PFGE) DNA macrorestriction analysis, 291–292, 293 molecular subtyping role, 309 PulseNet clusters of listeriosis, 345 deli meat and frankfurters, 321, 324–326 DNA macrorestriction analysis, 292 epidemiological patterns, 94 FDA method, 238 as surveillance system, 309–310 PurePulse system, 189

Q Quaternary ammonium compounds, 194 Quebec, Canada, 314–315 Queen Anne (of England), 2 Queso blanco cheese, see Mexican-style cheese Queso de los Ibores cheese, 464 Queso fresco cheese, see Mexican-style cheese Quinto studies, 383, 389

R Rabbits, 41 Rainbow trout, 71, 340–341 Rajkowski studies, 380, 485 Ralovich studies, 221, 225, 228 Random amplification of polymorphic DNA (RAPD), 292–294 Rapid method detection advances, 265–266 antibody-based methods, 264–267 automation, 259 biosensors, 269–271, 270 cell separation and concentration, 266–267 chip-based microanalytical systems, 271, 272 commercial availability, 258–259 diagnostic targets, 259 DNA microarrays, 262–263 flow cytometry, 267–269, 268–269 fluorescence in situ hybridization, 263–264 fundamentals, 257–259, 274 generic species vs. specific species, 259 ideal detection, 258 nucleic-acid-based methods, 259–264 polymerase chain reaction, 260–262 potential pitfalls, 265 recombinant bateriophage, 264 spectroscopic methods, 271–274, 273 traditional methods, 258

Listeria, Listeriosis, and Food Safety Rasmussen studies, 295 Rats, 41 Raw chicken, 587–590, 588, see also Poultry products; Raw poultry Raw cow’s milk, 358–366, see also Milk Raw ewe’s milk, 366, 366–367, see also Sheep Raw fish, 242, see also Fish and seafood products Raw goats milk, 366, 366–367, see also Goats and goat’s milk Raw ground meat, 226, 557–558, see also Meats and meat products Rawles studies, 639 Raw meat, see also Meats and meat products meat products, 505, 514–523, 530–533 moxalactam, 223 USDA-FSIS method, 241 Raw milk, see also Chocolate milk; Milk cheese and fermented products, 481 FDA method, 238 fraser broth, 226 hydrogen peroxide, 183 PALCAM agar, 229 selective enrichment and plating, 224–225 unfermented dairy products, 377–379, 378–380 Raw-milk fresh, unripened cheese, 315–317, see also Fermented products and cheese Raw-milk soft cheese, 314–315, see also Fermented products and cheese Raw poultry, see also Poultry products; Raw chicken direct plating media, 232 fraser broth, 227 incidence, 580–586 Raw turkey, 578–580, 579, see also Poultry products; Turkey and turkey products Raw vegetables, see also Vegetables epidemic listeriosis, 94 isolation methods for, 234 live fermentate, 185 McBride Listeria agar, 228 PALCAM agar, 229 plant origin products, 657–663, 658 Razavilar studies, 478 REA, see Restriction endonuclease analysis (REA) Read, Ralston, 814 Ready-to-eat meat and poultry products, see also Deli meats; Luncheon meats; Meats and meat products; Poultry products epidemic listeriosis, 96–97 febrile gastroenteritis, 341–342 foodborne infection, 321–327, 341–342 Ready-to-eat products meat products, 508, 513, 514–515, 523–527, 534–538 poultry products, 586–587, 590–592, 597–599 risk assessment, regulatory control, and economic impact, 783 REB, see Repetitive element-based (REB) typing Rebagged products, 755 Recalls fermented products, 407–419 meat products, 507–508, 509–513, 514, 516 poultry products, 574, 575–576 unfermented products, 370, 371–373

DK3089_C020.fm Page 867 Tuesday, February 20, 2007 7:13 PM

Index Reclaimed and reworked products, 753 Recombinant bateriophage, 264 Record-keeping procedures, 718–719 Recovering injured Listeria, 244–248 Reduction, 333 Reglier-Poupet studies, 125 Regulation, gene expression, 136 Regulations, see also Risk assessment, regulatory control, and economic impact fish and seafood products, 634–635 tolerance, 836–837 Reichel, Gill and, studies, 548 Reindeer, 70 Repair practices, 693, 693–697 Repetitive element-based (REB) typing, 294 Research advances, 244 Research needs academic perspective, 830–835 behavior in foods, 817 consumer education, 839–840 consumer exposure, 816–817, 821–827 contamination prevention, 821–823 control options, 837–838 deli meats, 838 destruction, 818–819 detection, 818 elimination, 818–819 enumeration, 818 FAO/WHO risk assessment significance, 819–820 FDA/FSIS risk assessment significance, 819–820 FDA perspective, 815–819 food safety perspective, 835–840 foods of concern, 839 fundamentals, 814–815 gene expression, 833–834 genomic endowment, 831–833 growth prevention, 824–826 industry perspective, 819–827 labeling, 839–840 outbreaks vs. sporadic cases, 820–821 pathogenicity, 817–818 postpasteurization, 823–824 processed food risks, 838–839 proteomic endowment, 831–833 regulatory tolerance, 836–837 retail product focus, 838–839 risk assessment significance, 819–820 Restriction endonuclease analysis (REA), 287–288 Restriction fragment length polymorphism, 288–291, 289–290 Retail product focus, 838–839 Rho studies, 523 Ribeiro, Morris and, studies, 543, 586 Ribeiro and Carminati studies, 444 Ribotyping, 288–290, 288–291 Rice salad, 37, 338 Richard, Siswanto and, studies, 382 Richard studies, 452 Richter, Al-Sheddy and, studies, 579 Ridley studies, 290 Riedo studies, 337 Rijpens and Herman studies, 230

867 Rijpens studies, 574, 587 Rillettes epidemic listeriosis, 97 fluid milk similarity, 317 foodborne infection, 330–331 shelf life, 332 Ripabelli studies, 294 Risk assessment fish and seafood products, 635–636 plant origin products, 656–657, 657 significance, 819–820 Risk assessment, regulatory control, and economic impact Australia, 773–774, 791–793 Austria, 796 Canada, 775, 794–796, 795 characterization of risk, 785–789, 786–788 China, 775 Codex Alimentarius Commission, 783–790 consumption, 784 costs, 801–807, 802–803, 805–806 criteria and standards, 790–807 Denmark, 796–797 dose-response assessment, 784–785 European countries, 796–799 European Union, 799–800, 800 France, 775–776, 797 fundamentals, 768–771 Germany, 777, 797 growth, 784 Hong Kong, 775 initial contamination, 783–784 internationally, 781–790, 800–801 Italy, 798 Netherlands, 777, 798 New Zealand, 791–793 North America, 793–796 process, 771–773 ready-to-eat foods, 783 Slovenia, 798 standards and criteria, 790–807 summary, 790 Sweden, 777–778 United Kingdom, 798–799 United States, 778–781, 782, 793–794 Rivera-Betancourt studies, 724 Roast beef, 535, 755, see also Beef Robbins studies, 129 Robertson studies, 527, 661 Robinson, Higgins and, studies, 10 Robinson studies, 160 Rocourt, Bille and, studies, 284, 297 Rocourt studies, 1–12, 285–286 Rodriguez-Saona studies, 274 Rodriguez studies, 222–223, 225, 231, 469 Roe deer, 70 Romania, 71 Rooks, 41 Rørvik studies, 631, 637, 640 Rosenow and Marth studies, 381, 383–385, 392 Roth and Donnelly studies, 248 rRNA sequencing, 4

DK3089_C020.fm Page 868 Tuesday, February 20, 2007 7:13 PM

868 Rudi studies, 296 Russell studies, 600 Russia, 66, 72 Rusul studies, 585 Ryser, Issa and, studies, 446 Ryser, Mathew and, studies, 437 Ryser and Marth studies brick cheese, 455–456 brine solutions, 484–485 Camembert cheese, 449–451 cheddar cheese, 459, 461 cold enrichment, 217–218 cold-pack cheese food, 476 International Dairy Federation method, 238 lithium chloride/phenylethanol, 221 selective enrichment and plating, 219 survival in cheddar cheese, 413 whey, 482–483 Ryser studies cold enrichment, 217 current characterization methods limitations, 24 fermented dairy products, 405–486 injured recovery, 246 poultry and egg products, 571–606 RiboPrinter, 290 unfermented dairy products, 357–394 Ryu studies, 522, 662

S Salamah studies, 662 Salcedo studies, 296 Sallam and Donnelly studies, 245 Salmon, 165 Saltijeral studies, 435 Salts, 174–178 Salvat studies, 743 Samelis and Metaxopoulos studies, 535 Samelis studies, 475, 547 Sampling plans, 713–715, 714 Sandine, Benkerroum and, studies, 474 San Francisco, 98 Sanitation, control method, 707–710 Sanitizers acid type, 194 calcinated calcium solution, 196 carvacrol, 196 chitosan, 196 chlorine and chlorinated compounds, 193–194 chlorohexidine, 196 fundamentals, 192–193 glutaraldehyde, 196 hydrogen peroxide, 196 iodophors, 195–196 listeriophages, 196 medicated soap, 196 monolaurin, 196 ozone, 194–195, 195 peptone water, 196 quaternary ammonium compounds, 194

Listeria, Listeriosis, and Food Safety sodium dichloroisocyanurate, 196 Ultra-Kleen, 196 Sarumehmetoglu and Kaymaz studies, 467 Sauders studies, 21–44 Saudi Arabia, 59 Saunders studies, 290 Sausages cooked smoked sausage, 540–542, 542 dry fermented sausage, 545–547 fermented sausage, 544, 558–559 fraser broth, 227 fresh sausage, 538–540, 539–540 fundamentals, 538–547, 549 incidence, 523–527 modified-atmosphere packaging, 549 plant extracts, 181–182 semidry fermented sausage, 544–545 smoked sausage, 540–543, 542 uncooked smoked sausage, 543 unfermented sausage, 538–547 Scandinavia, 61 Schaack and Marth studies, 436–439, 441–443, 446, 471 Schabinski, Urbach and, studies, 601, 604 Schaffer studies, 461 Schlech studies, 671 Schmidt, Kaya and, studies, 531 Schmidt, Marshall and, studies, 383, 389 Schmidt and Nyfeldt studies, 63 Schoeni, Doyle and, studies, 223–224, 226, 229, 238 Schönberg, Lehmann and, studies, 285 Schönberg studies, 284, 430 Schöpfer, Breer and, studies, 518 Schultz studies, 2, 64 Schuman, Sheldon and, studies, 603 Schwarte, Biester and, studies, 66, 217 Schwartz and Cantor studies, 291 Scotland, 31 Scott, Jenny, 827 Scott studies, 806 Seafood products, see Fish and seafood products Seeliger, Donker-Voet and, studies, 6 Seeliger, H.P.R., 5 Seeliger, Weiss and, studies, 28 Seeliger studies, 6, 221, 407, 814 Selective enrichment and plating acriflavine, 221–222 agents, 219–223, 220 caftazidime, 223 fundamentals, 219 lithium chloride, 220–221 moxalactam, 223 nalidixic acid, 221 phenylethanol, 220–221 polymyxin B, 222–223 potassium tellurite, 219–220 potassium thiocyanate, 222 thallous acetate, 222 trypaflavine, 221–222 Selective enrichment media ARS-MMLA, 229–230 blood-free plating, 229

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Index comparative evaluation, 230–232 Food and Drug Administration method, 234–238, 236–237 Fraser broth, 226–227 fundamentals, 223–225 gum base nalidixic acid medium, 229 β-hemolysis, 233–234 incubation conditions, 232 International Dairy Federation method, 238–239, 239 isolation media, 227–232 LPM agar, 228 McBride Listeria agar, 227–228 modified Oxford agar, 228 Netherlands Government Food Inspective Service, 243 oblique illumination, 232–234, 233 official methods, 234–243 Oxford agar, 228 PALCAM agar, 228–229 USDA-FSIS method, 239–243, 240 UVM broth, 225–226 Selective media, 223–243 Seman studies, 539, 825 Semidry fermented sausage, 544–545 Semihard soft cheese, 314–315 Semisoft cheeses, 457–463 Separators, 750 Serotyping historical developments, 6 subtyping, 284–285 Seskin, Landefeld and, studies, 803 Sewage, 30–31 Sexual transmission, 100 Shamsuzzaman studies, 590 Sheehan studies, 134 Sheep, see also Ewe’s milk; Lambs animal feeds, 33–34 animal listeriosis, 57–60 fecal material, 41 soil contamination, 30 water contamination, 32 Sheldon and Schuman studies, 603 Shelef, Ferguson and, studies, 675 Shelef, Sionkowski and, studies, 602 Shelef and Yang studies, 589 Shelef studies, 531 Shellfish, 630, see also Fish and seafood products Sheref, Peng and, studies, 244 Sheridan studies, 516, 549 Shineman and Harrison studies, 639 Shrimp, see also Fish and seafood products fatty acid monoesters, 178 febrile gastroenteritis, 337–338 water contamination, 32 Sielaff studies, 530 Sikes studies, 444 Silk studies, 246 Silliker and Gabis studies, 721 Sindoni studies, 56 Sionkowski and Shelef studies, 602 Sipka studies, 468 Siragusa, Cutter and, studies, 559 Siragusa and Johnson studies, 223

869 Siswanto and Richard studies, 382 Sizmur and Walker studies, 660, 666 Skovgaard and Morgen studies, 226, 584 Skovgaard studies, 67 Skunks, 41 Slabospits’ Kii studies, 66 Slade and Collins-Thompson studies, 222, 224–225, 362 Slade studies, 362 Slavchev studies, 393 Sleath, Watkins and, studies, 23, 30–31 Slovenia, 798 Slutsker studies, 85–102 Smoke, 181 Smoked fish products fish and seafood products, 630–631 processing plants, 38–39 Smoked mussels, 334 Smoked-salmon processing facilities, 743 Smoked sausage, 540–543, 542 Sodium benzoate, 175, 178 Sodium diacetate, 175, 176–177 Sodium dichloroisocyanurate, 196 Sodium nitrite, 180 Sodium propionate, 175, 177 Soft, unripened Mexican-style cheese, 310–312, see also Mexican-style cheese Soft cheese epidemic listeriosis, 96 FDA method, 238 human outbreaks, 37 PALCAM agar, 229 Soft Italian cheese, 457 Soft mold-ripened cheeses, 230 Soft unripened cheese, 471–474 Soil, 28, 29, 30 Solano-Lopez and Hernandez-Sanchez studies, 464–465 Sontakke and Farber studies, 291 Soonthoranant and Garland studies, 32 Soriano studies, 522, 661 Soultos studies, 581 Sows, see Swine Spahr, Bachmann and, studies, 456 Spain, 745 Specialty items, 543–544 Species identification, 10–12 Specific species vs. generic species, 259 Spectroscopic methods, 271–274, 273 Spices, 181–182, 182 Sporadic human cases dietary risk factors, 98–100 foodborne infection, 344–348 incidence, 97–98, 99 transmission dynamics, 38–40 Stajner studies, 441, 471 Standards and criteria, 790–807 Stanley, Foegeding and, studies, 606 Starlings, 41 Starter cultures, 436–445 Starter distillates, 448–449 Stecchini studies, 457

DK3089_C020.fm Page 870 Tuesday, February 20, 2007 7:13 PM

870 Stegeman studies, 534 Steinbruegge studies, 664 Stilton cheese, 186 Strain, resistance to heat, 167–168 Strangeways, Bendig and, studies, 661 Stress ecology, 27–35 elevated sublethal temperatures, 163 response mediators, 131–132 Strikwerda, de Vries and, studies, 63 Stuart and Welshimer studies, 6–7 Study methods, ecology, 22–27 Subtyping amplified fragment length polymorphism, 294–295 antimicrobial susceptibility testing, 286 bacteriocin typing, 286 bacteriophage typing, 285–286 chromosomal DNA restriction endonuclease analysis, 287–288 comparison of methods, 297, 298 conventional methods, 284–286 DNA macrorestriction analysis, 291–292, 293 DNA-sequence-based strategies, 295–297 ecology study methods, 25–27, 26 fundamentals, 283–284 methods comparison, 297, 298 molecular methods, 287–297 multilocus enzyme electrophoresis, 287 PFGE, 291–292, 293 phage typing, 285–286 random amplification of polymorphic DNA, 292–294 repetitive element-based subtyping, 294 restriction fragment length polymorphism, 288–291, 289–290 ribotyping, 288–290, 288–291 serotyping, 284–285 Sudanese white-pickled cheese, 467–468 Sulzer and Busse studies, 452 Sumner and Gallagher studies, 773 Superoxide dismutase, 131 Surendran, Fuchs and, studies, 630 Surrogate microorganisms, 168 Surveillance, see also Monitoring programs Canada, 419–421, 420 domestic cheese, 407–414 FDA method, 234 foodborne infection, 344–349, 346–347 France, 414–418, 421, 428 Germany, 428–430, 429 imported cheese, 414–419 Italy, 430 non-United States programs, 419–435 Switzerland, 430–432, 431 Western European countries, 418–419 Surveys, 618–632 Survival enrichment and isolation methods, 22 fish and seafood products, 638–640 high salt concentration, 174 low pH, 169 meat products, 530–533

Listeria, Listeriosis, and Food Safety plant origin products, 663–666, 664 stress conditions, 27–35 water activity, 171–172, 172 Sutherland and Porritt studies, 748 Swaminathan studies, 283–297 Swann, Murray, Web and, studies, 1 Sweden cold-smoked fish products, 334 foodborne infection, 342–343 food processing, facilities, 746–747 risk assessment, 777–778 transmission to animals, 63 Sweetened condensed milk, 387 Swine, 66–67, see also Pigs Swiss cheese, 462 Switzerland epidemic listeriosis, 96 fermented dairy products, 430–432, 431 foodborne infection, 312–313 food processing, facilities, 747 listeriosis outbreak, 329 monitoring programs, 430–432, 431 transmission to animals, 62, 71 Sword and Pickett studies, 286

T Tahon-Castel, Beerens and, studies, 221 Taleggio cheese, 456 Taormina and Beuchat studies, 556 Tasmania, smoked mussels, 334 Tatini, Mehta and, studies, 461 Tawfik studies, 467 Taxonomy, 5–9, see also Phylogenetic position Temperature cold tolerance, 161–163, 162 culture, 9 D-value, 165–167 elevated sublethal temperature, 163 engulfment, 167 extrinsic factors, 167 food composition, 167 freezing, 163–164 growth, 159–163 heat resistance, 167 intrinsic factors, 167–168 kinetics, thermal inactivation, 165–167 kinetics of growth, 160–161, 161 lethal temperature, 164–168 measurement, thermal inactivation kinetics, 165 mechanisms, thermal inactivation, 164–165 optimum, 159–160 physiological state, 168 range, 159–160 strain, 167–168 stress adaptation, 163 surrogate microorganisms, 168 thermal inactivation, 164–167 virulence variations, 163 z-value, 165–167 Temperature-dependent virulence, gene expression, 134–136

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Index Tennessee (United States) deli meat and frankfurters, 321 transmission to animals, 64 Ternstrom and Molin studies, 584 Terplan studies, 363, 450, 455, 483, 743 Texas (United States) Mexican-style cheese, 316 vegetables, 336 Thallous acetate, 222 Tham studies, 469 Thermal inactivation egg products, 604–606 kinetics, 165–167 meat products, 552–557, 553–556 mechanisms, 164–165 poultry products, 593–597, 594–596 Thermophilic starter cultures, 441–442, 441–443 Thimothe studies, 734, 737 Thompson, Daryl, 647 Thunberg studies, 660 Ticks, 41 Tilney and Portnoy studies, 126 Tilsiter cheese, 456 Tipparaju studies, 444 Tissue localization, 528–530 Todd studies, 767–807 Todorov, Ikonomov and, studies, 467–468 Todorov studies, 379 Tolerance, acid, 170–171 Tompkin studies, 690, 727, 819–827 Traditional methods control approaches, 704–710 enrichment, 22–23 fermented milks, 445–446 rapid method detection, 258 Traffic patterns, 710 Transcripts, 134–136 Transmission, to animals cattle, 62–66 crustaceans, 71–72 ecology, 40–42 fish, 71–72 fowl, 67–69 fundamentals, 55 goats, 60–62 incidence, 56 minor species, 69–71 predisposing factors, 56–57 sheep, 57–60 swine, 66–67 transmission dynamics, 40–42 treatment, 72 Transmission dynamics animals listeriosis, 40–42 food-processing environments, 36 global pathways and reservoirs, 42–44 human listeriosis, 35–40 human outbreaks, 37–38 modes, fish and seafood products, 636–638

871 within natural environments, 42 sporadic human cases, 38–40 Trappist cheese, 456 Treatment animal listeriosis, 72 human listeriosis, 101 Truscott and McNab studies, 227 Trüssel and Jemmi studies, 543, 545 Trypaflavine, 221–222 Tsigarda studies, 548 Tsiotsias studies, 475 Turkey and turkey products, see also Poultry products asymptomatic carriers, 67 dietary risk factors, 99–100 epidemic listeriosis, 96–97 fecal material, 41 human outbreaks, 37 organic acids and salts, 176–177 potassium sorbate, 177–178 Turkish white-brined cheese, 467 Turtles, 41 Two-component systems, 136

U Ukuku and Fett studies, 673 Ultrafiltered milk, 388 Ultra-Kleen, 196 Ultraviolet radiation, 188–189 Uncooked smoked sausage, 543 Unfermented dairy products, see also Dairy products autoclaved milk, 380–387 behavior, 376–388 butter, 392–393 chocolate milk, 380–387 cream, 380–387 evaporated milk, 387 fundamentals, 357–358 ice cream, 391–392 incidence, 358–376 intensively pasteurized milk, 379–380 mixed cultures, 388–389, 390 nonfat dry milk, 393–394 nonfluid dairy products, 391–394 other products, 367–376 pasteurized milk, 367–376, 379–380 raw cow’s milk, 358–366 raw ewe’s milk, 366, 366–367 raw goats milk, 366, 366–367 raw milk, 377–379, 378–380 sweetened condensed milk, 387 ultrafiltered milk, 388 Unfermented sausage, 538–547 United Kingdom asymptomatic carriage, 93 fluid milk, 317 food processing, facilities, 742–743 poultry products, 580–584, 582–583 predisposing factors, 57 standards and criteria, 798–799 water contamination, 31

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872 United States alternative processing technologies, 187 animal feeds, 33 asymptomatic carriage, 91 cheese and fermented products, 407–419 chocolate milk, 338–339 epidemic listeriosis, 96 foodborne infection, 321–327 food processing, facilities, 719–738 frankfurters and deli meat, 321–327 human outbreaks, 37 meningitis relationship, 88–89 neonatal disease, 88 paté, 328 plant origin products, 658–660 poultry products, 577–579 predisposing factors, 56 prevention, 101 ready-to-eat meat and poultry products, 341–342 risk assessment, 778–781, 782 shrimp, 337–338 sporadic incidence, 98 standards and criteria, 793–794 surveillance, 344–349, 407–419 thermal inactivation, 165 transmission to animals, 63–64, 66 ultraviolet radiation, 188 University of Vermont (UVM) broth detection and isolation, 225–226 enrichment and isolation, 22 Unnerstad studies, 297 Unpasteurized cheese, 96, see also specific type Urbach and Schabinski studies, 601, 604 USDA control guidelines, 597–599 USDA-FSIS detection and isolation method, 239–243, 240 monitoring program, meat products, 504–508 testing program, poultry products, 572–574, 574 zero tolerance, 321 UVM, see University of Vermont (UVM) broth Uyttendaele studies, 522, 581, 585

V Vacherin Mont d’Or, 312–313 Valenti, Comi and, studies, 463 Validation, HACCP, 718 Vanderlinde, Grau and, studies, 527, 532, 535 Van Laack studies, 536 van Netten, De Boer and, studies, 543 van Netten studies, 223, 227–229, 661 Van Nierop studies, 585 van Renterghem studies, 23, 663 Van Schothorst studies, 777 Varabioff studies, 527 Various environments, ecology, 27 Vasquez-Salinas studies, 364 Vazquez-Boland, Kreft and, studies, 136 Vazquez-Boland studies, 24 VBNC, see Viable but not culturable (VBNC) state Vegetables, 335–336, see also Raw vegetables

Listeria, Listeriosis, and Food Safety Vegetables, processing facilities control methods, 758–759 Spain, 745 United States, 738 Vegetation, 28, 29, 30 Vela studies, 41 Venables studies, 435 Venkitanarayanan studies, 673 Verification procedures, HACCP, 718 Viable but not culturable (VBNC) state, 24 Virulence variations, 163 Vogel studies, 631 Voles, 41 von Holy, Mosupye and, studies, 662 Vorster studies, 527 Vorst studies, 576 Vos studies, 294

W Waak studies, 746 Wadsworth, Goldfine and, studies, 119 Wales epidemic listeriosis, 97 foodborne infection, 327 listeriosis outbreak, 327, 330 neonatal disease, 88 sporadic incidence, 98 Walker, Sizmur and, studies, 660, 666 Walker studies, 592, 722 Wang and Muriana studies, 523 Wang studies, 522 Wan studies, 452 Warburton studies, 243–244 Warriner studies, 530 Washington (United States), 315, 317 Water, survival in stress conditions, 31–32 Water activity food processing importance, 171–173 growth, 171–172, 172 osmotolerance factors, 172–173, 172–173 survival, 171–172, 172 Watkins and Sleath studies, 23, 30–31 Weagant studies, 620 Webb and Swann, Murray, studies, 1 Wederquist studies, 591 Weiner, Newman and, studies, 135 Weiss and Seeliger studies, 28 Weis studies, 23 Wekell studies, 617–646 Welshimer, Groves and, studies, 6 Welshimer, H.J., 5 Welshimer, Stuart and, studies, 6–7 Welshimer and Donker-Voet studies, 28 Welshimer studies, 30 Wendorff studies, 542 Wenzel and Marth studies, 377 Wernars studies, 293 Wesley and Ashton studies, 37 Wesley studies, 55–72, 579 Western European countries, 418–419

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Index Westöö, Lindqvist and, studies, 777–778 Wet processing areas, cleaning, 707–709 Whey cheese and fermented dairy products, 474–477, 482, 482–483 pulsed electric field processing, 190 Whittenbury studies, 28 Whole genome sequencing, 4, 5 Wiedmann, Hoffman and, studies, 242 Wiedmann studies, 21–44, 290 Wilkinson and Jones studies, 3 Williams, Palumbo and, studies, 577 Williams, T., 139 Wilson and Miles studies, 2 Wilson studies, 516 Wimpfheimer studies, 550, 577, 587–588 Wolyniak, Cecilia, 647 Wong and Kitts studies, 600 Wood and Woodbine studies, 163 Woodbine, Wood and, studies, 163 Wramby studies, 63

X Xenos, Karaioannoglou and, studies, 552

873

Y Yakima County, 317 Yang, Shelef and, studies, 589 Yan studies, 688, 690 Yen studies, 552 Yogurt, 443–444 Yousef and Marth studies, 221, 458, 462, 481 Yousef studies, 157–198, 449, 461, 557 Yugoslavia, 67 Yugoslavian white-pickled cheese, 468

Z Zaika and Fanelli studies, 160 Zaika studies, 541 Zeitoun and Debevere studies, 588 Zhang and Farber studies, 668 Zhu studies, 599 Zottola, Dean and, studies, 391 Zottola, Herald and, studies, 529 Zuniga-Estrada studies, 443 z-value, 165–167

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