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.

REFERENCES 1. Allerberger, F. J., A. Schulz, and M. P. Dierich. 1988. Microcalorimetric investigations on Listeria. Zbl. Bakteriol. Hyg. A 268:15–23. 2. Bannerman, E., M. N. Yersin, and J. Bille. l992. Evaluation of the Organon–Teknika MICRO-ID Listeria system. Appl. Environ. Microbiol. 58:2011–2015. 3. Bergey’s manual of determinative bacteriology, 4th ed. 1934. Ed. D. H. Bergey. Baltimore: Williams & Wilkins Co. 4. Bergey’s manual of determinative bacteriology, 6th ed. 1948. Eds. R. D. Breed, E. G. D. Murray, and A. P. Hitchens. Baltimore: Williams & Wilkins Co. 5. Bergey’s manual of determinative bacteriology, 7th ed. 1957. Eds. R. D. Breed, E. G. D. Murray, and N. R. Smith. Baltimore: Williams & Wilkins Co. 6. Bergey’s manual of determinative bacteriology, 8th ed. 1974. Eds. R. E. Buchanan and N. E. Gibbons. Baltimore: Williams & Wilkins Co. 7. Bergey’s manual of systematic bacteriology, vol. 2. 1986. Eds. P. H. A. Sneath, N. S. Mair, N. E. Sharpe, and J. G. Holt. Baltimore: Williams & Wilkins Co. 8. Beumer, R. R., M. C. T. Giffel, Kok, and F. M. Rombouts. 1996. Confirmation and identification of Listeria spp. Lett. Appl. Microbiol. 22:448–452.

DK3089_C001.fm Page 13 Tuesday, February 20, 2007 6:47 PM

The Genus Listeria and Listeria monocytogenes

13

9. Bille, J., B. Catimel, E. Bannerman, C. Jacquet, M. N. Yersin, I. Caniaux, D. Monget, and J. Rocourt. 1992. API Listeria, a new and promising one-day system to identify Listeria isolates. Appl. Environ. Microbiol. 58:1857–1860. 10. Boerlin, P., J. Rocourt, F. Grimont, P. A. D. Grimont, Ch. Jacquet, and J.-C. Piffaretti. l992. Listeria ivanovii subsp. londoniensis subsp. Int. J. Syst. Bacteriol. 15:42–46. 11. Boerlin, P., J. Rocourt, and J. C. Piffaretti. 1991. Taxonomy of the genus Listeria by using multilocus enzyme electrophoresis. Int. J. Syst. Bacteriol. 41:59–64. 12. Bosgiraud, C., A. Menudier, M. J. Cornuejols, N. Hangard-Vidaud, and J. A. Nicolas. 1989. Etude de la virulence de Listeria monocytogenes isolees d’aliments de I’homme. Microbiol. Alim. Nutr. 7:413–420. 13. Bousfield, I. 1972. A taxonomic study of some coryneform bacteria. J. Gen. Microbiol. 71:441–455. 14. Brosch, R., C. Buchrieser, and J. Rocourt. 1991. Subtyping of Listeria monocytogenes serovar 4b by use of low-frequency-cleavage restriction endonucleases and pulsed-field gel electrophoresis. Res. Microbiol. 142:667–675. 15. Brzin, B., and H. P. R. Seeliger. 1975. A brief note on the Camp phenomenon in Listeria. In Problems of listeriosis, ed. M. Woodbine. Leicester: University of Leicester, pp. 34–37. 16. Bubert, A., I. Hein, M. Rauch, A. Lehner, B. Yoon, W. Goebel, and M. Wagner. 1999. Detection and differentiation of Listeria spp. by a single reaction based on Multiplex. Appl. Environ. Microbiol. 65:4688–4692. 17. Buchrieser, C., C. Rusniok, the Listeria Consortium, F. Kunst, P. Cossart, and P. Glaser. 2003. Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua: Clues for evolution and pathogenicity. FEMS Immunol. Med. Microbiol. 35:207–213. 18. Chatelain, R., and L. Second. 1966. Taxonomie numerique de quelques Brevibacterium. Ann. Inst. Pasteur 111:630–644. 19. Christensen, D. P., and R. W. Hutkins. 1994. Glucose uptake by Listeria monocytogenes Scott A and inhibition by pediocin JD. Appl. Environ. Microbiol. 60:3870–3873. 20. Clark, A.G., and J. McLaughlin. 1997. Simple color tests based on an alanyl peptidase reaction which differentiate Listeria monocytogenes from other Listeria species. J. Clin. Microbiol. 35:2155–2156. 21. Cocolin, L., K. Rantsiou, K. L. Cantoni, and G. Comi. 2002. Direct identification in food samples of Listeria spp. and Listeria monocytogenes by molecular methods. Appl. Environ. Microbiol. 68:6273–6282. 22. Coffey, A., F. M. Rombouts, and T. Abee. 1996. Influence of environmental parameters on phosphatidylcholine phospholipase C production in Listeria monocytogenes: A convenient method to differentiate L. monocytogenes from other Listeria species. Appl. Environ. Microbiol. 62:1252–1256. 23. Cole, M. B., M. V. Jones, and C. Holyoak. 1990. The effect of pH, salt concentration and temperature on the survival and growth of Listeria monocytogenes. J. Appl. Bacteriol. 69:63–72. 24. Collins, M. D., D. Jones, M. Goodfellow, and D. E. Minnikin. 1979. Isoprenoid quinone composition as a guide to the classification of Listeria, Brochothrix, Erysipelothrix and Caryophanon. J. Gen. Microbiol. 111:453–457. 25. Collins, M. D., S. Feresu, and D. Jones. 1983. Cell wall, DMA base composition and lipid studies on Listeria denitrificans (Prevot). FEMS Microbiol. Lett. 18:131–134. 26. Collins, M. D., S. Wallbanks, D. J. Lane, J. Shah, R. Nietupski, J. Smida, M. Dorsch, and E. Stackebrandt. 1991. Phylogenic analysis of the genus Listeria based on reverse transcriptase sequencing of 16S rRNA. Int. J. Syst. Bacteriol. 41:240–246. 27. Conner, D. E., V. N. Scott, S. S. Summer, and D. T. Bernard. l989. Pathogenicity of foodborne, environmental and clinical isolates of Listeria monocytogenes in mice. J. Food Sci. 54:1553–1556. 28. Cotoni, L. 1942. A propos des bacteries denommees Listerella—rappel d’une observation ancienne de meningite chez l’homme. Ann. Inst. Pasteur 68:92–95. 29. Cowart, R. E., and B. G. Foster. 1985. Differential effects of iron in the growth of Listeria monocytogenes: Minimum requirements and mechanism of acquisition. J. Infect. Dis. 151:721–730. 30. Czajka, J., N. Bsat, M. Piani, W. Russ, K. Sultana, M. Wiedmann, R. Whitaker, and C. A. Batt. 1993. Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DMA polymorphisms. Appl. Environ. Microbiol. 59:304–308. 31. Da Silva, G. A. N., and J. G. Holt. 1965. Numerical taxonomy of certain coryneform bacteria. J. Bacteriol. 90:921–927.

DK3089_C001.fm Page 14 Tuesday, February 20, 2007 6:47 PM

14

Listeria, Listeriosis, and Food Safety 32. Daneshvar, M. I., J. B. Brooks, G. B. Malcolm, and L. Pine. 1989. Analyses of fermentation products of Listeria species by frequency-pulsed electron-capture gas-liquid chromatography. Can. J. Microbiol. 35:786–793. 33. Davis, G. H. G., L. Fomin, E. Wilson, and K. G. Newton. 1969. Numerical taxonomy of Listeria, streptococci and possibly related bacteria. J. Gen. Microbiol. 57:333–348. 34. Del Corral, F., R. L. Buchanan, M. M. Bencivengo, and P. H. Cooke. 1990. Quantitative comparison of selected virulence associated characteristics in food and clinical isolates of Listeria. J. Food Prot. 53:1003–1009. 35. Dominguez Rodriguez, L., J. A. Vasquez Boland, J. F. Fernandez-Garayzabal, P. Echalecu Tranchant, E. Gomez-Lucia, E. F. Rodriguez Ferri, and G. Suarez Fernandez. 1986. Microplate technique to determine hemolytic activity for routine typing of Listeria strains. J. Clin. Microbiol. 24:99–103. 36. Donker-Voet, J. 1972. Listeria monocytogenes: Some biochemical and serological aspects. Acta Microbiol. Acad. Sci. Hung. 19:287–291. 37. Doumith, M., C. Cazalet, N. Simoes, L. Frangeul, C. Jaquet, F. Kunst, P. Martin, P. Cossart, P. Glaser, and C. Buchrieser. 2004. New aspects regarding evolution and virulence of Listeria monocytogenes revealed by comparative genomics. Infect. Immun. 72:1072–1083. 38. Drancourt, M., C. Bollet, A. Carlioz, R. Martelin, J.-P. Gayral, and D. Raoult. 2000. 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J. Clin. Microbiol. 38:3623–3630. 39. Drebot, M., S. Neal, W. Schlech, and K. Rozee. 1996. Differentiation of Listeria isolates by PCR amplicon profiling and sequence analysis of 16S-23S rRNA internal transcribed spacer loci. J. Appl. Bacteriol. 80:174–178. 40. Farber, J. M., and C. J. Addison. 1994. RAPD typing for distinguishing species and strains in the genus Listeria. J. Appl. Bacteriol. 77:242–250. 41. Farber, J. M., F. Coates, and E. Daley. 1992. Minimum water activity requirements for the growth of Listeria monocytogenes. Lett. Appl. Microbiol. 15:103–105. 42. Farber, J. M., J. I. Speirs, R. Pontefract, and D. E. Conner. 1991. Characteristics of nonpathogenic strains of Listeria monocytogenes. Can. J. Microbiol. 37:647–650. 43. Feresu, S. B., and D. Jones. 1988. Taxonomic studies on Brochothrix, Erysipelothrix, Listeria and atypical lactobacilli. J. Gen. Microbiol. 134:1165–1183. 44. Fernandez-Garayzabal, J. F., G. Suarez, M. M. Blanco, A. Gibello, and L. Dominguez. l996. Taxonomic note: A proposal for reviewing the interpretation of the CAMP reaction between Listeria monocytogenes and Rhodococcus equi. Int. J. Syst. Bacteriol. 46:832–834. 45. Fernandez-Garayzabal, J. F., C. Delgado, M. M. Blanco, G. Suarez, and L. Dominguez. 1996. Cholesterol-oxidase from Rhodococcus equi is likely the major factor involved in the cooperative lytic process (CAMP reaction) with Listeria monocytogenes. Lett. Appl. Microbiol. 22:249–252. 46. Fiedler, F., and J. Seger. 1983. The murein types of Listeria grayi, Listeria murrayi, and Listeria denitrificans. Syst. Appl. Microbiol. 4:444–450. 47. Fiedler, F., J. Seger, A. Schrettenbrunner, and H. P. R. Seeliger. 1984. The biochemistry of murein and cell wall teichoic acids in the genus Listeria. Syst. Appl. Microbiol. 5:360–376. 48. Fraser, G. 1962. A plate method for the rapid identification of Listeria (Erysipelothrix) monocytogenes. Vet. Rec. 74:50–51. 49. Freney, J., S. Bland, J. Etienne, M. Desmonceaux, J. M. Boeufgras, and J. Fleurette. 1992. Description and evaluation of the semiautomated 4-hour rapid ID 32 strep method for identification of Streptococci and members of related genera. J. Clin. Microbiol. 30:2657–2661. 50. Fujisawa, T., and M. Mori. 1994. Evaluation of media for determining hemolytic activity and that of API Listeria system for identifying strains of Listeria monocytogenes. J. Clin. Microbiol. 32:1127–1129. 51. Galsworthy, S. B., S. Girdler, and S. F. Koval. 1990. Chemotaxis in Listeria monocytogenes. Acta Microbiol. Hung. 37:81–85. 52. Gavin, S. E., R. B. Leonard, A. M. Briselden, and M. B. Coyle. l992. Evaluation of the rapid CORYNE identification system for Corynebacterium species and other coryneforms. J. Clin. Microbiol. 30:1692–1695.

DK3089_C001.fm Page 15 Tuesday, February 20, 2007 6:47 PM

The Genus Listeria and Listeria monocytogenes

15

53. George, S. M., and B. M. Lund. 1992. The effect of culture medium and aeration on growth of Listeria monocytogenes at pH 4.5. Lett. Appl. Microbiol. 15:49–52. 54. Gibbons, N. E. 1972. Listeria pirie—whom does it honor? Int. J. Syst. Bacteriol. 22:1–3. 55. Gill, D. A. 1937. Ovine bacterial encephalitis (circling disease) and the bacterialgenus Listerella. Austral. Vet. J. 13:46–56. 56. Glaser, P., L. Frangeul, C. Buchrieser, C. Rusniok, F. Baquerd, P. Berce, H. Bloecker, P. Brandt, T. Chakraborty, A. Charbit, F. Cetouani, E. Couve, A. de Daruvar, P. Dehoux, E. Domann, G. Dominguez-Bernal, E. Duchaud, L. Durant, O. Dussurget, K. D. Entian, H. Fsihi, F. G. Portillo, P. Gerrido, L. Gautier, W. Goebe, N. Gomez-Lopez, T. Hain, J. Hauf, D. Jackson, L. M. Jones, U. Kaerst, J. Kreft, M. Kuhn, F. Kunst, G. Kurapkat, E. Madueno, A. Maitournam, J. M. Vicente, E. Ng, H. Nedjari, G. Nordsiek, S. Novella, B. de Pablos, J. C. Perez-Diaz, R. Purcell, B. Remmel, M. Rose, T. Schlueter, N. Simoes, A. Tierrez, J. A. Vazquez-Boland, H. Voss, J. Wehland, and P. Cossart. 2001. Comparative genetics of Listeria species. Science 294:849–852. 57. Gormon, T., and L. Phan-Thanh. 1995. Identification and classification of Listeria by two-dimensional protein mapping. Res. Microbiol. 146:143–154. 58. Gouin, E., J. Mengaud, and P. Cossart. 1994. The virulence gene cluster of Listeria monocytogenes is also present in Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species. Infect. Immunol. 62:3550–3553. 59. Graham, T., E. J. Golsteyn-Thomas, V. P. J. Gannon, and J. E. Thomas. 1996. Genus- and speciesspecific detection of Listeria monocytogenes using polymerase chain reaction assays targeting the 16S/23S intergenic spacer region of the rRNA operon. Can. J. Microbiol. 42:1155–1162. 60. Graham, T. A., E. J. Golsteyn-Thomas, J. E. Thomas, and V. P. J. Gannon. 1997. Inter- and intraspecies comparison of the 16-23S rRNA operon intergenic spacer regions of six Listeria spp. Int. J. Syst. Bacteriol. 47:863–869. 61. Gray, M. L. 1957. A rapid method for the detection of colonies of Listeria monocytogenes. Zbl. Bakteriol. Parasit. Infekt. Hyg. I Orig. 169:373–377. 62. Gray, M. L., and A. H. Killinger. 1966. Listeria monocytogenes and listeric infections. Bacteriol. Rev. 30:309–382. 63. Gray, M. L., H. J. Stafseth, and F. Thorp Jr. 1957. Colonial dissociation of Listeria monocytogenes. Zbl. Bakteriol. Parasit. Infekt. Hyg. I Orig. 169:378–392. 64. Gray, M., J. Stafseth, F. Thorp, L. B. Sholl, and W. F. Riley. 1948. A new technique for isolating Listerellae from the bovine brain. J. Bacteriol. 55:471–476. 65. Groves, R. D., and H. Welshimer. 1977. Separation of pathogenic from apathogenic Listeria monocytogenes by three in vitro reactions. J. Clin. Microbiol. 5:559–563. 66. Gutekunst, K. A., L. Pine, E. White, S. Kathariou, and G. M. Carlone. 1992. A filamentous-like mutant of Listeria monocytogenes with reduced expression of a 60-kilodalton extracellular protein invades and grows in 3T6 and Caco-2 cells. Can. J. Microbiol. 38:843–851. 67. Hartford, T., and P. H. A. Sneath. 1993. Optical DNA–DNA homology in the genus Listeria. Int. J. Syst. Bacteriol. 43:26–31. 68. Hether, N. W., and L. L. Jackson. 1983. Lipoteichoic acid from Listeria monocytogenes. J. Bacteriol. 156:809–817. 69. Higgins, D. L., and B. Robinson. 1993. Comparison of micro-ID Listeria method with conventional biochemical methods for identification of Listeria isolated from food and environmental samples: Collaborative study. J. AOAC 76:831–838. 70. Hof, H. 1984. Virulence of different strains of Listeria monocytogenes serovar 1/2. Med. Microbiol. Immunol. 173:207–218. 71. Holmberg, K., and H. O. Hollander. 1973. Numerical taxonomy and laboratory identification of Bacterionema matruchoti, Rothia dendocariosa, Actinomyces naeslundi, Actinomyces viscosus, and some related bacteria. J. Gen. Microbiol. 76:43–63. 72. 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. 73. Hülphers, G. 1911. Lefvernekros hos kanin orsakad af ej förut beskrifven bakterie. Svensk. Vet. Tidskrift. 2:265–273.

DK3089_C001.fm Page 16 Tuesday, February 20, 2007 6:47 PM

16

Listeria, Listeriosis, and Food Safety 74. Hunter, R. 1973. Observations on Listeria monocytogenes type 5 (Iwanow) isolated in New Zealand. Med. Lab. Technol. 30:51–56. 75. Ivanov, I. 1975. Establishment of non-motile strains of Listeria monocytogenes type 5. In Problems of listeriosis, ed. M. Woodbine. Leicester: University of Leicester, pp. 18–29. 76. Iwanow, I. 1962. Untersuchungen über die Listeriose der Schafe in Bulgarien. Mh. Vet. Med. 17:729–736. 77. Jacquet, C., S. Aubert, N. Elsolh, and J. Rocourt. 1992. Use of rRNA gene restriction patterns for the identification of Listeria species. Syst. Appl. Microbiol. 15:42–46. 78. Jersek, B., E. Tcherneva, N. Rijpens, and L. Herman. 1996. Repetitive element sequence-based PCR for species and strain discrimination in the genus Listeria. Lett. Appl. Microbiol. 23:55–60. 79. Jones, D. 1975. A numerical taxonomic study of coryneform and related bacteria. J. Gen. Microbiol. 87:52–96. 80. Jones, D., and H. P. R. Seeliger. 1983. Designation of a new type strain for Listeria monocytogenes—request for an opinion. Int. J. Syst. Bacteriol. 33:429. 81. Jones, D., M.D. Collins, M. Goodfellow, and D E. Minnikin. 1979. Chemical studies in the classification of the genus Listeria and possibly related bacteria. In Problems of listeriosis, ed. I. lvanov. Sofia: National Agroindustrial Union, Center for Scientific Information, pp. 17–23. 82. Jones, D., S. B. Feresu, and M. D. Gollins. 1986. Classification and identification of Listeria, Brochothrix and Erysipelothrix. In Listeriose, Listeria, listeriosis, eds. A. L. Courtieu, E. P. Espaze, and A. E. Reynaud. Nantes: Université de Nantes. 83. Jorgensen, F., P. J. Stephens, and S. Knoche. 1995. The effect of osmotic shock and subsequent adaptation on the thermotolerance and cell morphology of Listeria monocytogenes. J. Appl. Bacteriol. 79:274–281. 84. Julak, J., and M. Mara. 1973. Effect of glucose and glycerin in cultivation media on the fatty acid composition of Listeria monocytogenes. J. Hyg. Epidemiol. Microbiol. Immunol. 17:329–338. 85. Junttila, J. R., S. I. Niemelä, and J. Hirn. l988. Minimum growth temperatures of Listeria monocytogenes and non-haemolytic Listeria. J. Appl. Bacteriol. 65:321–327. 86. Kämpfer, P. 1992. Differentiation of Corynebacterium spp., Listeria spp., and related organisms by using fluorogenic substrates. J. Clin. Microbiol. 30:1067–1071. 87. Kämpfer, P., S. Böttcher, W. Dott, and H. Rüden. 1991. Physiological characterization and identification of Listeria species. Zbl. Bakteriol. 275:423–435. 88. Kämpfer, P., R. U. Dastis, and W. Dott. 1994. Characterization of Listeria by standardized cellular protein electrophoretic patterns. Syst. Appl. Microbiol. 17:211–215. 89. Kathariou, S., R. Kanenaka, R. D. Allen, A. K. Fok, and C. Mizumoto. 1995. Repression of motility and flagellin production at 37°C is stronger in Listeria monocytogenes than in the nonpathogenic species Listeria innocua. Can. J. Microbiol. 41:572–577. 90. Kathariou, S., and L. Pine. 1991. The type strain(s) of Listeria monocytogenes: A source of continuing difficulties. Int. J. Syst. Bacteriol. 41:328–330. 91. Kerr, K. G., P. M. Hawkey, and R. W. Lacey. 1993. Evaluation of the API Coryne System for identification of Listeria species. J. Clin. Microbiol. 31:749–750. 92. Kerr, K. G., N. A. Rotowa, P. M. Hawkey, and R. W. Lacey. 1990. Evaluation of the Mast ID and API 50CH systems for identification of Listeria spp. Appl. Environ. Microbiol. 56:657–660. 93. Kuhn, M., and W. Goebe. 1989. Identification of an extracellular protein of Listeria monocytogenes possibly involved in intracellular uptake by mammalian cells. Infect. Immunol. 57:55–61. 94. Lachica, R. V. 1996. Hemolytic activity reevaluation of putative nonpathogenic Listeria monocytogenes strains. Appl. Env. Microbiol. 62:4293–4295. 95. Lachica, R. V. 1990. Simplified Henry technique for initial recognition of Listeria colonies. Appl. Environ. Microbiol. 56:1164–1165. 96. Lammerding, A. M., K. A. Glass, A. Gendronfitzpatrick, and M. P. Doyle. 1992. Determination of virulence of different strains of Listeria monocytogenes and by Listeria innocua by oral inoculation of pregnant mice. Appl. Environ. Microbiol. 58:3991–4000. 97. Lament, R. J., D. T. Petrie, W. T. Melvin, and R. Postlethwaite. 1986. An investigation of the taxonomy of Listeria species by comparison of electrophoretic protein patterns. In Listeriose, Listeria, listeriosis, eds. A. L. Courtieu, E. P. Espaze, and A. E. Reynaud. Nantes: Université de Nantes, pp. 41–46. 98. Larsen, H. E., and H. P. R. Seeliger. 1966. A mannitol fermenting Listeria: Listeria grayi sp. In Proceedings of the third international symposium on listeriosis. Bilthoven, The Netherlands.

DK3089_C001.fm Page 17 Tuesday, February 20, 2007 6:47 PM

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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.

DK3089_C001.fm Page 18 Tuesday, February 20, 2007 6:47 PM

18

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124. Paterson, J. St. 1940. Studies on organisms of the genus Listerella. IV. An outbreak of abortion associated with the recovery of Listerella from the aborted fetuses. Vet. J. 96:1–6. 125. Petran, R. L., and E. A. Zpttola. 1989. A study of factors affecting growth and recovery of Listeria monocytogenes Scott A. J. Food Sci. 54:458–460. 126. Pine, L., S. Kathariou, F. Quinn, V. George, J. D. Wenger, and R. E. Weaver. 1991. Cytopathogenic effects in enterocytelike Caco-2 cells differentiate virulent from avirulent Listeria strains. J. Clin. Microbiol. 29:990–996. 127. 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. 128. Pine, L., R. E. Weaver, G. M. Carione, P. A. Pienta, J. Rocourt, W. Goebel, S. Kathariou, W. F. Bibb, and G. B. Malcolm. 1987. Listeria monocytogenes ATCC 35152 and NCTC 7973 contain a nonhemolytic, nonvirulent variant. J. Clin. Microbiol. 25:2247–2251. 129. Pirie, J. H. H. 1940. The genus Listerella pirie. Science 91:383. 130. Pirie, J. H. H. 1927. A new disease of veld rodents. “Tiger River disease.” S. Afr. Inst. Med. Res. 3:163–186. 131. Pontgratz, G., and H. P. R. Seeliger. 1984. Hämolysewirkungen durch Listeria innocua auf Schaferythrozyten. Zbl. Bakteriol. Hyg. 257:296–307. 132. Potel, J. 1951. Die Morphologie, Kultur und Tierpathogenität des Corynebacterium infantisepticum. Zbl. Bakt. Parasit. Infekt. Hyg. I Orig. 156:490–493. 133. Premaratne, R. J., W. J. Lin, and E. A. Johnson. 1991. Development of an improved chemically defined minimal medium for Listeria monocytogenes. Appl. Environ. Microbiol. 57:3046–3048. 134. Ralovich, B. S., M. Shahamat, and M. Woodbine. 1977. Further data on the characters of Listeria strains. Med. Microbiol. Immunol. 163:125–139. 135. Ralovich, B., M. Shahamat, and M. Woodbine. 1977. Further data on the characters of Listeria strains. Med. Microbiol. Immunol. 163:125–139. 136. Rainey, A., N. Weiss, and E. Stackebrand. 1995. Phylogenetic analysis of the genera Cellulomonas, Promicrospora, and Jonesia and proposal to exlude the genus Jonesia from the family Cellulomonadaceae. Int. J. Syst. Bacteriol. 45:649–652. 137. Ripio, M. T., C. Geoffroy, G. Dominguez, J. E. Alouf, and J. Vazquez-Boland. 1995. The sulphydrylactivated cytolysin and a sphingomyelinase C are the major membrane-damaging factors involved in cooperative (CAMP-like) hemolysis of Listeria spp. Res. Microbiol. 146:303–313. 138. Robinson, B. J., and C. P. Cunningham. 1991. Accuracy of micro-ID Listeria for identification of members of the genus Listeria. J. Food Prot. 54:798–800. 139. Rocourt, J., J. M. Alonso, and H. P. R. Seeligeri. 1983. Virulence comparée des cinq groupes génomiques de Listeria monocytogenes (sensu lato). Ann. Microbiol. (Inst. Pasteur) 134A:359–364. 140. Rocourt, J., P. Boerlin, F. Grimont, Ch. Jacquet, and J.-C. Piffaretti. 1992. Assignment of Listeria grayi and Listeria murrayi to a single species, Listeria grayi, with a revised description of Listeria grayi. Int. J. Syst. Bacteriol. 42:69–73. 141. Rocourt, J., and B. Catimel. 1985. Caracterization biochimique des especes du genre Listeria. Zbl. Bakteriol. Hyg. A. 260:221–231. 142. Rocourt, J., F. Grimont, P. A. D. Grionot, and H. P. R. Seeliger. 1982. DNA relatedness among serovars of Listeria monocytogenes sensu lato. Curr. Microbiol. 7:383–388. 143. Rocourt, J., and P. A. D. Grimont. 1983. Listeria welshimeri sp. nov. and Listeria seeligeri sp. nov. Int. J. Syst. Bacteriol. 33:866–869. 144. Rocourt, J., A. Schrettenbrunner, and H. P. R. Seeliger. 1983. Différentiation biochimique des groupes génomiques de Listeria monocytogenes (sensu lato). Ann. Microbiol. (Inst. Pasteur) 134A:65–71. 145. Rocourt, J., U. Wehmeyer, P. Cossart, and E. Stackebrandt. 1987. Proposal to retain Listeria murrayi and Listeria grayi in the genus Listeria. Int. J. Syst. Bacteriol. 37:298–300. 146. Rocourt, J., U. Wehmeyer, and E. Stackebrandt. 1987. Transfer of Listeria denitrificans to a new genus, Jonesia gen. nov., as Jonesia denitrificans comb, nov. Int. J. Syst. Bacteriol. 37:266–270. 147. Romick, T. L., H. P. Fleming, and R. F. McFeeters. 1996. Aerobic and anaerobic metabolism of Listeria monocytogenes in defined glucose medium. Appl. Environ. Microbiol. 62:304–307. 148. Ruhland, G. J., and F. Fiedler. 1987. Occurrence and biochemistry of lipoteichoic acids in the genus Listeria. Syst. Appl. Microbiol. 9:40–46. 149. Sallen, B., A. Rajoharison, S. Desvarenne, F. Quinn, and C. Mabilat. 1996. Comparative analysis of 16S and 23S rRNA sequences of Listeria species. Int. J. Syst. Bacteriol. 46:669–674.

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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.

DK3089_C001.fm Page 20 Tuesday, February 20, 2007 6:47 PM

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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.)

REFERENCES 1. Aarnisalo, K., T. Autio, A. M. Sjoberg, J. Lunden, H. Korkeala, and M. L. Suihko. 2003. Typing of Listeria monocytogenes isolates originating from the food processing industry with automated ribotyping and pulsed-field gel electrophoresis. J. Food Prot. 66:249–255. 2. Adams, C. J., T. E. Neff, and L. L. Jackson. 1979. Induction of Listeria monocytogenes infection by the consumption of ponderosa pine needles. Infect. Immun. 25:117–120. 3. al-Ghazali, M. R., and S. K. al-Azawi. 1988. Effects of sewage treatment on the removal of Listeria monocytogenes. J. Appl. Bacteriol. 65:203–208. 4. al-Ghazali, M. R., and S. K. al-Azawi. 1988. Storage effects of sewage sludge cake on the survival of Listeria monocytogenes. J. Appl. Bacteriol. 65:209–213. 5. Amtsberg, G. von. 1979. Epidemiology and diagnosis of Listeria infections. 1. Dtsch. Tierärztl. Wochenschr. 86:253–257. 6. Anonymous. 1999. Update: Multistate outbreak of listeriosis—United States, 1998–1999. MMWR 47:1117–1118. 7. Anonymous. 2000. Multistate outbreak of listeriosis—United States, 2000. MMWR 49:1129–1130. 8. Anonymous. 2002. Outbreak of listeriosis—northeastern United States, 2002. MMWR 51:950–951. 9. Antoniollo, P. C., S. Bandeira Fda, 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. 10. Arimi, S. M., E. T. Ryser, T. J. Pritchard, and C. W. Donnelly. 1997. Diversity of Listeria ribotypes recovered from dairy cattle, silage, and dairy processing environments. J. Food Prot. 60:811–816. 11. Armstrong, D. 1985. Listeria monocytogenes. In Principles and practices of infectious diseases. 2nd ed. eds. G. L. Mandell, R. G. Douglas, Jamie Robertson, and J. E. Bennett, New York: Wiley. 12. Arvanitidou, M., A. Papa, T. C. Constantinidis, V. Danielides, and V. Katsouyannopoulos. 1997. The occurrence of Listeria spp. and Salmonella spp. in surface waters. Microbiol. Res. 152:395–397. 13. Autio, T., J. Lunden, M. Fredriksson-Ahomaa, J. Bjorkroth, A. M. Sjoberg, and H. Korkeala. 2002. Similar Listeria monocytogenes pulsotypes detected in several foods originating from different sources. Int. J. Food Microbiol. 77:83–90. 13a. Barker, J., and M. R. Brown. 1994. Trojan horses of the microbial world: protozoa and the survival of bacterial pathogens in the environment. Microbiology 170:1253–1259. 14. Barnes, R., P. Archer, J. Strack, and G. R. Istre. 1989. Listeriosis associated with consumption of turkey franks. MMWR 38:267–268. 15. Basher, H. A., D. R. Fowler, F. G. Rodgers, A. Seaman, and M. Woodbine. 1984. Pathogenicity of natural and experimental listeriosis in newly hatched chicks. Res. Vet. Sci. 36:76–80. 16. Bauwens, L., F. Vercammen, and A. Hertsens. 2003. Detection of pathogenic Listeria spp. in zoo animal feces: use of immunomagnetic separation and a chromogenic isolation medium. Vet. Microbiol. 91:115–123. 17. Baxter, F., F. Wright, R. M. Chalmers, J. C. Low, and W. Donachie. 1993. Characterization by multilocus enzyme electrophoresis of Listeria monocytogenes isolates involved in ovine listeriosis outbreaks in Scotland from 1989 to 1991. Appl. Environ. Microbiol. 59:3126–3129.

DK3089_C002.fm Page 45 Tuesday, February 20, 2007 11:34 AM

Ecology of Listeria Species and L. monocytogenes in the Natural Environment

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18. Begley, M., C. G. Gahan, and C. Hill. 2002. Bile stress response in Listeria monocytogenes LO28: adaptation, cross-protection, and identification of genetic loci involved in bile resistance. Appl. Environ. Microbiol. 68:6005–6012. 19. Belding, R. C., and M. L. Mayer. 1959. Listeriosis in the turkey—two case reports. J. Am. Vet. Med. Assoc. 131:296–297. 20. Bernagozzi, M., F. Bianucci, R. Sacchetti, and P. Bisbini. 1994. Study of the prevalence of Listeria spp. in surface water. Zentralbl. Hyg. Umweltmed. 196:237–244. 21. Besnard, V., M. Federighi, E. Declerq, F. Jugiau, and J. M. Cappelier. 2002. Environmental and physico-chemical factors induce VBNC state in Listeria monocytogenes. Vet. Res. 33:359–370. 22. Bille, J. 1990. Epidemiology of human listeriosis in Europe, with special reference to the Swiss outbreak. In Foodborne listeriosis, eds. A. J. Miller, J. L. Smith, and G. A. Somkuti. New York: Elsevier. 23. Bille, J., J. Rocourt, and B. Swaminathan. 1999. Listeria, Erysipelothrix, and Kurthia. In Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C. 24. Boerlin, P., and J. C. Piffaretti. 1991. Typing of human, animal, food, and environmental isolates of Listeria monocytogenes by multilocus enzyme electrophoresis. Appl. Environ. Microbiol. 57:1624–1629. 25. Botzler, R. G., A. B. Cowan, and T. F. Wetzler. 1974. Survival of Listeria monocytogenes in soil and water. J. Wildl. Dis. 10:204–212. 26. Botzler, R. G., T. F. Wetzler, and A. B. Cowan. 1973. Listeria in aquatic animals. J. Wildl. Dis. 9:163–170. 27. Brosch, R., B. Catimel, G. Millon, C. Buchrieser, E. Vindel, and J. Rocourt. 1993. Virulence heterogeneity of Listeria monocytogenes strains from various sources (food, human, animal) in immunocompetent mice and its association with typing characteristics. J. Food Prot. 56:296–301. 28. Buchrieser, C., R. Brosch, B. Catimel, and J. Rocourt. 1993. Pulsed-field gel electrophoresis applied for comparing Listeria monocytogenes strains involved in outbreaks. Can. J. Microbiol. 39:395–401. 29. Buchrieser, C., C. Rusniok, F. Kunst, P. Cossart, and P. Glaser. 2003. Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua: Clues for evolution and pathogenicity. FEMS Immunol. Med. Microbiol. 35:207–213. 30. Cai, S., D. Y. Kabuki, A. Y. Kuaye, T. G. Cargioli, M. S. Chung, R. Nielsen, and M. Wiedmann. 2002. Rational design of DNA sequence-based strategies for subtyping Listeria monocytogenes. J. Clin. Microbiol. 40:3319–3325. 31. Capita, R., C. Alonso-Calleja, B. Moreno, and M. C. Garcia-Fernandez. 2001. Occurrence of Listeria species in retail poultry meat and comparison of a cultural/immunoassay for their detection. Int. J. Food Microbiol. 65:75–82. 32. Caro, M. R., E. Zamora, L. Leon, F. Cuello, J. Salinas, D. Megias, M. J. Cubero, and A. Contreras. 1990. Isolation and identification of Listeria monocytogenes in vegetable byproduct silages containing preservative additives and destined for animal feeding. Anim. Feed Sci. Technol. 31:285–291. 33. Chakraborty, T., F. Ebel, J. Wehland, J. Dufrenne, and S. Notermans. 1994. Naturally occurring virulence-attenuated isolates of Listeria monocytogenes capable of inducing long-term protection against infection by virulent strains of homologous and heterologous serotypes. FEMS Immunol. Med. Microbiol. 10:1–9. 34. Colburn, K. G., C. A. Kaysner, C. Abeyta, Jr., and M. M. Wekell. 1990. Listeria species in a California coast estuarine environment. Appl. Environ. Microbiol. 56:2007–2011. 35. Conte, M. P., C. Longhi, M. Polidoro, G. Petrone, V. Buonfiglio, S. Di Santo, E. Papi, L. Seganti, P. Visca, and P. Valenti. 1996. Iron availability affects entry of Listeria monocytogenes into the enterocytelike cell line Caco-2. Infect. Immun. 64:3925–3929. 36. Cook, L. V. 2003. Isolation and identification of Listeria monocytogenes from red meat, poultry, egg, and environmental samples. United States Department of Agriculture. http://www.fsis.usda.gov/ OPHS/microlab/mlgbook.htm (accessed 3/15/03). 37. Cox, L. J., T. Kleiss, J. L. Cordier, C. Cordellana, P. Konkel, C. Pedrazzini, R. Beumer, and A. Siebenga. 1989. Listeria spp. in food processing, nonfood and domestic environments. Food Microbiol. 6:49–61. 38. Cranfield, M., M. A. Eckhaus, B. A. Valentine, and J. D. Strandberg. 1985. Listeriosis in Angolan giraffes. J. Am. Vet. Med. Assoc. 187:1238–1240.

DK3089_C002.fm Page 46 Tuesday, February 20, 2007 11:34 AM

46

Listeria, Listeriosis, and Food Safety 39. Dahllof, I. 2002. Molecular community analysis of microbial diversity. Curr. Opin. Biotechnol. 13:213–217. 40. Dalton, C. B., C. C. Austin, J. Sobel, P. S. Hayes, W. F. Bibb, L. M. Graves, B. Swaminathan, M. E. Proctor, and P. M. Griffin. 1997. An outbreak of gastroenteritis and fever due to Listeria monocytogenes in milk. N. Engl. J. Med. 336:100–105. 41. De Luca, G., F. Zanetti, P. Fateh-Moghadm, and S. Stampi. 1998. Occurrence of Listeria monocytogenes in sewage sludge. Zentralbl. Hyg. Umweltmed. 201:269–277. 42. DeLong, E. F. 2002. Microbial population genomics and ecology. Curr. Opin. Microbiol. 5:520–524. 43. Destro, M. T., M. F. Leitao, and J. M. Farber. 1996. Use of molecular typing methods to trace the dissemination of Listeria monocytogenes in a shrimp processing plant. Appl. Environ. Microbiol. 62:705–711. 44. Dijkstra, R. G. 1971. Investigations on the survival times of Listeria bacteria in suspensions of brain tissue, silage and faeces and in milk. Zentralbl. Bakteriol. [Orig]. 216:92–95. 45. Dijkstra, R. G. 1975. The occurrence of Listeria encephalitis in cattle in a loose housing after the use of litter, infected with Listeria bacteria, from a broiler farm. To what extent do Listeria bacteria occur in the gut contents of broilers (author’s transl.)? Tijdschr. Diergeneeskd. 100:1154–1155. 46. Dijkstra, R. G. 1979. Listeria monocytogenes in intestinal contents and faeces from healthy broilers of different ages, in the litter and its potential danger for other animals including cattle. Proceedings of VII international symposium of problems in listeriosis, National Agroindustrial Union, Center for Scientific Information, Sofia, pp. 289–294. 47. Dijkstra, R. G. 1982. The occurrence of Listeria monocytogenes in surface water of canals and lakes, in ditches of one big polder and in the effluents and canals of a sewage treatment plant. Zentralbl. Bakteriol. Mikrobiol. Hyg. [B]. 176:202–205. 48. Dingle, K. E., F. M. Colles, D. R. Wareing, R. Ure, A. J. Fox, F. E. Bolton, H. J. Bootsma, R. J. Willems, R. Urwin, and M. C. Maiden. 2001. Multilocus sequence typing system for Campylobacter jejuni. J. Clin. Microbiol. 39:14–23. 49. Donachie, W., J. C. Low, F. Baxter, and F. Thompson-Carter. 1995. Typing of Listeria monocytogenes isolates with multilocus enzyme electrophoresis (MEE) and pulsed field gel electrophoresis (PFGE). Proceedings of XII international symposium on problems of listeriosis, Perth, Western Australia, Promaco Conventions Pty, Ltd., pp. 417–420. 50. Donald, A. S., D. R. Fenlon, and B. Seddon. 1995. The relationship between ecophysiology, indigenous microflora and growth of Listeria monocytogenes in grass silage. J. Appl. Bacteriol. 79:141–148. 51. Donnelly, C. W. 2002. Detection and isolation of Listeria monocytogenes from food samples: implications of sublethal injury. J. AOAC Int. 85:495–500. 52. Dutta, P. K., and B. S. Malik. 1981. Isolation and characterization of Listeria monocytogenes from animals and human beings. Indian J. Anim. Sci. 51:1045–1052. 53. Enright, M. C., K. Knox, D. Griffiths, D. W. Crook, and B. G. Spratt. 2000. Molecular typing of bacteria directly from cerebrospinal fluid. Eur. J. Clin. Microbiol. Infect. Dis. 19:627–630. 54. Enright, M. C., and B. G. Spratt. 1999. Multilocus sequence typing. Trends Microbiol. 7:482–487. 55. Fantelli, K., and R. Stephan. 2001. Prevalence and characteristics of shigatoxin-producing Escherichia coli and Listeria monocytogenes strains isolated from minced meat in Switzerland. Int. J. Food Microbiol. 70:63–69. 56. Farber, J. M., E. M. Daley, M. T. MacKie, and B. Limerick. 2000. A small outbreak of listeriosis potentially linked to the consumption of imitation crab meat. Lett. Appl. Microbiol. 31:100–104. 57. Farber, J. M., G. W. Sanders, and M. A. Johnston. 1989. A survey of various foods for the presence of Listeria species. J. Food Prot. 52:456–458. 58. Fenlon, D. R. 1985. Wild birds and silage as reservoirs of Listeria in the agricultural environment. J. Appl. Bacteriol. 59:537–543. 59. Fenlon, D.R. 1986. Growth of naturally occurring Listeria spp. in silage: A comparative study of laboratory and farm ensiled grass. Grass Forage Sci. 41:375–378. 60. Fenlon, D. R. 1986. Rapid quantitative assessment of the distribution of Listeria in silage implicated in a suspected outbreak of listeriosis in calves. Vet. Rec. 118:240–242. 61. Fenlon, D. R. 1988. Listeriosis. In Silage and health, 1st ed., eds. B. A. Stark and J. M. Wilkinson. Marlow, England: Chalcombe Publications. 62. Fenlon, D. R., T. Stewart, and W. Donachie. 1995. The incidence, numbers and types of Listeria monocytogenes isolated from farm bulk tank milks. Lett. Appl. Microbiol. 20:57–60.

DK3089_C002.fm Page 47 Tuesday, February 20, 2007 11:34 AM

Ecology of Listeria Species and L. monocytogenes in the Natural Environment

47

63. Fenlon, D. R., J. Wilson, and W. Donachie. 1996. The incidence and level of Listeria monocytogenes contamination of food sources at primary production and initial processing. J. Appl. Bacteriol. 81:641–650. 64. Fenlon, D. R., and J. R. Wilson. 1996. Enterobacteria as indicators of poor fermentation and Listeria contamination of silage. Proceedings of XI international silage conference, IGER, Aberystwyth, U.K., p. 103. 65. Fensterbank, R., A. Audurier, J. Godu, P. Guerrault, and N. Malo. 1984. Listeria strains isolated from sick animals and consumed silage. Ann. Rech. Vet. 15:113–118. 66. Finley, G. G., and J. R. Long. 1977. An epizootic of listeriosis in chinchillas. Can. Vet. J. 18:164–167. 67. Frances, N., H. Hornby, and P. R. Hunter. 1991. The isolation of Listeria species from fresh-water sites in Cheshire and North Wales. Epidemiol. Infect. 107:235–238. 68. Francis, M. S., and C. J. Thomas. 1996. Effect of multiplicity of infection on Listeria monocytogenes pathogenicity for HeLa and Caco-2 cell lines. J. Med. Microbiol. 45:323–330. 69. Frye, D. M., R. Zweig, J. Sturgeon, M. Tormey, M. LeCavalier, I. Lee, L. Lawani, and L. Mascola. 2002. An outbreak of febrile gastroenteritis associated with delicatessen meat contaminated with Listeria monocytogenes. Clin. Infect. Dis. 35:943–949. 70. Garcia, E., M. De Paz, J. L. Rodriguez, P. Gaya, M. Medina, and M. Nunez. 1996. Exogenous sources of Listeria contamination in raw ewe’s milk. J. Food Prot. 59:950–954. 71. Garrec, N., F. Picard-Bonnaud, and A. M. Pourcher. 2003. Occurrence of Listeria sp. and L. monocytogenes in sewage sludge used for land application: Effect of dewatering, liming and storage in tank on survival of Listeria species. FEMS Immunol. Med. Microbiol. 35:275–283. 72. Geuenich, H. H., and H. E. Muller. 1984. Isolation and germ count of Listeria monocytogenes in raw and biologically treated waste water. Zentralbl. Bakteriol. Mikrobiol. Hyg. (B). 179:266–273. 73. Gilbert, R. J., J. McLauchlin, and S. K. Velani. 1993. The contamination of pate by Listeria monocytogenes in England and Wales in 1989 and 1990. Epidemiol. Infect. 110:543–551. 74. Gitter, M. 1986. A changing pattern of ovine listeriosis in Great Britain. Proceedings of IX international symposium on problems of listeriosis, University of Nantes, Nantes, France, pp. 294–299. 75. Gitter, M., R. S. Stebbings, J. A. Morris, D. Hannam, and C. Harris. 1986. Relationship between ovine listeriosis and silage feeding. Vet. Rec. 118:207–208. 76. Glaser, P., L. Frangeul, C. Buchrieser, C. Rusniok, A. Amend, F. Baquero, P. Berche, H. Bloecker, P. Brandt, T. Chakraborty, A. Charbit, F. Chetouani, E. Couve, A. de Daruvar, P. Dehoux, E. Domann, G. Dominguez-Bernal, E. Duchaud, L. Durant, O. Dussurget, K. D. Entian, H. Fsihi, F. G. Portillo, P. Garrido, L. Gautier, W. Goebel, N. Gomez-Lopez, T. Hain, J. Hauf, D. Jackson, L. M. Jones, U. Kaerst, J. Kreft, M. Kuhn, F. Kunst, G. Kurapkat, E. Madueno, A. Maitournam, J. M. Vicente, E. Ng, H. Nedjari, G. Nordsiek, S. Novella, B. de Pablos, J. C. Perez-Diaz, R. Purcell, B. Remmel, M. Rose, T. Schlueter, N. Simoes, A. Tierrez, J. A. Vazquez-Boland, H. Voss, J. Wehland, and P. Cossart. 2001. Comparative genomics of Listeria species. Science 294:849–852. 77. 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. 78. Gouin, E., J. Mengaud, and P. Cossart. 1994. The virulence gene cluster of Listeria monocytogenes is also present in Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species. Infect. Immun. 62:3550–3553. 79. Graves, L. M., B. Swaminathan, and S. B. Hunter. 1999. Subtyping of Listeria monocytogenes. In Listeria, listeriosis, and food safety. 2nd ed. eds. E. T. Ryser and E. H. Marth, New York: Marcel Dekker, Inc., pp. 279–297. 80. Graves, L. M., B. Swaminathan, M. W. Reeves, S. B. Hunter, R. E. Weaver, B. D. Plikaytis, and A. Schuchat. 1994. Comparison of ribotyping and multilocus enzyme electrophoresis for subtyping of Listeria monocytogenes isolates. J. Clin. Microbiol. 32:2936–2943. 81. Gray, M. L. 1958. Listeriosis in fowls—a review. Avian Dis. 2:296–314. 82. Gray, M. L. 1960. Silage feeding and listeriosis. J. Am. Vet. Med. Assoc. 136:205–208. 83. Gray, M. L., and A. H. Killinger. 1966. Listeria monocytogenes and listeric infections. Bacteriol. Rev. 30:309–382. 84. Gray, M. L., C. Singh, and F. Thorp. 1956. Abortion and pre- or post-natal death of young due to Listeria monocytogenes. III. Studies in ruminants. Am. J. Vet. Res. 17:510–516. 85. Gray, M. L., H. J. Stafseth, F. Thorp, L. B. Scholl, and W. F. Riley. 1948. A new technique for isolating listerellae from the bovine brain. J. Bacteriol. 55:443–444.

DK3089_C002.fm Page 48 Tuesday, February 20, 2007 11:34 AM

48

Listeria, Listeriosis, and Food Safety

86. Grif, K., I. Hein, M. Wagner, E. Brandl, O. Mpamugo, J. McLauchlin, M. P. Dierich, and F. Allerberger. 2001. Prevalence and characterization of Listeria monocytogenes in the feces of healthy Austrians. Wien. Klin. Wochenschr. 113:737–742. 87. Grif, K., G. Patscheider, M. P. Dierich, and F. Allerberger. 2003. Incidence of fecal carriage of Listeria monocytogenes in three healthy volunteers: A one-year prospective stool survey. Eur. J. Clin. Microbiol. Infect. Dis. 22:16–20. 88. Grønstol, H. 1979. Listeriosis in sheep. Isolation of Listeria monocytogenes from grass silage. Acta Vet. Scand. 20:492–497. 89. Harvey, J., and A. Gilmour. 1994. Application of multilocus enzyme electrophoresis and restriction fragment length polymorphism analysis to the typing of Listeria monocytogenes strains isolated from raw milk, nondairy foods, and clinical and veterinary sources. Appl. Environ. Microbiol. 60:1547–1553. 90. Hatkin, J. M., W. E. Phillips, Jr., and G. A. Hurst. 1986. Isolation of Listeria monocytogenes from an eastern wild turkey. J. Wildl. Dis. 22:110–112. 91. Haydon, D. T., S. Cleaveland, L. H. Taylor, and M. K. Laurenson. 2002. Identifying reservoirs of infection: A conceptual and practical challenge. Emerg. Infect. Dis. 8:1468–1473. 92. Hayes, P. S., L. M. Graves, G. W. Ajello, B. Swaminathan, R. E. Weaver, J. D. Wenger, A. Schuchat, and C. V. Broome. 1991. Comparison of cold enrichment and U.S. Department of Agriculture methods for isolating Listeria monocytogenes from naturally contaminated foods. The Listeria Study Group. Appl. Environ. Microbiol. 57:2109–2113. 93. Hitchins, A. D., and R. C. Whiting. 2001. Food-borne Listeria monocytogenes risk assessment. Food Addit. Contam. 18:1108–1117. 94. Hofer, E. 1983. Bacteriologic and epidemiologic studies on the occurrence of Listeria monocytogenes in healthy cattle. Zentralbl. Bakteriol. Mikrobiol. Hyg. [A]. 256:175–183. 95. Hoffman, A. D., K. L. Gall, D. M. Norton, and M. Wiedmann. 2003. Listeria monocytogenes contamination patterns for the smoked fish processing environment and for raw fish. J. Food Prot. 66:52–60. 96. Hohne, K., B. Loose, and H. P. Seeliger. 1975. Isolation of Listeria monocytogenes in slaughter animals and bats of Togo (West Africa). Ann. Microbiol. (Paris). 126A:501–507. 97. Holah, J. T., J. H. Taylor, D. J. Dawson, and K. E. Hall. 2002. Biocide use in the food industry and the disinfectant resistance of persistent strains of Listeria monocytogenes and Escherichia coli. Symp. Ser. Soc. Appl. Microbiol. 111S–120S. 98. Huss, H. H., L. V. Jorgensen, and B. F. Vogel. 2000. Control options for Listeria monocytogenes in seafoods. Int. J. Food Microbiol. 62:267–274. 99. Husu, J. R. 1990. Epidemiological studies on the occurrence of Listeria monocytogenes in the feces of dairy cattle. Zentralbl. Veterinarmed. B. 37:276–282. 100. Husu, J. R., J. T. Beery, E. Nurmi, and M. P. Doyle. 1990. Fate of Listeria monocytogenes in orally dosed chicks. Int. J. Food Microbiol. 11:259–269. 101. Husu, J. R., J. T. Seppanen, S. K. Sivela, and A. L. Rauramaa. 1990. Contamination of raw milk by Listeria monocytogenes on dairy farms. Zentralbl. Veterinarmed. B. 37:268–275. 102. Husu, J. R., S. K. Sivela, and A. L. Rauramaa. 1990. Prevalence of Listeria species as related to chemical quality of farm-ensiled grass. Grass Forage Sci. 45:309–314. 103. 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. 104. Jacquet, C., B. Catimel, V. Goulet, A. Lepoutre, P. Veit, P. Dehaumont, and J. Rocourt. 1995. Typing of Listeria monocytogenes during epidemiological investigations of the French listeriosis outbreaks in 1992, 1993, and 1995. Proceedings of XII international symposium on problems of listeriosis, Perth, Western Australia, Promaco Conventions, pp. 61–176. 105. 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. 106. Jeffers, G. T., J. L. Bruce, P. L. McDonough, J. Scarlett, K. J. Boor, and M. Wiedmann. 2001. Comparative genetic characterization of Listeria monocytogenes isolates from human and animal listeriosis cases. Microbiology 147:1095–1104. 107. Jensen, L. B., Y. Agerso, and G. Sengelov. 2002. Presence of erm genes among macrolide-resistant Gram-positive bacteria isolated from Danish farm soil. Environ. Int. 28:487–491.

DK3089_C002.fm Page 49 Tuesday, February 20, 2007 11:34 AM

Ecology of Listeria Species and L. monocytogenes in the Natural Environment

49

108. Johnson, G. C., C. W. Maddox, W. H. Fales, W. A. Wolff, R. F. Randle, J. A. Ramos, H. Schwartz, K. M. Heise, A. L. Baetz, I. V. Wesley, and D. E. Wagner. 1996. Epidemiologic evaluation of encephalitic listeriosis in goats. J. Am. Vet. Med. Assoc. 208:1695–1699. 109. Kathariou, S. 2002. Listeria monocytogenes virulence and pathogenicity, a food safety perspective. J. Food Prot. 65:1811–1829. 110. Kikuhara, H., M. Ogawa, H. Miyamoto, Y. Nikaido, and S. Yoshida. 1994. Intracellular multiplication of Legionella pneumophila in Tetrahymena thermophila. J. Uoeh. 16:263–275. 111. Larsen, H. E. 1984. Investigation on the epidemiology of listeriosis—the distribution of Listeria monocytogenes in environments in which clinical outbreaks have not been diagnosed. Nord. Vet. Med. 16:890–909. 112. 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. 113. Lawrence, L. M., and A. Gilmour. 1995. Characterization of Listeria monocytogenes isolated from poultry products and from the poultry-processing environment by random amplification of polymorphic DNA and multilocus enzyme electrophoresis. Appl. Environ. Microbiol. 61:2139–2144. 114. Le Marc, Y., V. Huchet, C. M. Bourgeois, J. P. Guyonnet, P. Mafart, and D. Thuault. 2002. Modeling the growth kinetics of Listeria as a function of temperature, pH and organic acid concentration. Int. J. Food Microbiol. 73:219–237. 115. 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. 116. Linnan, M. J., L. Mascola, X. D. Lou, V. Goulet, S. May, C. Salminen, D. W. Hird, M. L. Yonekura, P. Hayes, R. Weaver, et al. 1988. Epidemic listeriosis associated with Mexican-style cheese. N. Engl. J. Med. 319:823–828. 117. Loken, T., E. Aspoy, and H. Gronstol. 1982. Listeria monocytogenes excretion and humoral immunity in goats in a herd with outbreaks of listeriosis and in a healthy herd. Acta Vet. Scand. 23:392–399. 118. Low, J. C., R. M. Chalmers, W. Donachie, R. Freeman, J. McLauchlin, and P. R. Sisson. 1992. Pyrolysis mass spectrometry of Listeria monocytogenes isolates from sheep. Res. Vet. Sci. 53:64–67. 119. Low, J. C., and W. Donachie. 1997. A review of Listeria monocytogenes and listeriosis. Vet. J. 153:9–29. 120. Low, J. C., W. Donachie, J. McLauchlin, and F. Wright. 1995. Characterization of Listeria monocytogenes strains from a farm environment. Proceedings of XII international symposium on problems of listeriosis, Perth, Western Australia, Promaco Conventions Pty, pp. 141–144. 121. Low, J. C., and C. P. Renton. 1985. Septicaemia, encephalitis and abortions in a housed flock of sheep caused by Listeria monocytogenes type 1/2. Vet. Rec. 116:147–150. 122. Lozniewski, A., A. Humbert, D. Corsaro, J. Schwartzbrod, M. Weber, and A. Le Faou. 2001. Comparison of sludge and clinical isolates of Listeria monocytogenes. Lett. Appl. Microbiol. 32:336–339. 123. Ly, T. M., and H. E. Muller. 1989. Heat tolerance of free living and of intracellular Listeria. Zentralbl. Hyg. Umweltmed. 189:272–276. 124. Ly, T. M., and H. E. Muller. 1990. Ingested Listeria monocytogenes survive and multiply in protozoa. J. Med. Microbiol. 33:51–54. 125. Lyytikainen, O., T. Autio, R. Maijala, P. Ruutu, T. Honkanen–Buzalski, M. Miettinen, M. Hatakka, J. Mikkola, V. J. Anttila, T. Johansson, L. Rantala, T. Aalto, H. Korkeala, and A. Siitonen. 2000. An outbreak of Listeria monocytogenes serotype 3a infections from butter in Finland. J. Infect. Dis. 181:1838–1841. 126. MacDonald, F., and A. D. Sutherland. 1994. Important differences between the generation times of Listeria monocytogenes and Listeria innocua in two Listeria enrichment broths. J. Dairy Res. 61:433–436. 127. MacGowan, A. P., K. Bowker, J. McLauchlin, P. M. Bennett, and D. S. Reeves. 1994. The occurrence and seasonal changes in the isolation of Listeria spp. in shop bought food stuffs, human feces, sewage and soil from urban sources. Int. J. Food. Microbiol. 21:325–334. 128. 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. 129. McDonald, P., A. R. Henderson, and S. J. E. Heron. 1991. The biochemistry of silage. Marlow, England: Chalcombe Publications. 130. McLauchlin, J. 1996. The relationship between Listeria and listeriosis. Food Control. 7.

DK3089_C002.fm Page 50 Tuesday, February 20, 2007 11:34 AM

50

Listeria, Listeriosis, and Food Safety

131. McLauchlin, J., and G. S. Nichols. 1994. Listeria and seafood. PHLS Microbiol. Dig. 11:151–154. 132. Miettinen, M. K., L. Palmu, K. J. Bjorkroth, and H. Korkeala. 2001. Prevalence of Listeria monocytogenes in broilers at the abattoir, processing plant, and retail level. J. Food Prot. 64:994–999. 133. Milohanic, E., P. Glaser, J. Y. Coppee, L. Frangeul, Y. Vega, J. A. Vazquez-Boland, F. Kunst, P. Cossart, and C. Buchrieser. 2003. Transcriptome analysis of Listeria monocytogenes identifies three groups of genes differently regulated by PrfA. Mol. Microbiol. 47:1613–1625. 133a. Mitscherlich, E., and E. H. Marth. 1984. Microbial Survival in the Environment—Bacteria and Rickettsiae Important in Human and Animal Health. Berlin: Springer-Verlag. 134. Motes, M. L. 1991. Incidence of Listeria species in shrimp, oysters, and estuarine waters. J. Food Prot. 54. 135. Nadon, C. A., D. L. Woodward, C. Young, F. G. Rodgers, and M. Wiedmann. 2001. Correlations between molecular subtyping and serotyping of Listeria monocytogenes. J. Clin. Microbiol. 39:2704–2707. 136. Nagi, M. S., and J. D. Verma. 1967. An outbreak of listeriosis in chickens. Ind. J. Vet. Med. 44:539–543. 137. Nesbakken, T., G. Kapperud, and D. A. Caugant. 1996. Pathways of Listeria monocytogenes contamination in the meat processing industry. Int. J. Food Microbiol. 31:161–171. 138. Nicolas, J. A., E. P. Espaze, J. Rocourt, M. J. Cornuejols, M. Lamachere, N. Vidaued, B. Catimel, and A. L. Courtieu. 1988. Listeriose animale et ensilage. Rec. de Med. Vet. 164:203–206. 139. Nilsson, A., and K. A. Karlsson. 1959. Listeria monocytogenes isolations from animals in Sweden during 1948 to 1957. Nord. Vet. Med. 11:305–315. 140. Nolan, D. A., D. C. Chamblin, and J. A. Troller. 1992. Minimal water activity levels for growth and survival of Listeria monocytogenes and Listeria innocua. Int. J. Food Microbiol. 16:323–335. 141. Norrung, B., and J. K. Andersen. 2000. Variations in virulence between different electrophoretic types of Listeria monocytogenes. Lett. Appl. Microbiol. 30:228–232. 142. Norrung, B., and N. Skovgaard. 1993. Application of multilocus enzyme electrophoresis in studies of the epidemiology of Listeria monocytogenes in Denmark. Appl. Environ. Microbiol. 59:2817–2822. 143. Norton, D. M., M. A. McCamey, K. L. Gall, J. M. Scarlett, K. J. Boor, and M. Wiedmann. 2001. Molecular studies on the ecology of Listeria monocytogenes in the smoked fish processing industry. Appl. Environ. Microbiol. 67:198–205. 144. Norton, D. M., J. M. Scarlett, K. Horton, D. Sue, J. Thimothe, K. J. Boor, and M. Wiedmann. 2001. Characterization and pathogenic potential of Listeria monocytogenes isolates from the smoked fish industry. Appl. Environ. Microbiol. 67:646–653. 145. Olafson, P. 1940. Listerella encephalitis (circling disease) of sheep, cattle, and goats. Cornell Vet. 30:141–150. 146. Park, S. F., and R. G. Kroll. 1993. Expression of listeriolysin and phosphatidylinositol-specific phospholipase C is repressed by the plant-derived molecule cellobiose in Listeria monocytogenes. Mol. Microbiol. 8:653–661. 147. Phan-Thanh, L., and T. Gormon. 1995. Analysis of heat and cold shock proteins in Listeria by twodimensional electrophoresis. Electrophoresis 16:444–450. 148. Piffaretti, J. C., H. Kressebuch, M. Aeschbacher, J. Bille, E. Bannerman, J. M. Musser, R. K. Selander, and J. Rocourt. 1989. Genetic characterization of clones of the bacterium Listeria monocytogenes causing epidemic disease. Proc. Natl. Acad. Sci. USA 86:3818–3822. 149. Pinto, B., and D. Reali. 1996. Prevalence of Listeria monocytogenes and other Listeria in Italianmade soft cheeses. Zentralbl. Hyg. Umweltmed. 199:60–68. 150. Plagemann, O., and A. Weber. 1988. Listeria monocytogenes als Abortursache bei Klippschliefern (Procavia capensis). Kleintierpraxis 33:317–318. 151. Pritchard, T. J., K. J. Flanders, and C. W. Donnelly. 1995. Comparison of the incidence of Listeria on equipment versus environmental sites within dairy processing plants. Int. J. Food Microbiol. 26:375–384. 152. Rahman, T., D. K. Sarma, B. K. Goswami, T. N. Upadhyaya, and B. Choudhury. 1985. Occurrence of listerial meningoencephalitis in pigs. Ind. Vet. J. 62:7–9. 153. Ralovich, B. 1984. Listeriosis research—present situation and perspective. Budapest: Akademiai Kiado. 154. Ramage, C. P., J. C. Low, J. McLauchlin, and W. Donachie. 1999. Characterization of Listeria ivanovii isolates from the U.K. using pulsed-field gel electrophoresis. FEMS Microbiol. Lett. 170:349–353. 155. Ranjard, L., F. Poly, and S. Nazaret. 2000. Monitoring complex bacterial communities using cultureindependent molecular techniques: Application to soil environment. Res. Microbiol. 151:167–177. 156. Razavilar, V., and C. Genigeorgis. 1998. Prediction of Listeria spp. growth as affected by various levels of chemicals, pH, temperature and storage time in a model broth. Int. J. Food Microbiol. 40:149–157.

DK3089_C002.fm Page 51 Tuesday, February 20, 2007 11:34 AM

Ecology of Listeria Species and L. monocytogenes in the Natural Environment

51

157. Restaino, L., E. W. Frampton, R. M. Irbe, G. Schabert, and H. Spitz. 1999. Isolation and detection of Listeria monocytogenes using fluorogenic and chromogenic substrates for phosphatidylinositolspecific phospholipase C. J. Food Prot. 62:244–251. 158. Richmond, M. 1990. The microbiological safety of foods, parts I & II. Report of the Committee on the Microbiological Safety of Foods. London: HMSO. 159. Rørvik, L. M., B. Aase, T. Alvestad, and D. A. Caugant. 2003. Molecular epidemiological survey of Listeria monocytogenes in broilers and poultry products. J. Appl. Microbiol. 94:633–640. 160. Rørvik, L. M., D. A. Caugant, and M. Yndestad. 1995. Contamination pattern of Listeria monocytogenes and other Listeria spp. in a salmon slaughterhouse and smoked salmon processing plant. Int. J. Food Microbiol. 25:19–27. 161. Ryser, E. T., S. M. Arimi, M. M. 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. 162. Ryser, E. T., S. M. Arimi, and C. W. Donnelly. 1997. Effects of pH on distribution of Listeria ribotypes in corn, hay, and grass silage. Appl. Environ. Microbiol. 63:3695–3697. 163. Sabanadesan, S., A. M. Lammerding, and M. W. Griffiths. 2000. Survival of Listeria innocua in salmon following cold-smoke application. J. Food Prot. 63:715–720. 164. Salamina, G., E. Dalle Donne, A. Niccolini, G. Poda, D. Cesaroni, M. Bucci, R. Fini, M. Maldini, A. Schuchat, B. Swaminathan, W. Bibb, J. Rocourt, N. Binkin, and S. Salmaso. 1996. A foodborne outbreak of gastroenteritis involving Listeria monocytogenes. Epidemiol. Infect. 117:429–436. 165. Salcedo, C., L. Arreaza, B. Alcala, L. De La Fuente, and J. A. Vazquez. 2003. Development of a multilocus sequence typing method for analysis of Listeria monocytogenes clones. J. Clin. Microbiol. 41:757–762. 166. Sauders, B.D. 2003. Unpublished data. 167. Sauders, B. D., E. D. Fortes, D. L. Morse, N. Dumas, J. A. Kiehlbauch, Y. Schukken, J. R. Hibbs, and M. Wiedmann. 2003. Molecular subtyping to detect human listeriosis clusters. Emerg. Infect. Dis. 9:672–680. 168. Schlech, W. F., III, 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. 169. Schneegurt, M. A., S. Y. Dore, and C. F. Kulpa, Jr. 2003. Direct extraction of DNA from soils for studies in microbial ecology. Curr. Issues Mol. Biol. 5:1–8. 170. 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. J. 10:183–189. 171. Schwartz, J. C. 1969. Attempted isolations of Listeria monocytogenes from diagnostic accessions. Am. J. Vet. Res. 30:483–484. 172. Seeliger, H. P. R. 1961. Listeriosis. New York: Hafner Publishing. 173. Senczek, D., R. Stephan, and F. Untermann. 2000. Pulsed-field gel electrophoresis (PFGE) typing of Listeria strains isolated from a meat processing plant over a 2-year period. Int. J. Food Microbiol. 62:155–159. 174. Skjerve, E., L. M. Rørvik, and O. Olsvik. 1990. Detection of Listeria monocytogenes in foods by immunomagnetic separation. Appl. Environ. Microbiol. 56:3478–3481. 175. Skovgaard, N., and C. A. Morgen. 1988. Detection of Listeria spp. in feces from animals, in feeds, and in raw foods of animal origin. Int. J. Food Microbiol. 6:229–242. 176. Sontakke, S., and J. M. Farber. 1995. The use of PCR ribotyping for typing strains of Listeria spp. Eur. J. Epidemiol. 11:665–673. 177. Soontharanont, S., and C. D. Garland. 1995. The occurrence of Listeria in temperate aquatic habitats. Proceedings of XII international symposium on problems of listeriosis, Perth, Western Australia, Promaco Conventions, pp. 145–146. 178. Sturgess, C. P. 1989. Listerial abortion in the bitch. Vet. Rec. 124:177. 179. Takai, S., F. Orii, K. Yasuda, S. Inoue, and S. Tsubaki. 1990. Isolation of Listeria monocytogenes from raw milk and its environment at dairy farms in Japan. Microbiol. Immunol. 34:631–634. 180. Thimothe, J., J. Walker, V. Suvanich, K. L. Gall, M. W. Moody, and M. Wiedmann. 2002. Detection of Listeria in crawfish processing plants and in raw, whole crawfish and processed crawfish (Procambarus spp.). J. Food Prot. 65:1735–1739.

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181. Tompkin, R. B. 2002. Control of Listeria monocytogenes in the food-processing environment. J. Food Prot. 65:709–725. 182. Trott, D. J., I. D. Robertson, and D. J. Hampson. 1993. Genetic characterization of isolates of Listeria monocytogenes from man, animals and food. J. Med. Microbiol. 38:122–128. 183. Troxler, R., A. von Graevenitz, G. Funke, B. Wiedemann, and I. Stock. 2000. Natural antibiotic susceptibility of Listeria species: L. grayi, L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri and L. welshimeri strains. Clin. Microbiol. Infect. 6:525–535. 184. Ueno, H., K. Yokota, T. Arai, Y. Muramatsu, H. Taniyama, T. Iida, and C. Morita. 1996. The prevalence of Listeria monocytogenes in the environment of dairy farms. Microbiol. Immunol. 40:121–124. 185. 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 retail market. J. Food Prot. 62:735–740. 186. van Netten, P., I. Perales, A. van de Moosdijk, G. D. Curtis, and D. 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. 187. 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. 188. Vazquez-Boland, J. A., L. Dominguez, M. Blanco, J. Rocourt, J. F. Fernandez-Garayzabal, C. B. Gutierrez, R. I. Tascon, and E. F. Rodriguez-Ferri. 1992. Epidemiologic investigation of a silageassociated epizootic of ovine listeric encephalitis, using a new Listeria-selective enumeration medium and phage typing. Am. J. Vet. Res. 53:368–371. 189. Vazquez-Boland, J. A., M. Kuhn, P. Berche, T. Chakraborty, G. Dominguez-Bernal, W. Goebel, B. Gonzalez-Zorn, J. Wehland, and J. Kreft. 2001. Listeria pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev. 14:584–640. 190. Vela, A. I., J. F. Fernandez-Garayzabal, J. A. Vazquez, M. V. Latre, M. M. Blanco, M. A. Moreno, L. de La Fuente, J. Marco, C. Franco, A. Cepeda, A. A. Rodriguez Moure, G. Suarez, and L. Dominguez. 2001. Molecular typing by pulsed-field gel electrophoresis of Spanish animal and human Listeria monocytogenes isolates. Appl. Environ. Microbiol. 67:5840–5843. 191. Vizcaino, L. L., M. J. Cubero, and A. Contreras. 1988. Listeric abortions in ewes and cows associated to orange peel and artichoke silage feeding. Proceedings of X international symposium on listeriosis, Pecs, Hungary, p. P29. 192. Vlaemynck, G., L. Herman, and K. Coudijzer. 1994. Isolation and characterization of two bacteriocins produced by Enterococcus faecium strains inhibitory to Listeria monocytogenes. Int. J. Food Microbiol. 24:211–225. 193. Volokhov, D., A. Rasooly, K. Chumakov, and V. Chizhikov. 2002. Identification of Listeria species by microarray-based assay. J. Clin. Microbiol. 40:4720–4728. 194. Walker, J. K., and J. H. Morgan. 1993. Ovine ophthalmitis associated with Listeria monocytogenes. Vet. Rec. 132:636. 195. Watkins, J., and K. P. Sleath. 1981. Isolation and enumeration of Listeria monocytogenes from sewage, sewage sludge and river water. J. Appl. Bacteriol. 50:1–9. 196. Weis, J., and H. P. Seeliger. 1975. Incidence of Listeria monocytogenes in nature. Appl. Microbiol. 30:29–32. 197. Welsh, R. D. 1983. Equine abortion caused by Listeria monocytogenes serotype 4. J. Am. Vet. Med. Assoc. 182:291. 198. Welshimer, H. J. 1960. Survival of Listeria monocytogenes in soil. J. Bacteriol. 80:316–320. 199. Welshimer, H. J., and J. Donker-Voet. 1971. Listeria monocytogenes in nature. Appl. Microbiol. 21:516–519. 200. Wesley, I. V., and F. Ashton. 1991. Restriction enzyme analysis of Listeria monocytogenes strains associated with food-borne epidemics. Appl. Environ. Microbiol. 57:969–975. 201. Whittenbury, R. 1968. Microbiology of silage grass. Proces. Biochem. 3:27–31. 202. Wiedmann, M. 2002. Molecular subtyping methods for Listeria monocytogenes. J. AOAC Int. 85:524–531. 203. Wiedmann, M., T. Arvik, J. L. Bruce, J. Neubauer, F. del Piero, M. C. Smith, J. Hurley, H. O. Mohammed, and C. A. Batt. 1997. Investigation of a listeriosis epizootic in sheep in New York state. Am. J. Vet. Res. 58:733–737.

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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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REFERENCES 1. Abou-Eleinin, A. A., 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. 2. Al-Dughaym, A. M., A. Fadl Elmula, G. E. Mohamed, A. A. Hegazy, Y. A. Radwan, F. M. T. Housawi, and A. A. Gameel. 2001. First report of an outbreak of ovine septicaemic listeriosis in Saudi Arabia. Rev. Sci. Tech. Off. Int. Epiz. 20:777–783. 3. Alexander, A. V., R. L. Walker, B. J. Johnson, B. R. Charlton, and L. W. Woods. 1992. Bovine abortions attributable to Listeria ivanovii: four cases (1988–1990). J. Am. Vet. Med. Assoc. 200:711–714. 4. Allcock, J. C. 1992. Cutaneous listeriosis. Vet. Rec. 130:18–19. 5. Anonymous. 1990. Pilotprojekt vedrorende integreret kontrol af maelk og mejeriprodukter. Report No. 201. Danish Dairy Organization. 6. Anonymous. 1991. The ecology of Listeria monocytogenes. Int. J. Food Microbiol. 14:194–199. 7. Anonymous. 1994. Veterinary investigation diagnosis and analysis III. Ministry of Agriculture Fisheries and Food, Weybridge, England. 8. Arimi, S. N. M., E. T. Ryser, T. J. Pritchard, and C. W. Donnelly. 1997. Diversity of Listeria ribotypes recovered from dairy cattle, silage, and dairy processing environments. J. Food Prot. 60: 811–816. 9. Arumugaswamy, R., and L. E. Gibson. 1999. Listeriosis in zoo animals and rivers. Aust. Vet J. 77:819–820. 10. Audurier, A., M. Gitter, and A. Raoult. 1986. Phage typing of Listeria strains isolated by the Veterinary Investigation Centres in Great Britain from 1981–1984. In Proceedings of the 9th International Symposium on the Problems of Listeriosis, eds. A. L. Courtieu, E. P. Espaze, and A. E. Reynaud. Nantes, France: University of Nantes, p. 410. 11. Avery, S. M., and S. Buncic. 1997. Differences in pathogenicity for chick embryos and growth kinetics at 37°C between clinical and meat isolates of Listeria monocytogenes previously stored at 4°C. Int. J. Food Microbiol. 34:319–327. 12. Baetz, A. L., and I. V. Wesley. 1995. Detection of anti-listeriolysin O in dairy cattle experimentally infected with Listeria monocytogenes. J. Vet. Diag. Invest. 7:82–86. 13. Baetz, A. L., I. V. Wesley, and M. G. Stevens. 1996. The use of listeriolysin O in an ELISA, a skin test and a lymphocyte blastogenesis assay on sheep experimentally infected with Listeria monocytogenes, Listeria ivanovii or Listeria innocua. Vet. Microbiol. 51:151–159. 14. Bailey, J. S., D. L. Fletcher, and N. A. Cox. 1989. Recovery and serotype distribution of Listeria monocytogenes from broiler chickens in the southeastern United States. J. Food Prot. 52:148–150. 15. Bailey, J. S., D. L. Fletcher, and N. A. Cox. 1990. Listeria monocytogenes colonization of broiler chickens. Poult. Sci. 6:457–461. 16. Barley, J. 1989. Listeria and listeriosis. Goats Today 82:145. 17. Barlow, R. M., and B. McGorum. 1995. Ovine listerial encephalitis: analysis, hypothesis and syntheses. Vet. Rec. 116:233–236. 18. Barnes, H. J. 2002. Miscellaneous and sporadic bacterial infections. In Diseases of poultry, ed. Y. M. Saif. Ames: Iowa State University Press. 19. Baum, K. 1994. Neurologic diseases. In Update on llama medicine. Vet. Clin. N. Am. 10:384–385. 20. Bauwens, L., F. Vercammen, and W. deMeurichy. 2001. Occurrence of Listeria spp. in captive antelope herds and their environment. J. Zool. Wildlife Med. 32:514–518. 21. Baxter, F., F. Wright, R. M. Chalmers, J. C. Low, and W. Donachie. 1993. Characterization by multilocus enzyme electrophoresis of Listeria monocytogenes isolates involved in ovine listeriosis outbreaks in Scotland from 1989 to 1991. Appl. Envir. Microbiol. 59:3126–3129. 22. Beauregard, M., and K. L. Malkin. 1971. Isolation of Listeria monocytogenes from brain specimens of domestic animals in Ontario. Can. Vet. J. 12:221–223. 23. Beer, J., W. Seffner, and J. Potel. 1957. Listerienfunde bei Tieren und ihre Bedeutung für die Epidemiologie der Listeriose. Arch. Exp. Vet. Med. 11:550–577. 24. Belding, R. C., and M. L. Mayer. 1957. Listeriosis in the turkey—two case reports. J. Am. Vet. Med. Assoc. 131:296–297. 25. Belin, M. 1946. La listerellose equine. Bull. Acad. Vet. Fr. 19:176–181.

DK3089_C003.fm Page 74 Saturday, February 17, 2007 5:04 PM

74

Listeria, Listeriosis, and Food Safety 26. Berche, P., K. A. Reich, M. Bonnichon, J. L. Beretti, C. Geoffroy, J. Faveneau, P. Cossart, J. L. Gaillard, P. Geslin, K. Kreis, and M. Veron. 1990. Detection of anti-listeriolysin O for serodiagnosis of human listeriosis. Lancet 335:624–627. 27. Bhunia, A. K. 1997. Antibodies to Listeria monocytogenes. Crit. Rev. Microbiol. 23:77–107. 28. Biester, H. E., and L. H. Schwarte. 1940. Listerella infection in swine. J. Am. Vet. Med. Assoc. 96:339–342. 29. Blenden, D. C. 1986. Listeriosis. In Diseases of swine, 6th ed., eds. A. D. Leman, B. Straw, R. D. Glock, W. I. Mengeling, R. H. C. Penny, and E. Scholl. Ames: Iowa State University Press, pp. 584–590. 30. Bocklisch, H., D. Wilhelms, C. Mirle, S. Lange, and U. Kucken. 1991. Listeria abortions in cattle: bacteriology, serology and epizootiology. Berl. Münch. Tierärztl. Wochenschr. 104:307–313. 31. Boerlin, P., and J. C. Piffaretti. 1991. Typing of human, animal food and environmental isolates of Listeria monocytogenes by multilocus enzyme electrophoresis. Appl. Environ. Microbiol. 57:1624–1629. 32. Boerlin, P., F. Boerlin-Petzold, and T. Jemmi. 2003. Use of listeriolysin O and internalin A in seroepidemiological study of listeriosis in Swiss dairy cows. J. Clin. Microbiol. 41:1055–1061. 33. Bourry, A., and B. Poutrel. 1996. Bovine mastitis caused by L. monocytogenes: Kinetics of antibody responses in serum and milk after experimental infection. J. Dairy Sci. 79:2189–2195. 34. Bourry, A., B. Poutrel, and J. Rocourt. 1995. Bovine mastitis caused by Listeria monocytogenes: characteristics of natural and experimental infections. J. Med. Microbiol. 43:125–132. 35. Bouttefroy, A., J. P. Lemaitre, and A. L. Rousset. 1997. Prevalence of Listeria sp. in droppings from urban rooks (Corvus frugilelgus). J. Appl. Microbiol. 82:641–647. 36. Brackett, R. E. 1988. Presence and persistence of Listeria monocytogenes in food and water. Food Technol. 42:162–164. 37. Braun, U., C. Stehle, and F. Ehrensperger. 2002. Clinical findings and treatment of listeriosis in 567 sheep and goats. Vet. Rec. 150:38–42. 38. Broadbent, D. W. 1972. Listeria as a cause of abortion and neonatal mortality in sheep. Aust. Vet. J. 48:391–394. 39. Bryner, J., I. Wesley, and M. van der Maaten. 1989. Research on listeriosis in milk cows with intramammary inoculation of Listeria monocytogenes. Acta Microbiol. Hung. 36:137–140. 40. Buncic, S. 1991. The incidence of Listeria monocytogenes in slaughtered animals, in meat and in meat products in Yugoslavia. Int. J. Food Microbiol. 112:173–180. 41. Bunning, V. K., R. G. Crawford, J. G. Bradshaw, J. T. Peeler, J. T. Tierney, and R. M. Twedt. 1986. Thermal resistance of intracellular Listeria monocytogenes cells suspended in raw bovine milk. Appl. Environ. Microbiol. 52:1398–1402. 42. Busch, R. H., D. M. Barnes, and J. H. Sautter. 1971. Pathogenesis and pathologic changes of experimentally induced listeriosis in newborn pigs. Am. J. Vet. Res. 32:1313–1320. 43. Butt, M., A. Weldon, D. Step, L. A. De, and C. Huxtable. 1991. Encephalitic listeriosis in two adult llamas (Lama glama): clinical presentations, lesions and immunofluorescence of L. monocytogenes in brainstem lesions. Cornell Vet. 31:251–258. 44. Cain, D. B., and V. McCann. 1986. An unusual case of cutaneous listeriosis. J. Clin. Microbiol. 23:976–977. 45. Carpenter, S. L., and M. Sevoian. 1983. Cellular immune response to Marek’s disease: Listeriosis as a model of study. Avian Dis. 27:344–356. 46. Carrique-Mas, J. J., I. Hokeberg, Y. Andersson, M. Arneborn, W. Tham, M.-L. Danielsson-Tham, B. Osterman, M. Leffler, M. Steen, E. Ericksson, G. Hedin, and J. Giesecke. 2003. Febrile gastroenteritis after eating on-farm manufactured fresh cheese—an outbreak of listeriosis? Epidemiol. Infect. 130:79–86. 47. Cavill, J. P. 1967. Listeriosis in chinchillas (Chinchilla laaniger). Vet. Rec. 80:592–594. 48. Chalifoux, L. V., and E. M. Hajema. 1981. Septicemia and meningoencephalitis caused by Listeria monocytogenes in a neonatal Macaca fascicularis. J. Med. Primatol. 10:336–339. 49. Charlton, K. M. 1977. Spontaneous listeric encephalitis in sheep. Electron microscopic studies. Vet. Pathol. 14:429–434. 50. Charlton, K. M., and M. M. Garcia. 1977. Spontaneous listeric encephalitis in sheep. Light microscopic studies. Vet. Pathol. 14:297–313. 51. Clark, E. G., A. S. Turner, B. G. Boysen, and B. T. Rouse. 1978. Listeriosis in an Arabian foal with combined immunodeficiency. J. Am. Vet. Med. Assoc. 172:363–366.

DK3089_C003.fm Page 75 Saturday, February 17, 2007 5:04 PM

Listeriosis in Animals

75

52. Colburn, K. G., C. A. Kaysner, C. Abeyta, and M. M. Wekell. 1990. Listeria species in a California coast estuarine environment. Appl. Environ. Microbiol. 56:2007–2011. 53. Colburn, K. G., C. A. Kaysner, M. M. Wekkell, J. R. Matches, C. Abeyta, and R. Stott. 1989. Microbiological quality of oysters (Crassostrea gigas) and water of live holding tanks in Seattle, WA markets. J. Food Prot. 52:100–104. 54. Cooper, G. L. 1989. A encephalitic form of listeriosis in broiler chickens. Avian Dis. 33:182–185. 55. Cooper, G., B. Charlton, A. Bickford, C. Cardona, J. Barton, S. Channing-Santiago, and R. Warker. 1992. Listeriosis in California broiler chickens. J. Vet. Diagn. Invest. 4:345–347. 56. Cooper, J., and R. D. Walker. 1998. Listeriosis. In Veterinary clinics of North America, ed. L. Toillefson. Philadelphia: W. B. Saunders Co., pp. 113–125. 57. Cummins, T. J., I. M. Orne, and R. E. Smith. 1988. Reduced in vivo nonspecific resistance to Listeria monocytogenes infection during avian retrovirus-induced immunosuppression. Avian Dis. 32:663–667. 58. Danielsson-Tham, M. L., M. Prag, J. Rocourt, H. Seeliger, W. Tham, and T. Vikerfors. 1997. A fatal case of Listeria endocarditis in a man following his tending of goats suggests an epidemiological link which is not supported by the results. Zentralbl. Veterinärmed. B 44:253–256. 59. DeValk, H., V. Vaillant, C. Jacquet, J. Rocourt, F. LeQuerrec, F. Stainer, N. Quelquejeu, O. Pierre, V. Pierre, J. C. Desenclos, and V. Goulet. 2001. Two consecutive nationwide outbreaks of listeriosis in France, October 1999–February 2000. Am. J. Epidemiol. 154:944–950. 60. de Vries, J., and R. Strikwerda. 1957. Ein Fall klinischer Euter-Listeriose beim Rind. Zbl. Bakteriol. Abt. I Orig. 167:229–232. 61. Dedie, K. 1955. Beitrag zur Epizootologie der Listeriose. Arch. Exptl. Veterinärmed. 9:251–264. 62. Dennis, S. M. 1975. Perinatal lamb mortality in Western Australia. 6. Listeric infection. Aust. Vet. J. 51:75–79. 63. Dijkstra, R. G. 1966. Een studie over listeriosis bij runderen. Tijdschr. Diergeneesk. 91:906–916. 64. Dijkstra, R. G. 1971. Investigations on the survival times of Listeria bacteria in suspensions of brain tissue, silage and feces and in milk. Zbl. Bakteriol. I Abt. Orig. 216:92–95. 65. Dijkstra, R. G. 1975. Recent experiences on the survival times of Listeria bacteria in suspensions of brain, tissue, silage, faeces and in milk. In Problems of listeriosis, ed. M. Woodbine. Leicester, U.K.: Leicester University Press, pp. 71–73. 66. Dijkstra, R. G. 1976. Listeria—encephalitis in cows through litter from a broiler farm. Zbl. Bakteriol. Hyg., I Abt. Orig. B 161:383–385. 67. Dijkstra, R. G. 1978. Incidence of Listeria monocytogenes in the intestinal contents of broilers on different farms. Tijdschr. Diergeneeskd. 103:229–231. 68. Dijkstra, R. G. 1986. A fifteen year survey of isolations of Listeria monocytogenes out of animals and the environment in northern Netherlands (1970–1984). In Listeriose, Listeria, listeriosis, 1985–1986, ed. A. L. Courtieu. Nantes, France: Université de Nantes, pp. 291–293. 69. Dijkstra, R. 1987. Listeriosis in animals—clinical signs, diagnosis and treatment. In Listeriosis—joint WHO/ROI consultation on prevention and control, ed. A. Schonberg. West Berlin, December 10–12, 1986, pp. 68–76. Institut für Veterinärmed. des Bundesgesundheitsamtes, Berlin. 70. Dominguez-Rodriguez, L., J. F. Fernandez-Garayzabal, J. A. Vazquez-Boland, E. Rodriguez-Ferri, and G. Suarez-Fernandez. 1985. Isolation of micro-organisms of the species Listeria from raw milk intended for human consumption. Can. J. Microbiol. 31:935–941. 71. Donker-Voet, J. 1962. My view on the epidemiology of Listeria infections. In Second symposium on listeric infection, ed. M. L. Gray. Bozeman: Montana State College, pp. 133–139. 72. Eilertz, I., M. L. Danielsson-Tham, K. E. Hammarberg, M. W. Reeves, J. Rocourt, H. P. R. Seeliger, B. Swaminathan, and W. Tham. 1993. Isolation of Listeria monocytogenes from goat cheese associated with a case of listeriosis in goat. Acta Vet. Scand. 34:145–149. 73. Elischerova, K., E. Cupkova, E. Urgeova, J. Lysy, and A. Sesevickova. 1990. Isolation of Listeria ivanovii in Slovakia. Cesk. Epidemiol. Mikrobiol. Imunol. 39:228–236. 74. Emerson, F. G., and A. A. Jarvis. 1968. Listeriosis in ponies. J. Am. Vet. Med. Assoc. 152:1645–1646. 75. Engeland, I. V., H. Waldeland, E. Ropstad, H. Kindahl, and O. Andresen. 1997. Effect of experimental infection with Listeria monocytogenes on the development of pregnancy and on concentrations of progesterone, oestrone sulphate and 15-ketodihydro-PGF2α in the goat. Anim. Reprod. Sci. 45:311–327. 76. Ericksen, L., H. E. Larsen, T. Christiansen, M. M. Jensen, and E. Erisksen. 1988. An outbreak of meningoencephalitis in fallow deer caused by Listeria monocytogenes. Vet. Rec. 122:274–276.

DK3089_C003.fm Page 76 Saturday, February 17, 2007 5:04 PM

76

Listeria, Listeriosis, and Food Safety

77. Ericsson, H., and P. Stalhandske. 1997. PCR detection of Listeria monocytogenes in “gravad” rainbow trout. Int. J. Food Microbiol. 34:281–285. 78. Evans, M., and G. Watson. 1987. Septicemic listeriosis in a reindeer calf. J. Wild Dis. 23:314–317. 79. Farber, J. M., E. Daley, F. Coates, N. Beausoleil, and J. Fournier. 1991. Feeding trials of Listeria monocytogenes with a nonhuman primate model. J. Clin. Microbiol. 29:2606–2608. 80. 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. 81. Fedio, W. M., and H. Jackson. 1992. On the origin of Listeria monocytogenes in raw bulk-tank milk. Int. Dairy J. 2:197–208. 82. Fenlon, D. 1985. Wild birds and silage as reservoirs of Listeria in the agricultural environment. J. Appl. Bacteriol. 59:537–543. 83. Fenlon, D. R. 1986. Rapid quantitative assessment of the distribution of Listeria in silage implicated in a suspected outbreak of listeriosis in calves. Vet. Rec. 118:240–242. 84. Fenlon, D. R., T. Stewart, and W. Donachie. 1995. The incidence, numbers and types of Listeria monocytogenes isolated from farm bulk tank milks. Lett. Appl. Microbiol. 20:57–60. 85. Fenlon, D. R., and J. Wilson. 1989. The incidence of Listeria monocytogenes in raw milk from farm bulk tanks in north-east Scotland. J. Appl. Bacteriol. 66:191–196. 86. Fenlon, D. R., J. Wilson, and W. Donachie. 1996. The incidence and level of Listeria monocytogenes contamination of food sources at primary production and initial processing. J. Appl. Bacteriol. 81:641–650. 87. Fensterbank, R. 1987. Vaccination with a Listeria strain of reduced virulence against experimental Listeria abortion in goats. Ann. Rech. Vet. 18:415–419. 88. Fernandez-Garayzabal, J. F., L. Dominguez, J. A. Vazquez, E. Gomez-Lucia, E. R. Rodriguez-Ferri, and G. Suarez. 1987. Occurrence of Listeria monocytogenes in raw milk. Vet. Rec. 120:258–259. 89. Finley, G. G., and J. R. Long. 1977. An epizootic of listeriosis in chinchillas. Can. Vet. J. 18:164–167. 90. Fraser, C. M., J. A. Bergeron, A. Mays, and S. E. Aiello, eds. 1991. The Merck veterinary manual: A handbook of diagnosis, therapy, and disease prevention and control for the veterinarian, 7th ed. Rahway, N.J.: Merck & Co., Inc., p. 358. 91. Fraser, C. M., J. A. Bergeron, A. Mays, and S. E. Aiello, eds. 1991. The Merck veterinary manual: A handbook of diagnosis, therapy, and disease prevention and control for the veterinarian, 7th ed. Rahway, N.J.: Merck & Co., Inc., p. 1579. 92. Gaya, P., C. Saralegui, M. Medina, and M. Munez. 1996. Occurrence of Listeria monocytogenes and other Listeria spp. in raw caprine milk. J. Dairy Sci. 79:1936–1941. 93. 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. 94. Gill, D. A. 1931. Circling disease of sheep in New Zealand. Vet. J. 87:60–74. 95. Gill, P. A., J. G. Boulton, G. C. Fraser, A. E. Stevenson, and L. A. Reddacliff. 1997. Bovine abortion caused by Listeria ivanovii. Aust. Vet. J. 75:214. 96. Gitter, M. 1985. Listeriosis in farm animals in Great Britain. In Isolation and identification of microorganisms of medical and veterinary importance, eds. C. H. Collins and J.M. Grange. London: Academic Press. 97. Gitter, M., R. Bradley, and P. H. Blarnpied. 1980. Listeria monocytogenes infection in bovine mastitis. Vet. Rec. 107:390–393. 98. Gitter, M., C. Richardson, and E. Boughton. 1986. Experimental infection of pregnant ewes with Listeria monocytogenes. Vet. Rec. 118: 575–578. 99. Graham, R. 1939. Listerella from a premature bovine fetus. Science 90:336–337. 100. Gray, M. L. 1958. Listeriosis in fowls—a review. Avian Dis. 2:296–314. 101. Gray, M. L. 1963. Epidemiological aspects of listeriosis. Am. J. Pub. Health 53:554–563. 102. Gray, M. L., and A. H. Killinger. 1966. Listeria monocytogenes and listeric infections. Bacteriol. Rev. 30:309–382. 103. Gray, M. L., C. Singh, and F. Thorp. 1956. Abortion and pre- or postnatal death of young due to Listeria monocytogenes. III. Studies in ruminants. Am. J. Vet. Res. 17:510–516. 104. Green, L. E., and K. L. Morgan. 1994. Descriptive epidemiology of listerial meningoencephalitis in housed lambs. Prev. Vet. Med. 18:79–87. 105. Greenwood, M. H., D. Roberts, and P. Burden. 1991. The occurrence of Listeria species in milk and dairy products: A national survey in England and Wales. Int. J. Food Microbiol. 12:197–206.

DK3089_C003.fm Page 77 Saturday, February 17, 2007 5:04 PM

Listeriosis in Animals

77

106. Grini, O. 1943. Listerella monocytogenes as a cause of septicemia in foals. Nord. Vet. Tidsskr. 55:97–104. 107. Grønstol, H. 1979. Listeriosis in sheep—Listeria monocytogenes excretion and immunological state in healthy sheep. Acta Vet. Scand. 20:168–179. 108. Grønstol, H. 1984. Listeriosis in goats. Les maladies de la chevre. Nirot (France) INRA 28:189–192. 109. Gudding, R., H. Grønstol, and H. J. Larsen. 1985. Vaccination against listeriosis in sheep. Vet. Rec. 117:89–90. 110. Gudding, R., L. L. Nesse, and H. Grønstol. 1989. Immunization against infections caused by Listeria monocytogenes in sheep. Vet. Rec. 125:111–114. 111. Guerden, L. M. G., and A. Devos. 1952. Listerellose bijpluimvee. Vlaams. Diergeneesk. Tschr. 21:165–175. 112. Harcourt, R. A. 1966. Listeria monocytogenes in a piglet. Vet. Rec. 78:735. 113. Harvey, J., and A. Gilmour. 1992. Occurrence of Listeria spp. in raw milk and dairy products produced in Northern Ireland. J. Appl. Bacteriol. 72:119–125. 114. Harvey, J., and A. Gilmour. 1994. Application of multilocus enzyme electrophoresis and restriction fragment length polymorphism analysis to the typing of Listeria monocytogenes strains isolated from raw milk, nondairy foods, and clinical and veterinary sources. Appl. Environ. Microbiol. 60:1547–1553. 115. Hassan, L. H. O. Mohammed, and P. L. McDonough. 2001. Farm-management and milking practices associated with the presence of Listeria monocytogenes in New York State dairy herds. Prev. Vet. Med. 51:63–73. 116. Hatkin, J. M., and W. E. Phillips, Jr. 1986. Isolation of Listeria monocytogenes from an eastern wild turkey. J. Wildlife Dis. 22:110–112. 117. Hayashidani, H., N. Kanzaki, Y. Kaneko, A. Okatani, T. Taniguchi, K. Kaneko, and M. Ogawa. 2002. Occurrence of yersiniosis and listeriosis in wild boars in Japan. J. Wildlife Dis. 38:202–205. 118. 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. 119. Hefnawy, Y., S. Moustafa, and R. S. Refai. 1989. Occurrence of Yersinia enterocolitica and Listeria monocytogenes in fresh water fish. Assiut Vet. Med. J. 21:135–139. 120. Heim, D., R. Fatzer, B. Hornlimann, and M. Vandevelde. 1997. Frequency of neurological disease in cattle. Schweiz. Arch. Tierheilkd. 139:354–362. 121. Hessen, L. 1957. Listeriose hos gris. Nord. Vet. Med. 9:951–958. 122. Higgins, R., G. Goyette, R. Sauvageau, and T. Lemaire. 1987. Septicemia due to Listeria monocytogenes in a newborn foal. Can. Vet. J. 28:63. 123. Hird, D., and C. Genigeorgis. 1990. Listeriosis in food animals: Clinical signs and livestock as a potential source of direct (nonfoodborne) infections in humans. In Foodborne listeriosis, eds. A. J. Miller, J. L. Smith, and G. A. Somkuti. Amsterdam: Elsevier Science Publishers, pp. 31–39. 124. Hofstad, M. S. 1984. Listeriosis. In Disease of poultry, 9th ed., eds. B. W. Calnek, H. J. Barnes, C. W. Beard, W. M. Reid, and H. W. Yoder, Jr. Ames: Iowa State University Press, pp. 261–263. 125. Hohne, K., B. Loose, and H. P. Seeliger. 1975. Isolation of Listeria monocytogenes in slaughter animals and bats of Togo (West Africa). Ann. Microbiol. Paris 126A:501–507. 126. Hunter, R. 1973. Observations on Listeria monocytogenes type 5 (Ivanov) isolated in New Zealand. Med. Lab. Technol. 30:51–56. 127. Husu, J. R. 1990. Epidemiological studies on the occurrence of Listeria monocytogenes in the feces of dairy cattle. Zentralbl. Veterinärmed. B 37:276–282. 128. Husu, J. R., J. T. Beery, E. Nurmi, and M. P. Doyle. 1990. Fate of Listeria monocytogenes in orally dosed chicks. Int. J. Food Microbiol. 11:259–270. 129. Husu, J. R., J. T. Seppanen, S. K. Sivela, and A. O. L. Raurama. 1990. Contamination of raw milk by Listeria monocytogenes on dairy farms. Zentralbl. Veterinärmed. B 37:268–275. 130. Hyslop, N. St. G. 1975. Epidemiologic and immunologic factors in listeriosis. In Problems of listeriosis, ed. M. Woodbine. Leicester, U.K.: Leicester University Press, pp. 94–105. 131. Hyslop, N. St. G., and A. D. Osborne. 1959. Listeriosis: A potential danger to public health. Vet. Rec. 71:1082–1091. 132. Iida, T., M. Kanzaki, T. Maruyama, S. Inoue, and C. Kaneuchi. 1991. Prevalence of Listeria monocytogenes in intestinal contents of healthy animals in Japan. J. Vet. Med. Sci. 53:873–875. 133. Ivanov, I. 1957. La listeriose chez les ovins et les caprins. Bull. Off. Int. Epizoot. 57:571–583.

DK3089_C003.fm Page 78 Saturday, February 17, 2007 5:04 PM

78

Listeria, Listeriosis, and Food Safety

134. 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. 135. Jayarao, B. M. J., and D. R. Henning. 2001. Prevalence of foodborne pathogens in bulk tank milk. J. Dairy Sci. 84:2157–2162. 136. Jemmi, T., and A. Keusch. 1994. Occurrence of Listeria monocytogenes in freshwater fish farms and fish-smoking plants. Food Microbiol. 11:309–316. 137. Jensen, A., W. Frederiksen, and P. Gerner-Smidt. 1994. Risk factors for listeriosis in Denmark, 1989–1990. Scand. J. Infect. Dis. 26:171–178. 138. Jensen, N. E., F. M. Aarestrup, J. Jensen, and H. C. Wegener. 1996. Listeria monocytogenes in bovine mastitis. Possible implication for human health. Int. J. Food Microbiol. 32:209–216. 139. Jensen, R., and D. R. Mackey. 1979. Diseases of feedlot cattle, 3rd ed. Philadelphia: Lea & Febiger, pp. 71–75. 140. Johnson, G. C., W. H. Fales, C. W. Maddox, and J. A. Ramos-Vara. 1995. Evaluation of laboratory tests for confirming the diagnosis of encephalitic listeriosis in ruminants. J. Vet. Diag. Invest. 7:223–228. 141. Johnson, G. C., C. W. Maddox, W. H. Fales, W. A. Wolff, R. F. Randle, et al. 1996. Epidemiologic evaluation of encephalitic listeriosis in goats. J. Am. Vet. Med. Assoc. 208:1695–1696. 142. Johnson, J. L., M. P. Doyle, R. G. Cassens, and J. L. Schoeni. 1988. Fate of Listeria monocytogenes in tissues of experimentally infected cattle and in hard salami. Appl. Environ. Microbiol. 54:497–501. 143. Jones, F. S., and R. B. Linle. 1934. Sporadic encephalitis in cows. Arch. Pathol. 18:580–581. 144. Jubb, K. V. F., and C. R. Huxtable. 1993. Listeriosis. In Pathology of domestic animals, 4th ed., eds. K. V. F. Jubb, P. C. Kennedy, and N. Palmer. San Diego, Calif.: Academic Press, Inc., pp. 393–397. 145. Kampelmacher, E. H. 1962. Animal products as a source of listeric infection in man. In Second symposium on listeric infection, ed. M. L. Gray. Bozeman: Montana State College, pp. 146–156. 146. Kanuganti, S., I. V. Wesley, P. G. Reddy, J. McKean, and H. S. Hurd. 2002. Detection of Listeria monocytogenes in pigs and pork. J. Food Prot. 65:1470–1474. 147. Kautter, D. A., S. J. Silverman, W. G. Roessler, and J. F. Drawdy. 1963. Virulence of Listeria monocytogenes for experimental animals. J. Infect. Dis. 112:167–180. 148. Kemenes, F., T. Antal, and F. Vetesi. 1971. Experimental listeriosis in pigs. Magyar Allatorv. Lapja. 26:39–42. 149. Kemenes, F., R. Glavits, E. lvanics, G. Kovacs, and A. Vanyi. 1983. Listeriosis of roe-deer in Hungary. Zentralbl. Veterinärmed. B 30:258. 150. Kerlin, D. I., and R. Graham. 1945. Studies of listerellosis. VI. Isolation of Listerella monocytogenes from the liver of a pig. Proc. Soc. Exp. Med. 58:351. 151. Killinger, A. H., and M. E. Mansfield. 1970. Epizootiology of listeric infection in sheep. J. Am. Vet. Med. Assoc. 157:1318–1324. 152. Kimberling, C. V. 1988. Diseases of the central nervous system. In Jensen and Swift’s diseases of sheep, 3rd ed. Philadelphia: Lea and Febiger, pp. 195–199. 153. Kirkbride, C. A. 1993. Bacterial agents detected in a 10-year study of bovine abortions and stillbirths. J. Vet. Diag. Invest. 5:64–68. 154. Kirkbride, C. A., E. J. Bicknell, D. E. Reed, M. G. Robl, W. U. Knudtson, and K. Wohlgemuth. 1973. A diagnostic survey of bovine abortion and stillbirth in the northern plains states. J. Am. Vet. Med. Assoc. 162:556–560. 155. Kloster, O., and R. Guidding. 1987. Prevention of listeric abortion in goats by vaccination. Vet. Rec. 120:563. 156. Kovincic, I., B. Stajner, S. Zakula, and M. Galic. 1979. The finding of L. monocytogenes in the milk of cows from infected herds. In Proceedings of the seventh international symposium on listeriosis, ed. I. Ivanov. Sofia: National Agroindustrial Union, Center for Scientific Information, pp. 221–224. 157. Krueger, N., C. Low, and W. Donachie. 1995. Phenotypic characterization of the cells of the inflammatory response in ovine encephalitic listeriosis. J. Comp. Pathol. 113:263–275. 158. Ladds, P. W., S. M. Dennis, and C. O. Njoku. 1974. Pathology of listeric infection in domestic animals. Vet. Bull. 44:67–74. 159. Lechner, W., F. Allerberger, A. Bergant, E. Solder, and M. P. Dierich. 1993. Effect of Listeria on contractibility of human uterine muscle. Z. Geburtshilfe-Perinatol. 197:179–183. 160. Lennon, D., B. Lewis, C. Mantell, D. Becroft, B. Dove, K. Farmer, S. Tonkin, N. Yeates, R. Stamp, and K. Mickleson. 1984. Epidemic perinatal listeriosis. Pediatr. Infect. Dis. 3:30–34.

DK3089_C003.fm Page 79 Saturday, February 17, 2007 5:04 PM

Listeriosis in Animals

79

161. Lessing, M. P. A., G. D. W. Curtis, and I. C. F. Bowler. 1994. Listeria ivanovii infection. J. Infect. 29:230–231. 162. Leung, C., Y. Huang, and O. C. Pancorbo. 1992. Bacterial pathogens and indicators in catfish and pond environments. J. Food Prot. 55:424–427. 163. L’hopital, S., J. Marly, P. Pardon, and P. Berche. 1993. Kinetics of antibody production against listeriolysin O in sheep with listeriosis. J. Clin. Microbiol. 31:1537–1540. 164. Liewen, M. B., and M. W. Plautz. 1988. Occurrence of Listeria monocytogenes in raw milk in Nebraska. J. Food Prot. 51:840–841. 165. Linde, K., G. C. Fthenakis, R. Lippmann, J. Kinne, and A. Abraham. 1995. The efficacy of a live Listeria monocytogenes combined serotype 1/2 and serotype 4b vaccine. Vaccine 13:923–926. 166. Loken, T., E. Aspoy, and H. Grønstol. 1982. Listeria monocytogenes excretion and humoral immunity in goats in a herd with outbreaks of listeriosis and in a dairy herd. Acta Vet. Scand. 23:392–399. 167. Loken, T., and H. Grønstol. 1982. Clinical investigations in a goat herd with outbreaks of listeriosis. Acta Vet. Scand. 23:380–391. 168. Loncarevic, S., A. Milanovic, F. Caklovica, W. Tham, and M.-L. Danielsson-Tham. 1994. Occurrence of Listeria species in an abattoir for cattle and pigs in Bosnia and Hercegovina. Acta Vet. Scand. 34:11–15. 169. Lopez, A., and R. Bildfell. 1989. Neonatal porcine listeriosis. Can. Vet. J. 30:828–829. 170. 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. 171. Low, C., and K. Linklater. 1985. Listeriosis in sheep. Practice 7:66–67. 172. Low, J. C., R. M. Chalmers, W. Donachie, R. Freeman, J. McLauchlin, and P. R. Sisson. 1992. Pyrolysis mass spectrometry of Listeria monocytogenes isolates from sheep. Res. Vet. Sci. 53:64–67. 173. Low, J. C., and W. Donachie. 1989. Listeria in food: a veterinary perspective. Lancet 1:322. 174. Low, J. C., and W. Donachie. 1991. Clinical and serum antibody responses of lambs to infection by Listeria monocytogenes. Res. Vet. Sci. 51:185–192. 175. Low, J. C., and W. Donachie. 1997. A review of Listeria monocytogenes and listeriosis. Vet. J. 153:9–29. 176. Low, J. C., and C. P. Renton. 1985. Septicemia, encephalitis and abortions in a housed flock of sheep caused by Listeria monocytogenes type 1/2. Vet. Rec. 116:147–150. 177. Low, J. C., F. Wright, J. McLauchlin, and W. Donachie. 1993. Serotyping and distribution of Listeria isolates from cases of ovine listeriosis. Vet. Rec. 133:165–166. 178. Macleod, N. S. M., and J. A. Watt. 1974. Listeria monocytogenes type 5 as a cause of abortion in sheep. Vet. Rec. 95:365–367. 179. Malik, S. V., S. B. Barbuddhe, and S. P. Chaudhari. 2002. Listeric infections in humans and animals in the Indian subcontinent: a review. Trop. Anim. Health Prod. 34:359–381. 180. Mason, R.W., R.G. Brennan, and A. Corbould. 1980. Listeria monocytogenes abortion in a mare. Aust. Vet. J. 56:613. 181. Mathews, F. P. 1928. Encephalitis in calves. J. Am. Vet. Assoc. 73:513–516. 182. Mayer, H., M. Kinzler, and E. Sickel. 1976. Listeriosis in a herd of saddle horses. Berl. Münch. Tierärztl. Wochenschr. 89:209–211. 183. Mazzette, R., E. Sanna, E. P. De Santis, S. Pisanu, and A. Leoni. 1991. Experimental listeriosis in chickens: Microbiological and anatomo-histopathological studies and health and hygiene considerations. Boll. Soc. Ital. Biol. Sper. 67:569–76. 184. McCain, C. S., and M. Robinson. 1976. Listeria monocytogenes in the equine. Proc. Am. Assoc. Vet. Lab. Diagn. 18:257–261. 185. McClure, H. M., and L. M. Strozier. 1975. Perinatal listeric septicemia in a Celebese black ape. Am. J. Vet. Res. 167: 637–638. 186. McLauchlin, J. 1987. Listeria monocytogenes, recent advances in the taxonomy and epidemiology of listeriosis in humans. J. Appl. Bacteriol. 63:1–11. 187. McLauchlin, J. 1997. Animal and human listeriosis: a shared problem? Vet. J. 153:3–5. 188. McLauchlin, J., and J. C. Low. 1994. Primary cutaneous listeriosis in adults: an occupational disease of veterinarians and farmers. Vet. Rec. 135:615–617. 189. Menudier, A., F. P. Rougier, and C. Bosgiraud. 1996. Comparative virulence between different strains of Listeria in zebrafish (Brachydonio rerio) and mice. Pathol. Biol. 44:783–789. 190. Meredith, C., and D. Schneider. 1984. An outbreak of ovine listeriosis associated with poor flock management practices. J. S. Afr. Vet. Med. Assoc. 55:55–56. 191. Meyer, E. P., and J. M. Gardner. 1970. A case of listeriosis in piglets. Aust. Vet. J. 46:514.

DK3089_C003.fm Page 80 Saturday, February 17, 2007 5:04 PM

80

Listeria, Listeriosis, and Food Safety

192. Miettinen, A., J. Husu, and J. Tuomi. 1990. Serum antibody response to Listeria monocytogenes, listerial excretion and clinical characteristics in experimentally infected goats. J. Clin. Microbiol. 28:340–343. 193. Miettinen, A., and J. Husu. 1991. Antibodies to listeriolysin O reflect the acquired resistance of Listeria monocytogenes in experimentally infected goats. FEMS Microbiol. Lett. 77:181–186. 194. Morgan, J. H. 1977. Infectious keratoconjunctivitis in cattle associated with Listeria monocytogenes. Vet. Rec. 100:113–114. 195. Morner, T. Listeriosis. In Diseases of wildlife, 3rd ed., eds. E. S. Williams and I. K. Barker. Ames: Iowa State University Press, pp. 502–505. 196. Motes, M. L. 1991. Incidence of Listeria spp. in shrimp, oysters and estuarine waters. J. Food Prot. 54:17–173. 197. Mouton, R. P., and E. H. Kampelmacher. 1966. Proceedings of the third international symposium on listeriosis, Bilthoven, The Netherlands, p. 425. 198. 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. 199. Nagi, M. S., and J. D. Verma. 1967. An outbreak of listeriosis in chickens. Indian J. Vet. 44:539–543. 200. Narucka, V., and J. F. Westendorp. 1973. Het voorkomen van Listeria monocytogenes by slachtvarkens. Tijdschr. Diergeneesk. 98:1208. 201. Nash, M. L., L. L. Hungerford, T. G. Nash, and G. M. Zinn. 1995. Epidemiology and economics of clinical listeriosis in a sheep flock. Prev. Vet. Med. 24:147–156. 202. Nedoluha, P. C., and D. Westhoff. 1997. Microbiological analysis of striped bass (Morone saxatilis) grown in a recirculating system. J. Food Prot. 60:948–953. 203. Nesbakken, T., E. Nerbrink, O. J. Rotterud, and E. Borch. 1994. Reduction of Yersinia enterocolitica and Listeria spp. on carcasses by enclosure of the rectum during slaughter. Int. J. Food Microbiol. 23:197–208. 204. Nilsson, A., and K. A. Karlsson. 1959. Listeria monocytogenes isolations from animals in Sweden during 1948 to 1957. Nord. Vet. Med. 11:305–315. 205. Norrung, B., M. Solve, M. Ovesen, and N. Skogaard. 1991. Evaluation of an ELISA test for the detection of Listeria spp. J. Food Prot. 54:752–755. 206. Odegaard, B., R. Grelland, and S. D. Henricksen. 1952. A case of Listeria infection in man, transmitted from sheep. Acta Med. Scand. 67:231–238. 207. Ojeniyi, B., H. C. Wegener, N. E. Hjensen, and M. Bisgaard. 1996. Listeria monocytogenes in poultry and poultry products: epidemiological investigations in seven Danish abattoirs. J. Appl. Bacteriol. 80:395–401. 208. Osebold, J. W., J. W. Kendrick, and A. Njoku-Obi. 1960. Abortion in cattle experimentally with Listeria monocytogenes. J. Am. Vet. Med. Assoc. 137:227–233. 209. Osebold, J. W., J. W. Kendrick, and A. Njoku-Obi. 1960. Cattle abortion associated with natural Listeria monocytogenes infections. J. Am. Vet. Med. Assoc. 137:221–226. 210. Owen, C. R., A. Meis, J. W. Jackson, and H. G. Stoenner. 1960. A case of primary cutaneous listeriosis. New Engl. J. Med. 262:1026–1028. 211. Paterson, J. S. 1937. Listerella infection in fowls—preliminary note on its occurrence in East Anglia. Vet. Rec. 49:1533–1534. 212. Paul-Murphy, J., J. E. Markovitz, I. V. Wesley, and J. A. Roberts. 1990. Listeriosis causing stillbirths and neonatal septicemia in outdoor housed macaques. 41st Ann. Mtg. Am. Assoc. Lab. Anim. Sci. 40:547. 213. Peters, M., and G. Scheele. 1996. Listeriosis in a rabbitry. Dtsch. Tierärztl. Wochenschr. 103:460–462. 214. Pohjanvirta, R., and T. Huttunen. 1985. Some aspects of murine experimental listeriosis. Acta Vet. Scand. 26:563–580. 215. Portnoy, D. A., V. Auerbuch, and I. J. Glomski. 2002. The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity. J. Cell Biol. 1258:409–414. 216. Potel, J. 1953/1954. Atiologie der Granulomatosis Infantiseptica. Wiss. Z. Martin Luther Univ.-Halle, Wittenberg 3:341. 217. Price, H. H. 1981. Outbreak of septicemic listeriosis in a dairy herd. Vet. Med. Small Anim. Clin. 76:73–74. 218. Pustovaia, L. F. 1970. Susceptibility of wild fowl to listeriosis. Vet. Bull. 41:533–534.

DK3089_C003.fm Page 81 Saturday, February 17, 2007 5:04 PM

Listeriosis in Animals

81

219. Radostits, O. M., D. C. Blood, and E. E. Gay (eds.). 2000. Diseases caused by Listeria spp. In Veterinary medicine: A textbook of the diseases of cattle, sheep, pigs, goats and horses. Philadelphia: W. B. Saunders, pp. 736–740. 220. Rahman, T., D. K. Sarma, B. K. Goswami, T. N. Upadlhyaya, and B. Choudhury. 1985. Occurrence of listerial meningoencephalitis in pigs. Indian Vet. J. 62:7–9. 221. Ralovich, B. 1984. Listeriosis research—present situation and perspective. Budapest: Akademiai Kiado. 222. Ralovich, B., and H. Domján-Kovács. 1996. Occurrence of Listeria and listeriosis in Hungary. Acta Vet. Hung. 44:277–285. 223. Ramos, J. A., M. Domingo, L. Dominguez, L. Ferrer, and A. Marco. 1988. Immunohistologic diagnosis of avian listeriosis. Avian Pathol. 17:227–233. 224. 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. 225. Rebhun, W. C. 1987. Listeriosis. Vet. Clin. North Am. Food Anim. Pract. 3:75–83. 226. Rebhun, W. C., and A. deLahunta. 1982. Diagnosis and treatment of bovine listeriosis. J. Am. Vet. Med. Assoc. 180:395–398. 227. Reece, R. L., P. C. Scott, and D. A. Barr. 1992. Some unusual diseases in the birds of Victoria, Australia. Vet. Rec. 130:178–85. 228. Reuter, R., M. Bowden, and M. Palmer. 1989. Ovine listeriosis in south coastal Western Australia. Aust. Vet. J. 66:223–224. 229. Roberts, A. J., and M. Wiedmann. 2003. Pathogen, host and environmental factors contributing to the pathogenesis of listeriosis. Cell Mol. Life Sci. 60:904–918. 230. Rodriquez, J. L., P. Gaya, M. Medina, and M. Nunez. 1994. Incidence of Listeria monocytogenes and other Listeria spp. in ewes’ raw milk. J. Food Prot. 57:571–575. 231. 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. 232. Rorvik, L. M., B. Aase, T. Alvestad, and D. A. Caugant. 2003. Molecular epidemiological survey of Listeria monocytogenes in broilers and poultry products. J. Appl. Microbiol. 94:633–640. 233. Sanchez, S., M. Studer, P. Currin, P. Barlett, and D. Bounus. 2001. Listeria keratitis in a horse. Vet. Ophthalmol. 4:217–219. 234. Schukken Y. H., Y. T. Grohn, and M. Wiedmann. 2003. Epidemiology of Listeriosis. In Microbial food safety in animal agriculture, eds. M. E. Torrence and R. E. Issacson. Ames: Iowa State University Press, pp. 221–232. 235. Schultz, G. 1967. Untersuchungen über das Vorkommen von Listerien in Rohmilch. Monatsh. Veterinärmed. 22:766–768. 236. Schwartz, J. C. 1967. Incidence of listeriosis in Pennsylvania livestock. J. Am. Vet. Med. Assoc. 151:1435–1437. 237. Scott, P. R. 1993. A field study of ovine listerial meningo-encephalitis with particular reference to cerebrospinal fluid analysis as an aid to diagnosis and prognosis. Br. Vet. J. 149:165–170. 238. Seastone, C. V. 1935. Pathogenic organisms of the genus Listerella. J. Exp. Med. 62:203–212. 239. Seeliger, H. P. R. 1961. Listeriosis. New York: Hafner Publishing Co. 240. Seeliger, H. P. 1984. Modern taxonomy of the Listeria group relationship to its pathogenicity. Clin. Invest. Med. 7:217–221. 241. Seimiya, Y., K. Ohshima, H. Itoh, and R. Murakami. 1992. Listeria septicemia with meningitis in a neonatal calf. J. Vet. Med. Sci. 54:1205–1207. 242. Sergeant, E. S. G., S. C. J. Love, and A. McInnes. 1991. Abortions in sheep due to Listeria ivanovii. Aust. Vet. J. 68:39. 243. Sharma, K. N., P. K. Mehrotra, and P. N. Mehrotra. 1983. Characterization of L. monocytogenes strains causing occulo-encephalitis in goats. Ind. J. Anim. Sci. 54:514–515. 244. Sharma, M., M. K. Batta, and R. C. Kataoch. 1996. Listeria monocytogenes abortions among migratory sheep and goats in Himachal Pradesh. Ind. J. Anim. Sci. 66:1117–1119. 245. Sharp, M. W. 1989. Bovine mastitis and Listeria monocytogenes. Vet. Rec. 125:512–1513. 246. Shigidi, M. T. A. 1979. Isolation of Listeria monocytogenes from animals in the Sudan. Br. Vet. J. 135:297–298.

DK3089_C003.fm Page 82 Saturday, February 17, 2007 5:04 PM

82

Listeria, Listeriosis, and Food Safety

247. Sindoni, L., V. Ciano, I. Picemo, A. Di Pietro, and W. Farina. 1983. Ricerche sulla epidemiologia della listeriosi—Nota II: Ulteriori risultati di un’ indagine sierologica sulla frequenza di anticorpi anti-Listeria in diverse specie animali dimoranti in alcune zone della Sicilia e della Calabria. Arch. Vet. Ital. 34:103–109. 248. Shlygina, K. N. 1959. Studies of variation in the causative organism of listeriosis. Zh. Mikrobiol. Epidemiol. Immunobiol. 30:68–75. 249. Siragusa, G. R., J. S. Dickson, and E. K. Daniels. 1993. Isolation of Listeria spp. from the feces of feedlot cattle. J. Food Prot. 56:102–105. 250. Sixl, W., Z. Sebek, M. Kock, E. Marth, and H. Withalm. 1989. Serological studies of domestic animals for listeriosis, Q-fever and brucellosis in Cairo. Georgr. Med. Suppl. 3:127–128. 251. Sixl, W., E. Wisidagama, D. Stunzner, H. Withalm, and B. Sixl-Vigt. 1988. Serological examinations of dogs in Colombo/Sri Lanka. Georgr. Med. Suppl. 1:89–92. 252. Skovgaard, N., and B. Noarrung. 1989. The incidence of Listeria spp. in feces of Danish pigs and in minced pork meat. Int. J. Food Microbiol. 8:59–63. 253. Slabospits’Kii, T. P. 1938. Pro novii mikroorganizm, vidilenii vid porosyat. (New microorganism isolated from piglets). Nauk. Zap. Kiev. Vet. Inst. 1:39. Vet. Bull. 12:367. 254. Slade, P. J., D. L. Collins-Thompson, and F. Fletcher. 1988. Incidence of Listeria species in Ontario raw milk. Can. Inst. Food Sci. Technol. 21:425–429. 255. Smith, M. A., K. Takeuchi, R. E. Brackett, H. M. Mcclure, R. B. Raybourne, K. M. Williams, U. S. Babu, G. O. Ware, J. R. Broderson, and M. P. Doyle. 2003. Nonhuman primate model for Listeria monocytogenes-induced stillbirths. Infect. Immun. 71:1574–1579. 256. Smyth, R. L., and M. F. M. Bamford. 1988. Neonatal listeriosis: experience in Suffolk. J. Infect. 17:65–70. 257. Srivastava, N. C., and S. S. Khera. 1980. The prevalence of Listeria antibodies in farm stock. Indian Vet. J. 57:270–272. 258. Stajner, B. 1975. Excretion of Listeria through milk of infected cows. Dairy Sci. Abstr. 37:180. 259. Stamatin, N., C. Ungureanu, E. Constantinescu, A. Solnitzky, and E. Vasilescu. 1957. Infectia naturala cu Listeria monocytogenes la pastravul curcubeu Salmo irideus. Annuar. Inst. Anim. Pathol. Hyg. Bucuresti 7:163–180. 260. Stone, D. L. 1987. A survey of raw whole milk for Campylobacter jejuni, Listeria monocytogenes, and Yersinia enterocolitica. N.Z. J. Dairy Sci. 22:257. 261. Sturgess, C.P. 1989. Listerial abortion in the bitch. Vet. Rec. 124:177. 262. Svabic-Vlahovic, M., N. D. Pantic, M. Pavicic, and J. H. Bryner. 1988. Transmission of Listeria monocytogenes from mother’s milk to her baby and to puppies. Lancet 2:2101. 263. Svenkerud, R. R. 1948. Listerella (erysipelothrix) monocytogenes infection, especially in horses. Nor. Vet. Tidsskr. 60:321–340. 264. Svenning, M., and V. Baverud. 1992. Listeriosis diagnosed in a live foal. First recorded case in Sweden. Svensk Veterinartiddning 44:551–554. 265. Terplan, G., R. Schoen, W. Springmeyer, I. Degle, and H. Becker. 1986. Occurrence, behavior and significance of Listeria in milk and dairy products. Arch. Lebensmittel. Hyg. 36:131–137. 266. Terplan, G., and S. Steinmeyer. 1989. Investigations on the pathogenicity of Listeria spp. by experimental infection of the chick embryo. Int. J. Food Microbiol. 8:277–280. 267. Timoney, J. F., J. H. Gillespie, F. W. Scott, and J. E. Barlough. 1988. Hagan and Brunner’s microbiology and infectious diseases of domestic animals, 8th ed. Ithaca, N.Y.: Comstock Publishing Associates, pp. 241–246. 268. Ugorski, L., J. Kaminski, and S. Strojna. 1959. Listeriosis in horses. Medycyna Wet. 15:153–156. 269. Unanue, E. R. 1997. Studies in listeriosis show the strong symbiosis between the innate cellular system and the T-cell response. Immun. Rev. 158:11–25. 270. van Metre, D., G. Barrington, S. Parish, and D. Tumas. 1991. Otitis media and suppurative meningoencephalomyelitis associated with L. monocytogenes infection in a llama. J. Am. Vet. Med. Assoc. 199:236–240. 271. 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.

DK3089_C003.fm Page 83 Saturday, February 17, 2007 5:04 PM

Listeriosis in Animals

83

272. Vazquez-Boland, H. J. A., L. Dominguez, M. Blanco, J. Rocourt, J. F. Fernandez-Garayzabal, C. B. Gutierrez, R. I. Tascon, and E. F. Rodriquez-Ferri. 1992. Epidemiologic investigation of a silageassociated epizootic of ovine listeric encephalitis, using a new Listeria selective enumeration medium and phage typing. Am. J. Vet. Res. 3:368–371. 273. Vetesi, F., A. Balsai, and F. Kemenes. 1972. Abortion in Gray’s monkey (Cercopithecus mona) associated with Listeria monocytogenes. Acta Microbiol. Acad. Sci., Hung. 19:441–443. 274. Vishinsky, Y., A. Grinberg, and R. Ozery. 1993. Listeria monocytogenes udder infection and carcass contamination. Vet. Rec. 133:484. 275. Visser, I. J. 1996. Pustular dermatitis in veterinarians following delivery in domestic animals: an occupational disease. Ned. Tijdschr. Geneeskd. 140:1186–1190. 276. Vizcaino, L. L., and M. A. Garcia. 1975. A note on Listeria milk excretion in sero-positive apparently healthy cows. In Problems of listeriosis, ed. M. Woodbine. Surrey, England: Leicester University Press, p. 74. 277. Vojinovic, G. 1992. The incidence of Listeria monocytogenes in slaughtered healthy animals and minced meat. Acta Vet. (Belgrad) 42:329–336. 278. von Amtsberg, G., A. Elsner, H. A. Grabbar, and W. Winkenwerder. 1969. Die epidemiologische und lebensmittelhygienische Bedeutung der Listerieninfektion des Rindes. Dtsch. Tierärztl. Wochenschr. 76:497–501. 279. von Amtsberg, G., A. Elsner, H. A. Grabbar, and W. Winkenwerder. 1969. The animal health yearbook. 1986. Food and Agriculture Organization of the United Nations, World Health Organization and the International Office of Epizootics, Rome. 280. von Arda, M., W. Bisping, N. Aydin, E. Istanbulluoglu, O. Akay, M. Izgur, Z. Karaer, S. Diker, and G. Kirpal. 1987. Ätiologische Untersuchungen über den Abort bei Schafen unter besonderer Berücksichtigung des Nachweises von Brucellen, Campylobacter, Salmonellen, Listerien, Leptospiren und Chlamydien. Berl. Münch. Tierärztl. Wochenschr. 100:405–408. 281. von Hartwigk, H. 1958. Zum Nachweis von Listerieninder Kuhmilch. Berl. Münch. Tierärztl. Wochenschr. 71:82–85. 282. von Selbitz, H.-J. 1986. Immunological principles for control of listeriosis. Monatsh. Veterinärmed. 41:217–219. 283. Walker, J. K., and J. H. Morgan. 1993. Ovine ophthalmitis associated with Listeria monocytogenes. Vet. Rec. 132:636. 284. Wallace, S., and T. Hathcock. 1995. L. monocytogenes septicemia in a foal. J. Am. Vet. Med. Assoc. 207:1325–1326. 285. Wardrope, D. D., and N. S. M. Macleod. 1983. Outbreak of Listeria meningoencephalitis in young lambs. Vet. Rec. 113:213–214. 286. Webb, D., and A. Rebar. 1987. Listeriosis in an immature black buck antelope (Antilope cervicapra). J. Wildlife Dis. 23:318–320. 287. Weber, A., C. Datzmann, and J. Potel. 1993. Prevalence of Listeria monocytogenes in fecal samples from dogs and cats. Tierärztl. Umsch. 48:727–730. 288. Weber, A., J. Potel, R. Schäfer-Schmidt, A. Prell, and C. Datzmann. 1995. Investigations on the occurrence of L. monocytogenes in fecal samples of domestic and companion animals. Zentralbl. Hyg. Umweltmed. 198:117–123. 289. Welsh, A. 1983. Equine abortion caused by Listeria monocytogenes serotype 4. J. Am. Vet. Med. Assoc. 182:291. 290. Wesley, I. V., and F. Ashton. 1991. Restriction enzyme analysis of Listeria monocytogenes strains associated with food-borne epidemics. Appl. Environ. Microbiol. 57:969–975. 291. Wesley, I. V., J. H. Bryner, and M. J. van der Maaten. l989. Effects of dexamethasone on shedding of Listeria monocytogenes in dairy cattle. Am. J. Vet. Res. 50:2009–2113. 292. Wesley, I. V., D. J. Larson, K. M. Harmon, J. B. Luchansky, and A. R. Schwartz. 2002. A case report of sporadic ovine listerial meningoencephalitis in Iowa with an overview of livestock and human cases. J. Vet. Diag. Invest. 14:314–321. 293. WHO Working Group. 1988. Foodborne listeriosis. Bull. WHO 66:421–428. 294. Wiedmann, M. 2002. Molecular subtyping methods for Listeria monocytogenes. J. AOAC Int. 85:524–531.

DK3089_C003.fm Page 84 Saturday, February 17, 2007 5:04 PM

84

Listeria, Listeriosis, and Food Safety

295. Wiedmann, M. 2003. ADSA Foundation scholar award—an integrated science-based approach to dairy food safety: Listeria monocytogenes as a model system. J. Dairy Sci. 86:1865–1875. 296. Wiedmann, M., T. Arvik, J. Bruce, J. Neubauer, F. Pierro, M. C. Smith, J. Hurley, H. O. Mohammed, and C. A. Batt. 1997. Investigation of a listeriosis epizootic in sheep in New York State. Am. J. Vet. Res. 58:733–737. 297. Wiedman, M., J. L. Bruce, C. Keang, A. E. Johson, 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. 298. 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. 299. Wilesmith, J. W., and M. Gitter. 1986. Epidemiology of ovine listeriosis in Great Britain. Vet. Rec. 119:467–470. 300. Wilkerson, M. J., A. Melendy, and E. Stauber. 1997. An outbreak of listeriosis in a breeding colony of chinchillas. J. Vet. Diag. Invest. 9:320–323. 301. Wohler, W. H., and C. L. Baugh. 1983. Pulmonary listeriosis in feeder cattle. Med. Vet. Pract. 64:736–739. 302. Wramby, G. O. 1944. Om Listerella monocytogenes bakteriologi och om forekomst av Listerella infectioner has djur. Skand. Vet. Tskr. 34:278–290. 303. Yndestad, M. 1987. Personal communication. 304. Yousif, Y. A., B. P. Joshi, and H. A. Ali. 1984. Ovine and caprine listeric encephalitis in Iraq. Trop. Anim. Health Prod. 16:27–28. 305. Zhu, M., I. Wesley, R. Nannapaneni, M. Cox, A. Mendonca, M. G. Johnson, and D. U. Ahn. 2003. The role of dietary vitamin E in experimental Listeria monocytogenes infections in turkeys. Poultry Sci. 82:2559–2564. 306. Zwart, P., and J. Donker-Voet. 1959. Listeriosis bij in gevangenschap gehouden dieren. Tijdschr. Diergeneesk. 84:712–716.

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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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Listeriosis in Humans

103

11. Bassler, H. A., S. J. A. Flood, K. J. Livak, J. Marmaro, R. Knorr, and C. A. Batt. 1995. Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes. Appl. Environ. Microbiol. 61:3724–3728. 12. Benshushan, A., A. Tsafrir, R. Arbel, G. Rahav, I. Ariel, and N. Rojansky. 2002. Listeria infection during pregnancy: A 10-year experience. Isr. Med. Assoc. J. 4:776–780. 13. Bille, J. 1990. Epidemiology of human listeriosis in Europe, with special reference to the Swiss outbreak. Amsterdam: Elsevier. 14. Bille, J., and J. Rocourt. 1996. WHO international multicenter Listeria monocytogenes subtyping study—rationale and set-up of the study. Int. J. Food Microbiol. 32:251–262. 15. Bizet, C., D. Mechali, J. Rocourt, and F. Fraisse. 1989. Listeria monocytogenes bacteremia in AIDS. Lancet 1:501. 16. Blaser, M. J., R. A. Miller, J. Lacher, and J. W. Singleton. 1984. Patients with active Crohn’s disease have elevated serum antibodies to antigens of seven enteric bacterial pathogens. Gastroenterology 87:888–894. 17. Bojsen-Moller, J. 1972. Human listeriosis: diagnostic, epidemiological, and clinical studies. Acta Pathol. Microbiol. Scand. 229 (Sect B. suppl):72–92. 18. Bortolussi, R. 1990. Neonatal listeriosis. Semin. Perinatol. 14(suppl.):44–48. 19. Boucher, M., and M. L. Yonekura. 1984. Listeria meningitis during pregnancy. Am. J. Perinatol. 1:312–318. 20. Brown, W. R. 1995. Listeria: The latest putative pathogenetic microorganism in Crohn’s disease. Gastroenterology 108:1589–1590. 21. Büla, C. J., J. Bille, and M. P. Glauser. 1995. An epidemic of foodborne listeriosis in Western Switzerland: Description of 57 cases involving adults. Clin. Infect. Dis. 20:66–72. 22. Carrique-Mas, J. J., I. Hokeberg, Y. Andersson, M. Arneborn, W. Tham, M. L. Danielsson-Tham, B. Osterman, M. Leffler, M. Steen, E. Eriksson, G. Hedin, and J. Giesecke. 2003. Febrile gastroenteritis after eating on-farm manufactured fresh cheese—an outbreak of listeriosis? Epidemiol. Infect. 130:79–86. 23. Centers for Disease Control and Prevention. 2005. Health Topics A–Z: Listeriosis. Published on the World Wide Web at http://www.cdc.gov/ncidod/dbmd/diseaseinfo/listeriosis_g.htm. 24. Centers for Disease Control and Prevention. 1998. Update: Multistate outbreak of listeriosis—United States, 1998–1999. MMWR 47(51&52):1117–1132. 25. Centers for Disease Control and Prevention. 2003. Outbreak of Listeria—northeastern United States. MMWR 51:950–951. 26. 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. 27. Centers for Disease Control and Prevention. 1988. Update—listeriosis and pasteurized milk. MMWR 37:764–766. 28. Centers for Disease Control and Prevention. 1989. Listeriosis associated with consumption of turkey franks. Morbidity Mortality Wkly. Rep. 38:267–268. 29. Centers for Disease Control and Prevention. 1992. Preventing foodborne illness: Listeriosis. Atlanta: Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, U.S. Centers for Disease Control and Prevention. 30. Centers for Disease Control and Prevention. 2005. Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food—10 sites, United States, 2004. MMWR 54:352–356. 31. Chen, W., D. Li, B. Paulus, I. Wilson, and V. S. Chadwick. 2000. Detection of Listeria monocytogenes by polymerase chain reaction in intestinal mucosal biopsies from patients with inflammatory bowel disease and controls. J. Gastroenterol. Hepatol. 15:1145–1150. 32. Chiba, M., T. Fukushima, S. Inoue, Y. Horie, M. Iizuka, and O. Masamune. 1998. Listeria monocytogenes in Crohn’s disease. Scand. J. Gastroenterol. 33:430–434. 33. Chomel, B. B., E. E. DeBess, D. M. Mangiamele, K. F. Reilly, T. B. Farver, R. K. Sun, and L. R. Barrett. 1994. Changing trends in the epidemiology of human brucellosis in California from 1973 to 1992: a shift toward foodborne transmission. J. Infect. Dis. 170:1216–1223. 34. Chougle, A., and V. Narayanaswamy. 2004. Delayed presentation of prosthetic joint infection due to Listeria monocytogenes. Int. J. Clin. Pract. 58:420–421.

DK3089_C004.fm Page 104 Tuesday, February 20, 2007 11:38 AM

104

Listeria, Listeriosis, and Food Safety

35. Ciesielski, C. A., A. W. Hightower, S. K. Parsons, and C. V. Broome. 1988. Listeriosis in the United States: 1980–1982. Arch. Intern. Med. 148:1416–1419. 36. Coffey, T., M. Nelson, M. Bower, and B. G. Gazzard. 1989. Listeria monocytogenes meningitis in an HIV-infected patient. Aids 3:614–615. 37. Dalton, C. B., C. C. Austin, J. Sobel, P. S. Hayes, W. F. Bibb, L. M. Graves, B. Swaminathan, M. E. Proctor, and P. M. Griffin. 1997. An outbreak of gastroenteritis and fever due to Listeria monocytogenes in milk. New Engl. J. Med. 336:100–132. 38. Decker, C. F., G. L. Simon, R. A. DiGioia, and C. U. Tuazon. 1991. Listeria monocytogenes infections in patients with AIDS: Report of five cases and review. Rev. Infect. Dis. 13:413–417. 39. Department of Agriculture, U.S. 2003. Control of Listeria monocytogenes in ready-to-eat meat and poultry products; Final Rule. Fed. Reg. 68:34208–34254. 40. Diseases, D. O. B. A. M. Public Health Laboratory Information System (PHLIS): Centers for Disease Control and Prevention. 41. Ewert, D. P., L. Lieb, P. S. Hayes, M. W. Reeves, and L. Mascola. 1995. Listeria monocytogenes infection and serotype distribution among HIV-infected persons in Los Angeles County, 1985–1992. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 8:461–465. 42. Farber, J. M., and P. I. Peterkin. 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55:476–511. 43. Fleming, D. W., S. L. Cochi, K. L. MacDonald, J. Brondum, P. S. Hayes, B. D. Plikaytis, M. B. Holmes, A. Audurier, C. V. Broome, and A. L. Reingold. 1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. New Engl. J. Med. 312:404–407. 44. FDA. 1992. Eating defensively: Food safety advice for persons with AIDS. Washington, D.C.: Food and Drug Administration. Report No.: FDA publication 92-2232. 45. Reducing the risk of Listeria monocytogenes: Joint response to the president. 2001. Accessed at http://www.foodsafety.gov/~dms/lmriplan.html. 46. Reducing the risk of Listeria monocytogenes: FDA/CDC 2003 update of the Listeria Action Plan, 2003. Accessed at http://www.foodsafety.gov/~dms/lmr2plan.html. 47. Franciosa, G., A. Maugliani, F. Floridi, and P. Aureli. 2005. Molecular and experimental virulence of Listeria monocytogenes strains isolated from cases with invasive listeriosis and febrile gastroenteritis. FEMS Immunol. Med. Microbiol. 43:431–439. 48. Frederiksen, B. 1992. Feto-maternal listeriosis in Denmark 1981–1988. J. Infect. 24:277–287. 49. Frederiksen, B. 1992. Maternal septicemia with Listeria monocytogenes in second trimester without infection of the fetus. Acta Obstet. Gynecol. Scand. 71:313–315. 50. Frye, D. M., R. Zweig, J. Sturgeon, M. Tormey, M. LeCavalier, I. Lee, L. Lawani, and L. Mascola. 2002. An outbreak of febrile gastroenteritis associated with delicatessen meat contaminated with Listeria monocytogenes. Clin. Infect. Dis. 35:943–949. 51. Gaillard, J. L., J. L. Beretti, M. Boulot-Tolle, J. M. Wilhelm, J. L. Bertrand, T. Herbelleau, and P. Berche. 1992. Serological evidence for culture negative listeriosis of the central nervous system. Lancet 340:560. 52. Gallagher, P. C., C. A. Amedia, and C. Watanakunakorn. 1986. Listeria monocytogenes endocarditis in a patient on chronic hemodialysis, successfully treated with vancomycin-gentamicin: Case report. Infection 14:125–128. 53. Gellin, B. G., and C. V. Broome. 1989. Listeriosis. JAMA 261:1313–1320. 54. Gellin, B. G., C. V. Broome, W. F. Bibb, R. E. Weaver, S. Gaventa, L. Mascola, and the Listeria Study Group. 1991. The epidemiology of listeriosis in the United States—1986. Am. J. Epidemiol. 133:392–401. 55. Giraud, J. R., F. Denis, F. Gargot, T. Fizazi, P. Babin, R. Y. Rautlin, A. Hoppeler, J. Brisou, and H. Tourris. 1973. La listeriose: Incidence dans les interruptions spontanees de la grossese. Nouv. Presse Med. 2:215–218. 56. Girmenia, C., A. P. Iori, S. Bernasconi, A. M. Testi, M. L. Moleti, W. Arcese, and P. Martino. 2000. Listeriosis in recipients of allogeneic bone marrow transplants from unrelated donors. Eur. J. Clin. Microbiol. Infect. Dis. 19:711–714. 57. 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.

DK3089_C004.fm Page 105 Tuesday, February 20, 2007 11:38 AM

Listeriosis in Humans

105

58. Goulet, V., H. de Valk, O. Pierre, F. Stainer, J. Rocourt, V. Vaillant, C. Jacquet, and J. C. Desenclos. 2001. Effect of prevention measures on incidence of human listeriosis, France, 1987–1997. Emerg. Infect. Dis. 7:983–989. 59. Goulet, V., C. Jacquet, V. Vaillant, I. Rebière, E. Mouret, C. Lorente, E. Maillot, F. Staïner, and J. Rocourt. 1995. Listeriosis from consumption of raw-milk cheese. Lancet 345:1581–1582. 60. Goulet, V., A. Lepoutre, J. Rocourt, A. L. Courtieu, P. Dehaumont, and P. Veit. 1993. Epidémie de listériose en France: Bilan final et résultats de l'enquête épidémiologique. Bull. Epidémiol. Hebdom. 4:13–14. 61. Graham, J. C., S. Lanser, G. Bignardi, S. Pedler, and V. Hollyoak. 2002. Hospital-acquired listeriosis. J. Hosp. Infect. 51:136–139. 62. Graves, L. M., and B. Swaminathan. 2001. PulseNet standardized protocol for subtyping Listeria monocytogenes by macrorestriction and pulsed-field gel electrophoresis. Int. J. Food Microbiol. 65:55–62. 63. Gray, J. W., J. F. R. Barrett, S. J. Pedler, and T. Lind. 1993. Fecal carriage of Listeria during pregnancy. Br. J. Obstet. Gynaecol. 100:873–874. 64. Gray, M. L., and A. H. Killinger. 1966. Listeria monocytogenes and listeric infections. Bacteriol. Rev. 30:309–382. 65. Grif, K., I. Hein, M. Wagner, E. Brandl, O. Mpamugo, J. McLauchlin, M. P. Dierich, and F. Allerberger. 2001. Prevalence and characterization of Listeria monocytogenes in the feces of healthy Austrians. Wien. Klin. Wochenschr. 113:737–742. 66. Grif, K., G. Patscheider, M. P. Dierich, and F. Allerberger. 2003. Incidence of fecal carriage of Listeria monocytogenes in three healthy volunteers: A one-year prospective stool survey. Eur. J. Clin. Microbiol. Infect. Dis. 22:16–20. 67. Harisdangkul, V., S. Songcharoen, and A. C. Lin. 1992. Listerial infections in patients with systemic lupus erythematosus. S. Med. J. 85:957–960. 68. Harvey, R. L., and P. H. Chandreskar. 1988. Chronic meningitis caused by Listeria in a patient infected with the human immunodeficiency virus. J. Infect. Dis. 157:1091–1092. 69. Havell, E. A. 1986. Synthesis and secretion of interferon by murine fibroblasts in response to intracellular Listeria monocytogenes. Infect. Immun. 54:787–792. 70. Ho, J. L., K. N. Shands, G. Friedland, P. Eckind, and D. W. Fraser. 1986. An outbreak of type 4b Listeria monocytogenes infection involving patients from eight Boston hospitals. Arch. Intern. Med. 146:520–524. 71. Hof, H. 2003. Listeriosis: therapeutic options. FEMS Immunol. Med. Microbiol. 35:203–205. 72. Hof, H. 2004. An update on the medical management of listeriosis. Expert Opin. Pharmacother. 5:1727–1735. 73. Hof, H., and R. Lampidis. 2001. Retrospective evidence for nosocomial Listeria infection. J. Hosp. Infect. 48:321–322. 74. Hof, H., R. Lampidis, and J. Bensch. 2000. Nosocomial Listeria gastroenteritis in a newborn, confirmed by random amplification of polymorphic DNA. Clin. Microbiol. Infect. 6:683–686. 75. Horl, M. P., M. Schmitz, K. Ivens, and B. Grabensee. 2002. Opportunistic infections after renal transplantation. Curr. Opin. Urol. 12:115–123. 76. Houang, E. T., C. J. Williams, and P. F. M. Wrigley. 1976. Acute Listeria monocytogenes osteomyelitis. Infection 4:113–114. 77. Hugot, J. P., C. Alberti, D. Berrebi, E. Bingen, and J. P. Cezard. 2003. Crohn’s disease: The cold chain hypothesis. Lancet 362:2012–2015. 78. Hume, O. S. 1976. Maternal L. monocytogenes septicemia with sparing of the fetus. Obstet. Gynecol. 48(suppl.):33S–34S. 79. Hussein, A. S., and S. D. Shafran. 2000. Acute bacterial meningitis in adults. A 12-year review. Medicine (Baltimore) 79:360–368. 80. Jacquet, C., B. Catimel, R. Brosch, C. Buchreiser, 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. 81. Jean, D., J. Croize, P. Hirtz, C. Legeais, I. Pelloux, M. Favier, M. R. Mallaret, P. L. Noc, and P. Rambaud. 1991. Infection nosocomiale à Listeria monocytogenes en maternité. Arch. Fr. Pediatr. 48:419–422.

DK3089_C004.fm Page 106 Tuesday, February 20, 2007 11:38 AM

106

Listeria, Listeriosis, and Food Safety

82. Jensen, A., W. Frederiksen, and P. Gerner-Smidt. 1994. Risk factors for listeriosis in Denmark, 1989–1990. Scan. J. Infect. Dis. 26:171–178. 83. Jurado, R. L., M. M. Farley, E. Pereira, R. C. Harvey, A. Schuchat, J. D. Wenger, and D. S. Stephens. 1993. Increased risk of meningitis and bacteremia due to Listeria monocytogenes in patients with human immunodeficiency virus infection. Clin. Infect. Dis. 17:224–227. 84. Kampelmacher, E. H., and L. M. v. N. Jansen. 1972. Further studies on the isolation of Listeria monocytogenes in clinically healthy individuals. Zentralb. Bakteriol. Mikrobiol. Hyg. Abt. I Orig. Reihe A 2221.70–77. 85. Kraus, A., A. R. Cabral, J. Sifuentes-Osornio, and D. Alarcón-Segovia. 1994. Listeriosis in patients with connective tissue diseases. J. Rheumatol. 21:635–638. 86. Lamont, R. J., and R. Postlethwaite. 1986. Carriage of Listeria monocytogenes and related species in pregnant and nonpregnant women in Aberdeen, Scotland. J. Infect. 13:187–193. 87. Larsson, S., A. Cederberg, S. I. L. Svanberg, and S. Cronberg. 1978. Listeria monocytogenes causing hospital acquired enterocolitis and meningitis in newborn infants. Br. Med. J. 2:473–474. 88. Lay, J., J. Varma, R. Marcus, et al. 2002. Higher incidence of Listeria infection among Hispanics: FoodNet, 1996–2000 [abstract 86]. In: Program and abstracts of the International Conference on Emerging Infectious Diseases; 2002; Atlanta: Centers for Disease Control and Prevention. 89. Linnan, M. J., L. Mascola, X. D. Lou, V. Goulet, S. May, C. Salminen, D. W. Hird, M. L. Yonkura, P. Hayes, R. Weaver, A. Audurier, B. D. Plikaytis, S. L. Fannin, A. Kleks, and C. V. Broome. 1988. Epidemic listeriosis associated with Mexican-style cheese. New Engl. J. Med. 319:823–828. 90. Lohmann, C. P., V. P. Gabel, M. Heep, H. J. Linde, and U. Reischl. 1999. Listeria monocytogenesinduced endogenous endophthalmitis in an otherwise healthy individual: Rapid PCR-diagnosis as the basis for effective treatment. Eur. J. Ophthalmol. 9:53–57. 91. Lorber, B. 1997. Listeriosis. Clin. Infect. Dis. 24:1–11. 92. Louria, D. B., T. Hensle, D. Armstrong, H. S. Collins, A. Blevins, D. Krugman, and M. Buse. 1967. Listeriosis complicating malignant disease: A new association. Ann. Intern. Med. 67:261–268. 93. MacDonald, P. D., R. Whitwam, J. Boggs, J. MacCormack, K. Anderson, J. Reardon, J. Saah, L. Graves, S. Hunter, and J. Sobel. 2005. Outbreak of listeriosis among Mexican immigrants as a result of consumption of illicitly produced Mexican-style cheese. Clin. Infect. Dis. 40:677–682. 94. MacGowan, A. P., R. J. Marshall, I. M. MacKay, and D. S. Reeves. 1991. Listeria fecal carriage by renal transplant recipients, haemodialysis patients and patients in general practice: Its relation to season, drug therapy, foreign travel, animal exposure, and diet. Epidemiol. Infect. 106:157–166. 95. Maijala, R., O. Lyytikainen, T. Autio, T. Aalto, L. Haavisto, and T. Honkanen-Buzalski. 2001. Exposure of Listeria monocytogenes within an epidemic caused by butter in Finland. Int. J. Food. Microbiol. 70:97–109. 96. Makaryus, A. N., R. Yang, R. Cohen, D. Rosman, J. Mangion, and S. Kort. 2004. A rare case of Listeria monocytogenes presenting as prosthetic valve bacterial endocarditis and aortic root abscess. Echocardiography 21:423–427. 97. Mascola, L., D. P. Ewert, and A. Eller. 1994. Listeriosis: A previously unreported medical complication in women with multiple gestations. Am. J. Obstet. Gynecol. 170:1328–1332. 98. Mascola, L., L. Lieb, J. Chiu, S. L. Fannin, and M. J. Linnan. 1988. Listeriosis: An uncommon opportunistic infection in patients with the acquired immunodeficiency syndrome. Am. J. Med. 84:162–164. 99. Mascola, L., F. Sorvillo, V. Goulet, B. Hall, R. Weaver, and M. Linnan. 1992. Fecal carriage of Listeria monocytogenes—observations during a community-wide, common-source outbreak. Clin. Infect. Dis. 15:557–558. 100. Mascola, L., F. Sorvillo, J. Neal, K. Iwakoshi, and R. Weaver. 1989. Surveillance of listeriosis in Los Angeles County, 1985–1986. A first year’s report. Arch. Intern. Med. 149:1569–1572. 101. Mazzuli, T., and I. E. Salit. 1991. Pleural fluid infection caused by Listeria monocytogenes: Case report and review. Rev. Infect. Dis. 13:564–570. 102. McLauchlin, J., S. M. Hall, S. K. Velani, and R. J. Gilbert. 1991. Human listeriosis and pate: A possible association. Br. Med. J. 303:773–775. 103. McLauchlin, J. S. 1967. Human listeriosis in Britain. Epidemiol. Infect. 104:181–189. 104. 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. 5:607–625.

DK3089_C004.fm Page 107 Tuesday, February 20, 2007 11:38 AM

Listeriosis in Humans

107

105. Miettinen, M. K., A. Siitonen, P. Heiskanen, H. Haajanen, K. J. Bjorkroth, and H. J. Korkeala. 1999. Molecular epidemiology of an outbreak of febrile gastroenteritis caused by Listeria monocytogenes in cold-smoked rainbow trout. J. Clin. Microbiol. 37:2358–2360. 106. Mossey, R. T., and J. Sondheimer. 1985. Listeriosis in patients with long-term hemodialysis and transfusional iron overload. Am. J. Med. 79:397–400. 107. Müller, H. E. 1990. Listeria isolations from feces of patients with diarrhea and from healthy food handlers. Infection 18:97–100. 108. Mylonakis, E., E. L. Hohmann, and S. B. Calderwood. 1998. Central nervous system infection with Listeria monocytogenes. 33 Years’ experience at a general hospital and review of 776 episodes from the literature. Medicine (Baltimore) 77:313–336. 109. Mylonakis, E., M. Paliou, E. L. Hohmann, S. B. Calderwood, and E. J. Wing. 2002. Listeriosis during pregnancy: A case series and review of 222 cases. Medicine (Baltimore) 81:260–269. 110. Nelson, K. E., D. Warren, A. M. Tomasi, T. N. Raju, and D. Vidyasagar. 1985. Transmission of neonatal listeriosis in a delivery room. Am. J. Dis. Child. 139:903–905. 111. Newman, J. H., S. Waycott, and J. L. M. Cooney. 1979. Arthritis due to Listeria monocytogenes. Arthritis Rheum. 22:1139–1140. 112. Nguyen, M. H., and V. L. Yu. 1994. Listeria monocytogenes peritonitis in cirrhotic patients. Dig. Dis. Sci. 39:215–218. 113. Nieman, R. E., and B. Lorber. 1980. Listeriosis in adults: A changing pattern. Rev. Infect. Dis. 2:207–227. 114. Nyfeldt, A. 1929. Etiologie de la mononucleose infectieuse. Soc. Biol. 101:590–592. 115. Okutani, A., Y. Okada, S. Yamamoto, and S. Igimi. 2004. Nationwide survey of human Listeria monocytogenes infection in Japan. Epidemiol. Infect. 132:769–72. 116. Olsen, S., M. Patrick, S. Hunter, V. Reddy, L. Kornstein, W. MacKenzie, K. Lane, S. Bidol, G. Stoltman, D. Frye, I. Lee, S. Hurd, T. Jones, T. LaPorte, W. Dewitt, L. Graves, M. Wiedmann, D. Schoonmaker-Bopp, A. Huang, C. Vincent, A. Bugenhagen. J. Corby, E. Carloni, M. Holcomb, R. Woron, S. Zansky, G. Dowdle, F. Smith, S. Ahrabi-Fard, A. Rea Ong, N. Tucker, N. Hynes, and P. Mead. 2005. Multistate outbreak of Listeria monocytogenes infection linked to delicatessen turkey meat. Clin. Infect. Dis. 40:962–967. 117. Ooi, S. T., and B. Lorber. 2005. Gastroenteritis due to Listeria monocytogenes. Clin. Infect. Dis. 40:1327–1332. 118. Owen, C. R., A. Meis, J. W. Jackson, and H. G. Stoenner. 1960. A case of primary cutaneous listeriosis. New Engl. J. Med. 262:1026–1028. 119. Pinner, R. W., and C. V. Broome. 1992. Listeria monocytogenes. Philadelphia: W. B. Saunders. 120. Pinner, R. W., A. Schuchat, B. Swaminathan, P. S. Hayes, K. Deaver, R. E. Weaver, B. D. Plikaytis, M. Reeves, C. V. Broome, J. D. Wenger, and the Listeria Study Group. 1992. Role of foods in sporadic listeriosis. II: Microbiologic and epidemiologic investigation. JAMA 267:2046–2050. 121. Rappaport, F., M. Rabinovitz, R. Toaff, and N. Krochic. 1960. Genital listeriosis as a cause of repeated abortions. Lancet 1:1273–1275. 122. Rettally, C. A., and K. V. Speeg. 2003. Infection with Listeria monocytogenes following orthotopic liver transplantation: case report and review of the literature. Transplant Proc. 35:1485–1487. 123. Riedo, F. X., R. W. Pinner, M. L. Tosca, M. L. Cartter, L. M. Graves, M. W. Reeves, R. E. Weaver, B. D. Plikaytis, and C. V. Broome. 1994. A point-source foodborne listeriosis outbreak: documented incubation period and possible mild illness. J. Infect. Dis. 170:693–696. 124. Rohde, H., M. A. Horstkotte, S. Loeper, J. Aberle, L. Jenicke, R. Lampidis, and D. Mack. 2004. Recurrent Listeria monocytogenes aortic graft infection: confirmation of relapse by molecular subtyping. Diagn. Microbiol. Infect. Dis. 48:63–67. 125. Ryser, E. T., and E. H. Marth. 1991. Conventional methods to detect and isolate Listeria monocytogenes. In Listeria, listeriosis, and food safety. New York: Marcel Dekker, pp. 120–193. 126. Ryser, E. T., and E. H. Marth. 1991. Rapid methods to detect Listeria monocytogenes in food and environmental samples. In Listeria, listeriosis, and food safety. New York: Marcel Dekker, pp. 94–239. 127. Sagoo, S. K., C. L. Little, L. Ward, I. A. Gillespie, and R. T. Mitchell. 2003. Microbiological study of ready-to-eat salad vegetables from retail establishments uncovers a national outbreak of salmonellosis. J. Food Prot. 66:403–409. 128. Salamina, G., E. Dalle Donne, A. Niccolini, G. Poda, D. Cesaroni, M. Bucci, R. Fini, M. Maldini, A. Schuchat, B. Swaminathan, W. Bibb, J. Rocourt, N. Binkin, and S. Salmaso. 1996. A foodborne outbreak of gastroenteritis involving Listeria monocytogenes. Epidemiol. Infect. 117:429–436.

DK3089_C004.fm Page 108 Tuesday, February 20, 2007 11:38 AM

108

Listeria, Listeriosis, and Food Safety

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).

REFERENCES 1. Abachin, E., C. Poyart, E. Pellegrini, E. Milohanic, F. Fiedler, P. Berche, and P. Trieu-Cuot. 2002. Formation of D-alanyl-lipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes. Mol. Microbiol. 43:1–14. 2. Adams, T. J., S. Vartivarian, and R. E. Cowart. 1990. Iron acquisition systems of Listeria monocytogenes. Infect. Immun. 58:2715–2718. 3. Alvarez-Dominguez, C., A. M. Barbieri, W. Beron, A. Wandinger-Ness, and P. D. Stahl. 1996. Phagocytosed live Listeria monocytogenes influences Rab5-regulated in vitro phagosome–endosome fusion. J. Biol. Chem. 271:13834–13843.

DK3089_C005.fm Page 140 Tuesday, February 20, 2007 11:40 AM

140

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4. Alvarez-Dominguez, C., E. Carrasco-Marin, and F. Leyva-Cobian. 1993. Role of complement component C1q in phagocytosis of Listeria monocytogenes by murine macrophage-like cell lines. Infect. Immun. 61:3664–3672. 5. Alvarez-Dominguez, C., R. Roberts, and P. D. Stahl. 1997. Internalized Listeria monocytogenes modulates intracellular trafficking and delays maturation of the phagosome. J. Cell Sci. 110:731–743. 6. Alvarez-Dominguez, C., and P. D. Stahl. 1999. Increased expression of Rab5a correlates directly with accelerated maturation of Listeria monocytogenes phagosomes. J. Biol. Chem. 274:11459–11462. 7. Alvarez-Dominguez, C., J. A. Vazquez-Boland, E. Carrasco-Marin, P. Lopez-Mato, and F. LeyvaCobian. 1997. Host cell heparan sulfate proteoglycans mediate attachment and entry of Listeria monocytogenes, and the listerial surface protein ActA is involved in heparan sulfate receptor recognition. Infect. Immun. 65:78–88. 8. Arnold, R., J. Scheffer, B. König, and W. König. 1993. Effects of Listeria monocytogenes and Yersinia enterocolitica on cytokine gene expression and release from human polymorphonuclear granulocytes and epithelial (Hep-2) cells. Infect. Immun. 61:2545–2552. 9. Auerbuch, V., J. J. Loureiro, F. B. Gertler, J. A. Theriot, and D. A. Portnoy. 2003. Ena/VASP proteins contribute to Listeria monocytogenes pathogenesis by controlling temporal and spatial persistence of bacterial actin-based motility. Mol. Microbiol. 49:1361–1375. 10. Autret, N., C. Raynaud, I. Dubail, P. Berche, and A. Charbit. 2003. Identification of the agr locus of Listeria monocytogenes: Role in bacterial virulence. Infect. Immun. 71:4463–4471. 11. Bannam, T., and H. Goldfine. 1999. Mutagenesis of active-site histidines of Listeria monocytogenes phosphatidylinositol-specific phospholipase C: Effects on enzyme activity and biological function. Infect. Immun. 67:182–186. 12. Barchini, E., and R. E. Cowart. 1966. Extracellular iron reductase activity produced by Listeria monocytogenes. Arch. Microbiol. 166:51–57. 13. Bauer, S., T. Pfeuffer, and M. Kuhn. 2003. Identification and characterisation of regions in the cellular protein LaXp180 and the Listeria monocytogenes protein ActA necessary for the interaction of the two proteins. Mol. Genet. Genom. 268:607–617. 14. Bear, J. E., M. Krause, and F. B. Gertler. 2001. Regulating cellular actin assembly. Curr. Opin. Cell Biol. 13:158–166. 15. Beauregard, K. E., K. D. Lee, R. J. Collier, and J. A. Swanson. 1997. pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes. J. Exp. Med. 186:1159–1163. 16. Behari, J., and P. Youngman. 1998. Regulation of hly expression in Listeria monocytogenes by carbon sources and pH occurs through separate mechanisms mediated by PrfA. Infect. Immun. 66:3635–3642. 17. Bergmann, B., D. Raffelsbauer, M. Kuhn, M. Goetz, S. Hom, and W. Goebel. 2002. InlA- but not InlB-mediated internalization of Listeria monocytogenes by nonphagocytic mammalian cells needs the support of other internalins. Mol. Microbiol. 43:557–570. 18. Bielecki, J., P. Youngman, P. Connelly, and D. A. Portnoy. 1990. Bacillus subtilis expressing a haemolysin gene from Listeria monocytogenes can grow in mammalian cells. Nature (London) 345:175–176. 19. Bierne, H., and P. Cossart. 2002. InlB, a surface protein of Listeria monocytogenes that behaves as an invasin and a growth factor. J. Cell Sci.115:3357–3367. 20. Bierne, H., E. Gouin, P. Roux, P. Caroni, H. L. Yin, and P. Cossart. 2001. A role for cofilin and LIM kinase in Listeria-induced phagocytosis. J. Cell Biol. 155:101–112. 21. Bierne, H., S. K. Mazmanian, M. Trost, M. G. Pucciarelli, G. Liu, P. Dehoux, L. Jansch, F. Garciadel Portillo, O. Schneewind, and P. Cossart. 2002. Inactivation of the srtA gene in Listeria monocytogenes inhibits anchoring of surface proteins and affects virulence. Mol. Microbiol. 43:869–881. 22. Böckmann, R., C. Dickneite, B. Middendorf, W. Goebel, and Z. Sokolovic. 1996. Specific binding of the Listeria monocytogenes transcriptional regulator PrfA to target sequences requires additional factor(s) and is influenced by iron. Mol. Microbiol. 22:643–653. 23. Bohne, J., H. Kestler, C. Uebele, Z. Sokolovic, and W. Goebel. 1996. Differential regulation of the virulence genes of Listeria monocytogenes by the transcriptional activator PrfA. Mol. Microbiol. 20:1189–1198. 24. Bohne, J., Z. Sokolovic, and W. Goebel. 1994. Transcriptional regulation of prfA and PrfA-regulated virulence genes in Listeria monocytogenes. Mol. Microbiol. 11:1141–1150. 25. Borezee, E., T. Msadek, L. Durant, and P. Berche. 2000. Identification in Listeria monocytogenes of MecA, a homologue of the Bacillus subtilis competence regulatory protein. J. Bacteriol. 182:5931–5934.

DK3089_C005.fm Page 141 Tuesday, February 20, 2007 11:40 AM

Molecular Virulence Determinants of Listeria monocytogenes

141

26. Boujemaa-Paterski, R., E. Gouin, G. Hansen, S. Samarin, C. Le Clainche, D. Didry, P. Dehoux, P. Cossart, C. Kocks, M. F. Carlier, and D. Pantaloni. 2001. Listeria protein ActA mimics WASp family proteins: it activates filament barbed end branching by Arp2/3 complex. Biochemistry 40:11390–11404. 27. Braun, L., S. Dramsi, P. Dehoux, H. Bierne, G. Lindahl, and P. Cossart. 1997. InlB: An invasion protein of Listeria monocytogenes with a novel type of surface association. Mol. Microbiol. 25:285–294. 28. Braun, L., B. Ghebrehiwet, and P. Cossart. 2000. gC1q-R/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes. EMBO J. 19:1458–1466. 29. Brehm, K., A. Haas, W. Goebel, and J. Kreft. 1993. A gene encoding a superoxide dismutase of the facultative intracellular bacterium Listeria monocytogenes. Gene 118:121–125. 30. Brehm, K., J. Kreft, M. T. Ripio, and J.-A. Vazquez-Boland. 1996. Regulation of virulence gene expression in pathogenic Listeria. Microbiol. Sem. 12:219–236. 31. Brehm, K., M. T. Ripio, J. Kreft, and J. A. Vazquez-Boland. 1999. The bvr locus of Listeria monocytogenes mediates virulence gene repression by beta-glucosides. J. Bacteriol. 181:5024–5032. 32. Brown, J.S., and D.W. Holden. 2002. Iron acquisition by Gram-positive bacterial pathogens. Microbes Infect. 4:1149–1156. 33. Brundage, R. A., G. A. Smith, A. Camilli, J. A. Theriot, and D. A. Portnoy. 1993. Expression and phosphorylation of the Listeria monocytogenes ActA protein in mammalian cells. Proc. Natl. Acad. Sci. USA 90:11890–11894. 34. Bubert, A., M. Kuhn, W. Goebel, and S. Köhler. 1992. Structural and functional properties of the p60 proteins from different Listeria species. J. Bacteriol. 174:8166–8171. 35. Bubert, A., J. Riebe, N. Schnitzler, A. Schönberg, W. Goebel, and P. Schubert. 1997. Isolation of catalase-negative Listeria monocytogenes strains from listeriosis patients and their rapid identification by anti-p60 antibodies and/or PCR. J. Clin. Microbiol. 35:179–183. 36. Bubert, A., Z. Sokolovic, S. K. Chun, L. Papatheodorou, A. Simm, and W. Goebel. 1999. Differential expression of Listeria monocytogenes virulence genes in mammalian host cells. Mol. Gen. Genet. 261:323–336. 37. Buchrieser, C., C. Rusniok, F. Kunst, P. Cossart, P. Glaser, and the Listeria Consortium. 2003. Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua: Clues for evolution and pathogenicity. FEMS Immunol. Med. Microbiol. 35:207–213. 38. Cabanes, D., P. Dehoux, O. Dussurget, L. Frangeul, and P. Cossart. 2002. Surface proteins and the pathogenic potential of Listeria monocytogenes. Trends Microbiol. 10:238–245. 39. Cameron, L. A., M. J. Footer, A. van Oudenaarden, and J. A. Theriot. 1999. Motility of ActA proteincoated microspheres driven by actin polymerization. Proc. Natl. Acad. Sci. USA 96:4908–4913. 40. Cameron, L. A., P. A. Giardini, F. S. Soo, and J. A. Theriot. 2000. Secrets of actin-based motility revealed by a bacterial pathogen. Nat. Rev. Mol. Cell Biol. 1:110–119. 41. Camilli, A., H. Goldfine, and D. A. Portnoy. 1991. Listeria monocytogenes mutants lacking phosphaditylinositol-specific phospholipase C are avirulent. J. Exp. Med. 173:751–754. 42. Camilli, A., L. G. Tilney, and D. A. Portnoy. 1993. Dual roles of PlcA in Listeria monocytogenes pathogenesis. Mol. Microbiol. 8:143–157. 43. Carroll, S. A., T. Hain, U. Technow, A. Darji, P. Pashalidis, S. W. Joseph, and T. Chakraborty. 2003. Identification and characterization of a peptidoglycan hydrolase, MurA, of Listeria monocytogenes, a muramidase needed for cell separation. J. Bacteriol. 185:6801–6808. 44. Chakraborty, T., F. Ebel, E. Domann, K. Niebuhr, B. Gerstel, S. Pistor, C. J. Temm-Grove, B. M. Jockusch, M. Reinhard, U. Walter, and J. Wehland. 1995. A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells. EMBO J. 14:1314–1321. 45. Chakraborty, T., T. Hain, and E. Domann. 2000. Genome organization and the evolution of the virulence gene locus in Listeria species. Int. J. Med. Microbiol. 290:167–174. 46. Chakraborty, T., and T. Hain. Unpublished results. 47. Chakraborty, T., M. Leimeister-Wächter, E. Domann, M. Hartl, W. Goebel, T. Nichterlein, and S. Notermans. 1992. Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene. J. Bacteriol. 174:568–574. 48. Chico-Calero, I., M. Suarez, B. Gonzalez-Zorn, M. Scortti, J. Slaghuis, W. Goebel, and J. A. VazquezBoland. 2002. Hpt, a bacterial homolog of the microsomal glucose-6-phosphate translocase, mediates rapid intracellular proliferation in Listeria. Proc. Natl. Acad. Sci. USA 99:431–436.

DK3089_C005.fm Page 142 Tuesday, February 20, 2007 11:40 AM

142

Listeria, Listeriosis, and Food Safety

49. Cicchetti, G., P. Maurer, P. Wagener, and C. Kocks. 1999. Actin and phosphoinositide binding by the ActA protein of the bacterial pathogen Listeria monocytogenes. J. Biol. Chem. 274:33616–33626. 50. Coffey, A., B. van den Burg, R. Veltman, and T. Abee. 2000. Characteristics of the biologically active 35-kDa metalloprotease virulence factor from Listeria monocytogenes. J. Appl. Microbiol. 88:132–141. 51. Conte, M. P., G. Petrone, C. Longhi, P. Valenti, R. Morelli, F. Superti, and L. Seganti. 1996. The effects of inhibitors of vacuolar acidification on the release of Listeria monocytogenes from phagosomes of Caco-2 cells. J. Med. Microbiol. 44:418–424. 52. Copp, J., M. Marino, M. Banerjee, P. Ghosh, and P. van der Geer. 2003. Multiple regions of internalin B contribute to its ability to turn on the Ras-mitogen-activated protein kinase pathway. J. Biol. Chem. 278:7783–7789. 53. Cossart, P. 2000. Actin-based motility of pathogens: the Arp2/3 complex is a central player. Cell. Microbiol. 2:195–205. 54. Cossart, P., J. Pizarro-Cerda, and M. Lecuit. 2003. Invasion of mammalian cells by Listeria monocytogenes: functional mimicry to subvert cellular functions. Trends Cell. Biol. 13:23–31. 55. Cossart, P., M. F. Vicente, J. Mengaud, F. Baquero, J. C. Perez-Diaz, and P. Berche. 1989. Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation. Infect. Immun. 57:3629–3636. 56. Cotter, P. D., N. Emerson, C. G. Gahan, and C. Hill. 1999. Identification and disruption of lisRK, a genetic locus encoding a two-component signal transduction system involved in stress tolerance and virulence in Listeria monocytogenes. J. Bacteriol. 181:6840–6843. 57. Coulanges, V., P. Andre, and D. J. Vidon. 1998. Effect of siderophores, catecholamines, and catechol compounds on Listeria spp. growth in iron-complexed medium. Biochem. Biophys. Res. Commun. 249:526–530. 58. Cowart, R. E., and B. G. Foster. 1985. Differential effects of iron on the growth of Listeria monocytogenes: Minimum requirements and mechanism of acquisition. J. Infect. Dis. 151:721–730. 59. Croize, J., J. Arvieux, P. Berche, and M. G. Colomb. 1993. Activation of the human complement alternative pathway by Listeria monocytogenes: evidence for direct binding and proteolysis of the C3 component on bacteria. Infect. Immun. 61:5134–5139. 60. Dabiri, G. A., J. M. Sanger, D. A. Portnoy, and F. S. Southwick. 1990. Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly. Proc. Natl. Acad. Sci. USA 87:6068–6072. 61. Dancz, C. E., A. Haraga, D. A. Portnoy, and D. E. Higgins. 2002. Inducible control of virulence gene expression in Listeria monocytogenes: temporal requirement of listeriolysin O during intracellular infection. J. Bacteriol. 184:5935–5945. 62. Datta, A. R., and M. H. Kothary. 1993. Effects of glucose, growth temperature, and pH on listeriolysin O production in Listeria monocytogenes. Appl. Environ. Microbiol. 59:3495–3497. 63. David, V., E. Gouin, M. V. Troys, A. Grogan, A. W. Segal, C. Ampe, and P. Cossart. 1998. Identification of cofilin, coronin, Rac and capZ in actin tails using a Listeria affinity approach. J. Cell Sci. 111:2877–2884. 64. Davies, W. A. 1983. Kinetics of killing of Listeria monocytogenes by macrophages: Rapid killing accompanying phagocytosis. J. Reticuloendothel. Soc. 34:131–141. 65. Decatur, A. L., and D. A. Portnoy. 2000. A PEST-like sequence in listeriolysin O essential for Listeria monocytogenes pathogenicity. Science 290:992–995. 66. De Chastellier, C., and P. Berche. 1994. Fate of Listeria monocytogenes in murine macrophages: evidence for simultaneous killing and survival of intracellular bacteria. Infect. Immun. 62:543–553. 67. Deneer, H. G., V. Healey, and I. Boychuk. 1995. Reduction of exogenous ferric iron by a surfaceassociated ferric reductase of Listeria spp. Microbiology 141:1985–1992. 68. Dhar, G., K. F. Faull, and O. Schneewind. 2000. Anchor structure of cell wall surface proteins in Listeria monocytogenes. Biochemistry 39:3725–3733. 69. Dickneite, C., R. Böckmann, A. Spory, W. Goebel, and Z. Sokolovic. 1998. Differential interaction of the transcription factor PrfA and the PrfA-activating factor (Paf) of Listeria monocytogenes with target sequences. Mol. Microbiol. 27:915–928. 70. Dold, F. G., J. M. Sanger, and J. W. Sanger. 1994. Intact α-actinin molecules are needed for both the assembly of actin into tails and the locomotion of Listeria monocytogenes inside infected cells. Cell Motil. Cytoskel. 28:97–107.

DK3089_C005.fm Page 143 Tuesday, February 20, 2007 11:40 AM

Molecular Virulence Determinants of Listeria monocytogenes

143

71. Domann, E., and T. Chakraborty. 1989. Nucleotide sequence of the listeriolysin gene from a Listeria monocytogenes serotype 1/2a strain. Nucl. Acids Res. 17:6406. 72. Domann, E., M. Leimeister-Wächter, W. Goebel, and T. Chakraborty. 1991. Molecular cloning, sequencing, and identification of a metalloprotease gene from Listeria monocytogenes that is species specific and physically linked to the listeriolysin gene. Infect. Immun. 59:65–72. 73. Domann, E., J. Wehland, K. Niebuhr, C. Haffner, M. Leimeister-Wächter, and T. Chakraborty. 1993. Detection of a prfA-independent promoter responsible for listeriolysin gene expression in mutant Listeria monocytogenes strains lacking the PrfA regulator. Infect. Immun. 61:3073–3075. 74. Domann, E., J. Wehland, M. Rohde, S. Pistor, M. Hartl, W. Goebel, M. Leimeister-Wächter, M. Wuenscher, and T. Chakraborty. 1992. A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. EMBO J. 11:1981–1990. 75. Domann, E., S. Zechel, A. Lingnau, T. Hain, A. Darji, T. Nichterlein, J. Wehland, and T. Chakraborty. 1997. Identification and characterization of a novel PrfA-regulated gene in Listeria monocytogenes whose product, IrpA, is highly homologous to internalin proteins, which contain leucin-rich repeats. Infect. Immun. 65:101–109. 76. Dramsi, S., I. Biswas, E. Maguin, L. Braun, P. Mastroeni, and P. Cossart. 1995. Entry of Listeria monocytogenes into hepatocytes requires expression of InlB, a surface protein of the internalin multigen family. Mol. Microbiol. 16:251–261. 77. Dramsi, S., P. Dehoux, and P. Cossart. 1993. Common features of Gram-positive bacterial proteins involved in cell recognition. Mol. Microbiol. 9:1119–1122. 78. Dramsi, S., P. Dehoux, M. Lebrun, P. L. Goossens, and P. Cossart. 1997. Identification of four new members of the internalin multigene family of Listeria monocytogenes EGD. Infect. Immun. 65:1615–1625. 79. Dramsi, S., C. Kocks, C. Forestier, and P. Cossart. 1993. Internalin-mediated invasion of epithelial cells by Listeria monocytogenes is regulated by the bacterial growth state, temperature and the pleiotropic activator prfA. Mol. Microbiol. 9:931–941. 80. Drevets, D. A., and P. A. Campbell. 1991. Roles of complement and complement receptor type 3 in phagocytosis of Listeria monocytogenes by inflammatory mouse peritoneal macrophages. Infect. Immun. 59:2645–2652. 81. Drevets, D. A., P. J. M. Leenen, and P. A. Campbell. 1993. Complement receptor type 3 (CD11b/CD18) involvement is essential for killing of Listeria monocytogenes by mouse macrophages. J. Immunol. 151:5431–5439. 82. Drevets, D. A., R. T. Sawyer, T. A. Potter, and P. A. Campbell. 1995. Listeria monocytogenes infects human endothelial cells by two distinct mechanisms. Infect. Immun. 63:4268–4276. 83. Dubail, I., N. Autret, J. L. Beretti, S. Kayal, P. Berche, and A. Charbit. 2001. Functional assembly of two membrane-binding domains in listeriolysin O, the cytolysin of Listeria monocytogenes. Microbiology 147:2679–2688. 84. Dubail, I., P. Berche, and A. Charbit. 2000. Listeriolysin O as a reporter to identify constitutive and in vivo-inducible promoters in the pathogen Listeria monocytogenes. Infect. Immun. 68:3242–3250. 85. Dunne, D. W., D. Resnick, J. Greenberg, M. Krieger, and K. A. Joiner. 1994. The type I macrophage scavenger receptor binds to Gram-positive bacteria and recognizes lipoteichoic acid. Proc. Natl. Acad. Sci. USA 91:1863–1867. 86. Dussurget, O., D. Cabanes, P. Dehoux, M. Lecuit, C. Buchrieser, P. Glaser, and P. Cossart; European Listeria Genome Consortium. 2002. Listeria monocytogenes bile salt hydrolase is a PrfA-regulated virulence factor involved in the intestinal and hepatic phases of listeriosis. Mol. Microbiol. 45:1095–1106. 87. Eckmann, L., M. F. Kagnoff, and J. Fierer. 1993. Epithelial cells secrete the chemokine interleukin8 in response to bacterial entry. Infect. Immun. 61:4569–4574. 88. Elsner, H. A., I. Sobottka, A. Bubert, H. Albrecht, R. Laufs, and D. Mack. 1996. Catalase-negative Listeria monocytogenes causing lethal sepsis and meningitis in an adult hematologic patient. Eur. J. Clin. Microbiol. Infect. Dis. 15:965–967. 89. Engelbrecht, F., S.-K. Chun, C. Ochs, J. Hess, F. Lottspeich, W. Goebel, and Z. Sokolovic. 1996. A new PrfA-regulated gene of Listeria monocytogenes encoding a small, secreted protein which belongs to the family of internalins. Mol. Microbiol. 21:823–837.

DK3089_C005.fm Page 144 Tuesday, February 20, 2007 11:40 AM

144

Listeria, Listeriosis, and Food Safety

90. Falzano, L., C. Fiorentini, G. Donelli, E. Michel, C. Kocks, P. Cossart, L. Cabanie, E. Oswald, and P. Boquet. 1993. Induction of phagocytic behaviour in human epithelial cells by Escherichia coli cytotoxic necrotizing factor type 1. Mol. Microbiol. 9:1247–1254. 91. Fiedler, F. 1988. Biochemistry of the cell surface of Listeria strains: A locating general view. Infection 16 Suppl 2:92–97. 92. Finlay, B. B., and S. Falkow. 1997. Common themes in microbial pathogenicity revisited. Microbiol. Mol. Biol. Rev. 61:136–169. 93. Flanary, P. L., R. D. Allen, L. Dons, and S. Kathariou. 1999. Insertional inactivation of the Listeria monocytogenes cheYA operon abolishes response to oxygen gradients and reduces the number of flagella. Can. J. Microbiol. 45:646–652. 94. Fradelizi, J., V. Noireaux, J. Plastino, B. Menichi, D. Louvard, C. Sykes, R. M. Golsteyn, and E. Friederich. 2001. ActA and human zyxin harbour Arp2/3-independent actin-polymerization activity. Nat. Cell Biol. 3:699–707. 95. Freitag, N. E., and K. E. Jacobs. 1999. Examination of Listeria monocytogenes intracellular gene expression by using the green fluorescent protein of Aequorea victoria. Infect. Immun. 67:1844–1852. 96. Freitag, N. E. and D. A. Portnoy. 1994. Dual promoters of the Listeria monocytogenes prfA transcriptional activator appear essential in vitro but are redundant in vivo. Mol. Microbiol. 12:845–853. 97. Freitag, N. E., L. Rong, and D. A. Portnoy. 1993. Regulation of the prfA transcriptional activator in Listeria monocytogenes: Multiple promoter elements contribute to intracellular growth and cell-tocell spread. Infect. Immun. 61:2537–2544. 98. Freitag, N. E., P. Youngman, and D. A. Portnoy. 1992. Transcriptional activation of the Listeria monocytogenes haemolysin gene in Bacillus subtilis. J. Bacteriol. 174:1293–1298. 99. Friederich, E., E. Gouin, R. Hellio, C. Kocks, P. Cossart, and D. Louvard. 1995. Targeting of Listeria monocytogenes ActA protein to the plasma membrane as a tool to disect both actin-based cell morphogenesis and ActA function. EMBO J. 14:2731–2744. 100. Fuzi, M., and I. Pillis. 1962. Production of opacity in egg yolk medium by Listeria monocytogenes. Nature (London) 196:195. 101. Gahan, C. G., and C. Hill. 2000. The use of listeriolysin to identify in vivo induced genes in the Gram-positive intracellular pathogen Listeria monocytogenes. Mol. Microbiol. 36:498–507. 102. Gaillard, J. L., P. Berche, C. Frehel, E. Gouin, and P. Cossart. 1991. Entry of Listeria monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from Grampositive cocci. Cell 65:1127–1141. 103. Gaillard, J. L., P. Berche, J. Mounier, S. Richard, and P. J. Sansonetti. 1987. In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect. Immun. 55:2822–2829. 104. Gaillard, J. L., P. Berche, and P. J. Sansonetti. 1986. Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes. Infect. Immun. 52:50–55. 105. Gaillard, J. L., F. Jaubert, and P. Berche. 1996. The inlAB locus mediates the entry of Listeria monocytogenes into hepatocytes in vivo. J. Exp. Med. 183:359–369. 106. Gaillot, O., S. Bregenholt, F. Jaubert, J. P. Di Santo, and P. Berche. 2001. Stress-induced ClpP serine protease of Listeria monocytogenes is essential for induction of listeriolysin O-dependent protective immunity. Infect. Immun. 69:4938–4943. 107. Gaillot, O., E. Pellegrini, S. Bregenholt, S. Nair, and P. Berche. 2000. The ClpP serine protease is essential for the intracellular parasitism and virulence of Listeria monocytogenes. Mol. Microbiol. 35:1286–1294. 108. Gandhi, A. J., B. Perussia, and H. Goldfine. 1993. Listeria monocytogenes phosphatidylinositol (PI)-specific phospholipase C has low activity on glycosyl-PI-anchored proteins. J. Bacteriol. 175: 8014–8017. 109. Garandeau, C., H. Reglier-Poupet, I. Dubail, J. L. Beretti, P. Berche, and A. Charbit. 2002. The sortase SrtA of Listeria monocytogenes is involved in processing of internalin and in virulence. Infect. Immun. 70:1382–1390. 110. Gedde, M. M., D. E. Higgins, L. G. Tilney, and D. A. Portnoy. 2000. Role of listeriolysin O in cellto-cell spread of Listeria monocytogenes. Infect. Immun. 68:999–1003. 111. Geoffroy, C., J. L. Gaillard, J. E. Alouf, and P. Berche. 1987. Purification, characterization, and toxicity of the sulfhydyl-activated hemolysin listeriolysin O from Listeria monocytogenes. Infect. Immun. 55:1641–1646.

DK3089_C005.fm Page 145 Tuesday, February 20, 2007 11:40 AM

Molecular Virulence Determinants of Listeria monocytogenes

145

112. Geoffroy, C., J. Raveneau, J. L. Beretti, A. Lecroisey, J. A. Vazquez-Boland, J. E. Alouf, and P. Berche. 1991. Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes. Infect. Immun. 59:2382–2388. 113. Gertler, F. B., K. Niebuhr, M. Reinhard, J. Wehland, and P. Soriano. 1996. Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87:227–239. 114. Gilot, P., P. Andre, and J. Content. 1999. Listeria monocytogenes possesses adhesins for fibronectin. Infect. Immun. 67:6698–6701. 115. Gilot, P., Y. Jossin, and J. Content. 2000. Cloning, sequencing and characterisation of a Listeria monocytogenes gene encoding a fibronectin-binding protein. J. Med. Microbiol. 49:887–896. 116. Glaser, P., C. Buchrieser, and P. Cossart. Unpublished results. 117. Glaser, P., L. Frangeul, C. Buchrieser, C. Rusniok, A. Amend, F. Baquero, P. Berche, H. Bloecker, P. Brandt, T. Chakraborty, A. Charbit, F. Chétouani, E. Couvé, A. de Daruvar, P. Dehoux, E. Domann, G. Domínguez-Bernal, E. Duchaud, L. Durant, O. Dussurget, K.-D. Entian, H. Fsihi, F. Garcia-Del Portillo, P. Garrido, L. Gautier, W. Goebel, N. Gomez-Lopez, T. Hain, J. Hauf, D. Jackson, L.-M. Jones, U. Kaerst, J. Kreft, M. Kuhn, F. Kunst, G. Kurapkat, E. Madueno, A. Maitournam, J. Mata Vicente, E. Ng, H. Nedjari, G. Nordsiek, S. Novella, B. de Pablos, J.-C. Pérez-Diaz, R. Purcell, B. Remmel, M. Rose, T. Schlueter, N. Simoes, A. Tierrez, J. A. Vázquez-Boland, H. Voss, J. Wehland, and P. Cossart. 2001. Comparative genomics of Listeria species. Science 294:849–852. 118. Glomski, I. J., M. M. Gedde, A. W. Tsang, J. A. Swanson, and D. A. Portnoy. 2002. The Listeria monocytogenes hemolysin has an acidic pH optimum to compartmentalize activity and prevent damage to infected host cells. J. Cell Biol. 156:1029–1038. 119. Glomski, I. J., A. L. Decatur, and D. A. Portnoy. 2003. Listeria monocytogenes mutants that fail to compartmentalize listerolysin O activity are cytotoxic, avirulent, and unable to evade host extracellular defenses. Infect. Immun. 71:6754–6765. 120. Goebel, W., and M. Kuhn. 2000. Bacterial replication in the host cell cytosol. Curr. Opin. Microbiol. 3:49–53. 121. Goebel, W., M. Leimeister-Wächter, M. Kuhn, E. Domann, T. Chakraborty, S. Köhler, A. Bubert, M. Wuenscher, and Z. Sokolovic. 1993. Listeria monocytogenes—a model system for studying the pathomechanisms of an intracellular microorganism. Zbl. Bakt. 278:334–347. 122. Goetz, M., A. Bubert, G. Wang, I. Chico-Calero, J. A. Vazquez-Boland, M. Beck, J. Slaghuis, A. A. Szalay, and W. Goebel. 2001. Microinjection and growth of bacteria in the cytosol of mammalian host cells. Proc. Natl. Acad. Sci. USA 98:12221–12226. 123. Goldfine, H., N. C. Johnston, and C. Knob. 1993. Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixes micelles and in biological membranes. J. Bacteriol. 175:4298–4306. 124. Goldfine, H., and C. Knob. 1992. Purification and characterization of Listeria monocytogenes phosphatidylinositol-specific phospholipase C. Infect. Immun. 60:4059–4067. 125. Goldfine, H., and S. J. Wadsworth. 2002. Macrophage intracellular signaling induced by Listeria monocytogenes. Microbes Infect. 4:1335–1343. 126. Gormley, E., J. Mengaud, and P. Cossart. 1989. Sequences homologous to the listeriolysin O gene region of Listeria monocytogenes are present in virulent and avirulent haemolytic species of the genus Listeria. Res. Microbiol. 140:631–643. 127. Gouin, E., J. Mengaud, and P. Cossart. 1994. The virulence gene cluster of Listeria monocytogenes is also present in Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species. Infect. Immun. 62:3550–3553. 128. Greenberg, J. W., W. Fischer, and K. A. Joiner. 1996. Influence of lipoteichoic acid structure on recognition by the macrophage scavenger receptor. Infect. Immun. 64:3318–3325. 129. Greiffenberg, L., W. Goebel, K. S. Kim, J. Daniels, and M. Kuhn. 2000. Interaction of Listeria monocytogenes with human brain microvascular endothelial cells: an electron microscopic study. Infect. Immun. 68:3275–3279. 130. Greiffenberg, L., W. Goebel, K. S. Kim, I. Weiglein, A. Bubert, F. Engelbrecht, M. Stins, and M. Kuhn. 1998. Interaction of Listeria monocytogenes with human brain microvascular endothelial cells: InlB-dependent invasion, long-term intracellular growth and spread from macrophages to endothelial cells. Infect. Immun. 66:5260–5267. 131. Greiffenberg, L., Z. Sokolovic, H. J. Schnittler, A. Spory, R. Böckmann, W. Goebel, and M. Kuhn. 1997. Listeria monocytogenes-infected human umbilical vein endothelial cells: Internalin-independent invasion, intracellular growth, movement, and host cell responses. FEMS Microbiol. Lett. 157:163–170.

DK3089_C005.fm Page 146 Tuesday, February 20, 2007 11:40 AM

146

Listeria, Listeriosis, and Food Safety

132. Gross, R., B. Arico, and R. Rappuoli. 1989. Families of bacterial signal-transducing proteins. Mol. Microbiol. 3:1661–1667. 133. Gründling, A., M. D. Gonzalez, and D. E. Higgins. 2003. Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells. J. Bacteriol. 185:6295–6307. 134. Gunn, J. S. 2000. Mechanisms of bacterial resistance and response to bile. Microbes Infect. 2:907–913. 135. Guzman, C., M. Rhode, T. Chakraborty, E. Domann, M. Hudel, J. Wehland, and K. Timmis. 1995. Interaction of Listeria monocytogenes with mouse dentritic cells. Infect. Immun. 63:3665–3673. 136. Haas, A., M. Dumbsky, and J. Kreft. 1992. Listeriolysin genes: complete sequence of ilo from Listeria ivanovii and of lso from Listeria seeligeri. Biochim. Biophys. Acta 1130:81–84. 137. Haas, A., and W. Goebel. 1992. Microbial strategies to prevent oxygen-dependent killing by phagocytes. Free Rad. Res. Comms. 16:137–157. 138. Hackstadt, T. 2000. Redirection of host vesicle trafficing pathways by intracellular parasites. Traffic 1:93–99. 139. Hanawa, T., T. Yamamoto, and S. Kamiya. 1995. Listeria monocytogenes can grow in macrophages without the aid of proteins induced by environmental stresses. Infect. Immun. 63:4595–4599. 140. Hartford, T., S. O’Brien, P. W. Andrew, D. Jones, and I. S. Roberts. 1993. Utilization of transferrinbound iron by Listeria monocytogenes. FEMS Microbiol. Lett. 108:311–318. 141. Hauf, N., W. Goebel, F. Fiedler, Z. Sokolovic, and M. Kuhn. 1997. Listeria monocytogenes infection of P388D1 macrophages results in a biphasic NF-κB (RelA/p50) activation induced by lipoteichoic acid and bacterial phospholipases and mediated by IκBα and IκBβ degradation. Proc. Natl. Acad. Sci. USA 94:3994–3999. 142. Havell, E. A. 1986. Synthesis and secretion of interferon by murine fibroblasts in response to intracellular Listeria monocytogenes. Infect. Immun. 54:787–792. 143. Heesemann, J., D. Heinz, H. Rüssmann, J. Wehland, W. Goebel, and M. Kuhn. 2003. Lektionen aus der Bakterienwelt: Wie Krankheitserreger Wirtszellprozesse ausnützen. Biospektrum 9:486–489. 144. Herler, M., A. Bubert, M. Goetz, Y. Vega, J. A. Vazquez-Boland, and W. Goebel. 2001. Positive selection of mutations leading to loss or reduction of transcriptional activity of PrfA, the central regulator of Listeria monocytogenes virulence. J. Bacteriol. 183:5562–5570. 145. Hertzig, T., M. Weber, L. Greiffenberg, B. Schulte Holthausen, W. Goebel, K. S. Kim, and M. Kuhn. 2003. Antibodies present in normal human serum inhibit invasion of human brain microvascular endothelial cells by Listeria monocytogenes. Infect. Immun. 71:95–100. 146. Hess, J., I. Gentschev, G. Szalay, C. Ladel, A. Bubert, W. Goebel, and S. H. E. Kaufmann. 1995. Listeria monocytogenes p60 supports host cell invasion by and in vivo survival of attenuated Salmonella typhimurium. Infect. Immun. 63:2047–2053. 147. Hof, H., and P. Hefner. 1988. Pathogenicity of Listeria monocytogenes in comparison to other Listeria species. Infection 16 (Suppl. 2):141–144. 148. Huillet, E., S. Larpin, P. Pardon, and P. Berche. 1999. Identification of a new locus in Listeria monocytogenes involved in cellobiose-dependent repression of hly expression. FEMS Microbiol. Lett. 174:265–272. 149. Hurme, R., and M. Rhen. 1998. Temperature sensing in bacterial gene regulation—what it all boils down to. Mol. Microbiol. 30:1–6. 150. Ireton, K., B. Payrastre, H. Chap, W. Ogawa, H. Sakaue, M. Kasuga, and P. Cossart. 1996. A role for phosphoinositide 3-kinase in bacterial invasion. Science 274:780–782. 151. Ireton, K., B. Payrastre, and P. Cossart. 1999. The Listeria monocytogenes protein InlB is an agonist of mammalian phosphoinositide 3-kinase. J. Biol. Chem. 274:17025–17032. 152. Ito, Y., I. Kawamura, C. Kohda, H. Baba, T. Kimoto, I. Watanabe, T. Nomura, and M. Mitsuyama. 2001. Difference in cholesterol-binding and cytolytic activities between listeriolysin O and seeligeriolysin O: A possible role of alanine residue in tryptophan-rich undecapeptide. FEMS Microbiol. Lett. 203:185–189. 153. Jacobs, T., A. Darji, N. Frahm, M. Rohde, J. Wehland, T. Chakraborty, and S. Weiss. 1998. Listeriolysin O: Cholesterol inhibits cytolysis but not binding to cellular membranes. Mol. Microbiol. 28:1081–1089. 154. Johansson, J., and P. Cossart. 2003. RNA-mediated control of virulence gene expression in bacterial pathogens. Trends Microbiol. 11:280–285.

DK3089_C005.fm Page 147 Tuesday, February 20, 2007 11:40 AM

Molecular Virulence Determinants of Listeria monocytogenes

147

155. Johansson, J., P. Mandin, A. Renzoni, C. Chiaruttini, M. Springer, and P. Cossart. 2002. An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell 110:551–561. 156. Jones, S., and D. A. Portnoy. 1994. Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O instead of listeriolysin O. Infect. Immun. 62:5608–5613. 157. Jones, S., K. Preiter, and D. A. Portnoy. 1996. Conversion of an extracellular cytolysin into a phagosome-specific lysin which supports the growth of an intracellular pathogen. Mol. Microbiol. 21:1219–1225. 158. Jonquieres, R., H. Bierne, F. Fiedler, P. Gounon, and P. Cossart. 1999. Interaction between the protein InlB of Listeria monocytogenes and lipoteichoic acid: A novel mechanism of protein association at the surface of Gram-positive bacteria. Mol. Microbiol. 34:902–914. 159. Jonquieres, R., J. Pizarro-Cerda, and P. Cossart. 2001. Synergy between the N- and C-terminal domains of InlB for efficient invasion of non-phagocytic cells by Listeria monocytogenes. Mol. Microbiol. 42:955–965. 160. Kallipolitis, B. H., and H. Ingmer. 2001. Listeria monocytogenes response regulators important for stress tolerance and pathogenesis. FEMS Microbiol. Lett. 204:111–115. 161. Kallipolitis, B. H., H. Ingmer, C. G. Gahan, C. Hill, and L. Sogaard–Andersen. 2003. CesRK, a twocomponent signal transduction system in Listeria monocytogenes, responds to the presence of cell wall-acting antibiotics and affects β-lactam resistance. Antimicrob. Agents Chemother. 47:3421–3429. 162. Karunasagar, I., G. Krohne, and W. Goebel. 1993. Listeria ivanovii is capable of cell-to-cell spread involving actin polymerization. Infect. Immun. 61:162–169. 163. Kathariou, S., P. Metz, H. Hof, and W. Goebel. 1987. Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes. J. Bacteriol. 169:1291–1297. 164. Kathariou, S., L. Pine. V. George, G. M. Carlone, and B. P. Holloway. 1990. Nonhemolytic Listeria monocytogenes mutants that are also noninvasive for mammalian cells in culture: evidence for coordinate regulation of virulence. Infect. Immun. 58:3988–3995. 165. Kazmierczak, M. J., S. C. Mithoe, K. J. Boor, and M. Wiedmann. 2003. Listeria monocytogenes sigma B regulates stress response and virulence functions. J. Bacteriol. 185:5722–5734. 166. Klarsfeld, A. D., P. L. Goossens, and P. Cossart. 1994. Five Listeria monocytogenes genes preferentially expressed in infected mammalian cells: plcA, purH, purD, pyrE and an arginine ABC transporter gene arp. J. Mol. Microbiol. 13:585–597. 167. Kocks, C. 1994. Directional actin assembly by Listeria monocytogenes at the site of polar surface expression of the actA gene product involving the actin-binding protein plastin (fimbrin). Inf. Agents. Dis. 2:207–209. 168. Kocks, C., E. Gouin, M. Tabouret, P. Berche, H. Ohayon, and P. Cossart. 1992. Listeria monocytogenesinduced actin assembly requires the actA gene product, a surface protein. Cell 68:521–531. 169. Kocks, C., R. Hellio, P. Gounon, H. Ohayon, and P. Cossart. 1993. Polarized distribution of Listeria monocytogenes surface protein ActA at the site of directional actin assembly. J. Cell Sci. 3:699–710. 170. Kocks, C., J. B. Marchand, E. Gouin, H. d’Hauteville, P. J. Sansonetti, M. F. Carlier, and P. Cossart. 1995. The unrelated surface proteins ActA of Listeria monocytogenes and IcsA of Shigella flexneri are sufficient to confer actin-based motility on Listeria innocua and Escherichia coli, respectively. Mol. Microbiol. 18:413–423. 171. Köhler, S., M. Leimeister-Wächter, T. Chakraborty, F. Lottspeich, and W. Goebel. 1990. The gene coding for protein p60 of Listeria monocytogenes and its use as a species-specific probe for Listeria monocytogenes. Infect. Immun. 58:1943–1950. 172. Kohda, C., I. Kawamura, H. Baba, T. Nomura, Y. Ito, T. Kimoto, I. Watanabe, and M. Mitsuyama. 2002. Dissociated linkage of cytokine-inducing activity and cytotoxicity to different domains of listeriolysin O from Listeria monocytogenes. Infect. Immun. 70:1334–1341. 173. Kolb-Mäurer, A., I. Gentschev, H. W. Fries, F. Fiedler, E. B. Bröcker, E. Kämpgen, and W. Goebel. 2000. Listeria monocytogenes-infected human dendritic cells: uptake and host cell response. Infect. Immun. 68:3680–3688. 174. Kolb-Mäurer, A., S. Pilgrim, E. Kämpgen, A. D. McLellan, E. B. Bröcker, W. Goebel, and I. Gentschev. 2001. Antibodies against listerial protein 60 act as an opsonin for phagocytosis of Listeria monocytogenes by human dendritic cells. Infect. Immun. 69:3100–3109.

DK3089_C005.fm Page 148 Tuesday, February 20, 2007 11:40 AM

148

Listeria, Listeriosis, and Food Safety

175. Konkel, M. E., and K. Tilly. 2000. Temperature-regulated expression of bacterial virulence genes. Microbes Infect. 2:157–66. 176. Kreft, J., J. Bohne, R. Gross, H. Kestler, Z. Sokolovic, and W. Goebel. 1995. Control of Listeria monocytogenes virulence by the transcriptional regulator PrfA. In Signal transduction and bacterial virulence, eds. R. Rappuoli, V. Scarlato, and B. Arico. Austin, Tex.: R. G. Landes Company, pp. 129–142. 177. Kreft, J., M. Dumbsky, and S. Theiß. 1995. The actin-polymerization protein from Listeria ivanovii is a large repeat protein which shows only limited amino acid sequence homology to ActA from Listeria monocytogenes. FEMS Microbiol. Lett. 126:113–122. 178. Kreft, J., and J. A. Vazquez-Boland. 2001. Regulation of virulence genes in Listeria. Int. J. Med. Microbiol. 291:145–157. 179. Kreft, J., J. A. Vazquez-Boland, S. Altrock, G. Dominguez-Bernal, and W. Goebel. 2002. Pathogenicity islands and other virulence elements in Listeria. Cur. Top. Microbiol. Immunol. 264:109–125. 180. Kügler, S., S. Schüller, and W. Goebel. 1997. Involvement of MAP-kinases and -phosphatases in uptake and intracellular replication of Listeria monocytogenes in J774 macrophage cells. FEMS Microbiol. Lett. 157:131–136. 181. Kuhn, M. 1998. The microtubule depolymerizing drugs nocodazole and colchicine inhibit the uptake of Listeria monocytogenes by P388D1 macrophages. FEMS Microbiol. Lett. 160:87–90. 182. Kuhn, M., and W. Goebel. 1989. Identification of an extracellular protein of Listeria monocytogenes possibly involved in the intracellular uptake by mammalian cells. Infect. Immun. 57:55–61. 183. Kuhn, M., S. Kathariou, and W. Goebel. 1988. Hemolysin supports survival but not entry of the intracellular bacterium Listeria monocytogenes. Infect. Immun. 56:79–82. 184. Kuhn, M., M. C. Prevost, J. Mounier, and P. J. Sansonetti. 1990. A nonvirulent mutant of Listeria monocytogenes does not move intracellularly but still induces polymerization of actin. Infect. Immun. 58:3477–3486. 185. Lampidis, R., R. Gross, Z. Sokolovic, W. Goebel, and J. Kreft. 1994. The virulence regulator protein of Listeria ivanovii is highly homologous to PrfA from Listeria monocytogenes and both belong to the Crp-Fnr family of transcriptional regulators. Mol. Microbiol. 13:141–151. 186. Lasa, I., V. David, E. Gouin, J. B. Marchand, and P. Cossart. 1995. The amino-terminal part of ActA is critical for the actin-based motility of Listeria monocytogenes; the central proline-rich region acts as a stimulator. Mol. Microbiol. 18:425–436. 187. Lasa, I., E. Gouin, M. Goethals, K. Vancompernolle, V. David, J. Vandekerckhove, and P. Cossart. 1997. Identification of two regions in the N-terminal domain of ActA involved in the actin comet tail formation by Listeria monocytogenes. EMBO J. 16:1531–1540. 188. Lauer, P., J. A. Theriot, J. Skoble, M. D. Welch, and D. A. Portnoy. 2001. Systematic mutational analysis of the amino-terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin-based motility. Mol. Microbiol. 42:1163–1177. 189. Laurent, V., T. P. Loisel, B. Harbeck, A. Wehman, L. Grobe, B. M. Jockusch, J. Wehland, F. B. Gertler, and M. F. Carlier. 1999. Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes. J. Cell Biol. 144:1245–1258. 190. Leblond-Francillard, M., J. L. Gaillard, and P. Berche. 1989. Loss of catalase activity in Tn1545induced mutants does not reduce growth of Listeria monocytogenes in vivo. Infect. Immun. 57:2569–2573. 191. Lecuit, M., S. Dramsi, C. Gottardi, M. Fedor-Chaiken, B. Gumbiner, and P. Cossart. 1999. A single amino acid in E-cadherin responsible for host specificity towards the human pathogen Listeria monocytogenes. EMBO J. 18:3956–3963. 192. Lecuit, M., and P. Cossart. 2002. Genetically modified animal models for human infections: The Listeria paradigm. Trends Mol. Med. 8:537–542. 193. Lecuit, M., R. Hurme, J. Pizarro-Cerda, H. Ohayon, B. Geiger, and P. Cossart. 2000. A role for α- and β-catenins in bacterial uptake. Proc. Natl. Acad. Sci. USA 97:10008–100013. 194. Lecuit, M., S. Vandormael-Pournin, J. Lefort, M. Huerre, P. Gounon, C. Dupuy, C. Babinet, and P. Cossart. 2001. A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier. Science 292:1722–1725. 195. Leimeister-Wächter, M., E. Domann, and T. Chakraborty. 1991. Detection of a gene encoding a phosphatidylinositol-specific phospholipase C that is coordinately expressed with listeriolysin in Listeria monocytogenes. Mol. Microbiol. 5:361–366.

DK3089_C005.fm Page 149 Tuesday, February 20, 2007 11:40 AM

Molecular Virulence Determinants of Listeria monocytogenes

149

196. Leimeister-Wächter, M., E. Domann, and T. Chakraborty. 1992. The expression of virulence genes in Listeria monocytogenes is thermoregulated. J. Bacteriol. 174:947–952. 197. Leimeister-Wächter, M., W. Goebel, and T. Chakraborty. 1989. Mutations affecting hemolysin production located otside the listeriolysin gene. FEMS Microbiol. Lett. 65:23–30. 198. Leimeister-Wächter, M., C. Haffner, E. Domann, W. Goebel, and T. Chakraborty. 1990. Identification of a gene that positively regulates listeriolysin, the major virulence factor of Listeria monocytogenes. Proc. Natl. Acad. Sci. USA 87:8336–8340. 199. Lenz, L. L., S. Mohammadi, A. Geissler, and D. A. Portnoy. 2003. SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis. Proc. Natl. Acad. Sci. USA 100:12432–12437. 200. Lenz, L. L., and D. A. Portnoy. 2002. Identification of a second Listeria secA gene associated with protein secretion and the rough phenotype. Mol. Microbiol. 45:1043–1056. 201. Lety, M. A., C. Frehel, P. Berche, and A. Charbit. 2002. Critical role of the N-terminal residues of listeriolysin O in phagosomal escape and virulence of Listeria monocytogenes. Mol. Microbiol. 46:367–379. 202. Lety, M. A., C. Frehel, I. Dubail, J. L. Beretti, S. Kayal, P. Berche, and A. Charbit. 2001. Identification of a PEST-like motif in listeriolysin O required for phagosomal escape and for virulence in Listeria monocytogenes. Mol. Microbiol. 39:1124–1139. 203. Lingnau, A., E. Domann, M. Hudel, M. Bock, T. Nichterlein, J. Wehland, and T. Chakraborty. 1995. Expression of the Listeria monocytogenes EGD inlA and inlB genes, whose products mediate bacterial entry into tissue culture cell lines, by PrfA-dependent and -independent mechanisms. Infect. Immun. 63:3896–3903. 204. Loessner, M. J., and S. Scherer. 1995. Organization and transcriptional analysis of the Listeria phage A511 late gene region comprising the major capsid and tail sheath protein genes cps and tsh. J. Bacteriol. 177:6601–6609. 205. Machesky, L. M., S. J. Atkinson, C. Ampe, J. Vandekerckhove, and T. D. Pollard. 1994. Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose. J. Cell Biol. 127:107–115. 206. Machner, M. P., S. Frese, W. D. Schubert, V. Orian-Rousseau, E. Gherardi, J. Wehland, H. H. Niemann, and D. Heinz. 2003. Aromatic amino acids at the surface of InlB are essential for host cell invasion by Listeria monocytogenes. Mol. Microbiol. 48:1525–1536. 207. Machner, M. P., C. Urbanke, M. Barzik, S. Otten, A. S. Sechi, J. Wehland, and D. W. Heinz. 2001. ActA from Listeria monocytogenes can interact with up to four Ena/VASP homology 1 domains simultaneously. J. Biol. Chem. 276:40096–40103. 208. Mackaness, G. B. 1962. Cellular resistance to infection. J. Exp. Med. 116:381–406. 209. Marchand, J. B., P. Moreau, A. Paoletti, P. Cossart, M. F. Carlier, and D. Pantaloni. 1995. Actin-based movement of Listeria monocytogenes: Actin assembly results from the local maintenance of uncapped filament barbed ends at the bacterium surface. J. Cell. Biol. 130:331–343. 210. Marino, M., M. Banerjee, R. Jonquieres, P. Cossart, and P. Ghosh. 2002. GW domains of the Listeria monocytogenes invasion protein InlB are SH3-like and mediate binding to host ligands. EMBO J. 21:5623–5634. 211. Marino, M., L. Braun, P. Cossart, and P. Ghosh. 1999. Structure of the lnlB leucine-rich repeats, a domain that triggers host cell invasion by the bacterial pathogen L. monocytogenes. Mol. Cell. 4:1063–1072. 212. Marquis, H., H. G. Bouwer, D. J. Hinrichs, and D. A. Portnoy. 1993. Intracytoplasmic growth and virulence of Listeria monocytogenes auxotrophic mutants. Infect. Immun. 61:3756–3760. 213. Marquis, H., V. Doshi, and D. A. Portnoy. 1995. The broad-range phospholipase C and a metalloprotease mediate listeriolysin O-independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells. Infect. Immun. 63:4531–4534. 214. Marquis, H., H. Goldfine, and D. A. Portnoy. 1997. Proteolytic pathways of activation and degradation of a bacterial phospholipase C during intracellular infection by Listeria monocytogenes. J. Cell Biol. 137:1381–1392. 215. Mengaud, J., C. Braun-Breton, and P. Cossart. 1991. Identification of phosphatidylinositol-specific phospholipase C activity in Listeria monocytogenes: a novel type of virulence factor? Mol. Microbiol. 5:367–372.

DK3089_C005.fm Page 150 Tuesday, February 20, 2007 11:40 AM

150

Listeria, Listeriosis, and Food Safety

216. Mengaud, J., J. Chenevert, C. Geoffroy, J. L. Gaillard, and P. Cossart. 1987. Identification of the structural gene encoding the SH-activated hemolysin in Listeria monocytogenes: Listeriolysin O is homologous with streptolysin O and pneumolysin. Infect. Immun. 55:3225–3227. 217. Mengaud, J., S. Dramsi, E. Gouin, J. A. Vazquez-Boland, G. Milon, and P. Cossart. 1991. Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated. Mol. Microbiol. 5:2273–2283. 218. Mengaud, J., C. Geoffroy, and P. Cossart. 1991. Identification of a new operon involved in Listeria monocytogenes virulence: its first gene encodes a protein homologous to bacterial metalloproteases. Infect. Immun. 59:1043–1049. 219. Mengaud, J., H. Ohayon, P. Gounon, R. M. Mege, and P. Cossart. 1996. E-cadherin is the receptor for internalin, a surface protein required for entry of Listeria monocytogenes into epithelial cells. Cell 84:923–932. 220. Mengaud, J., M. F. Vicente, J. Chenevert, J. M. Pereira, C. Geoffroy, B. Gicquel-Sanzey, F. Baquero, J. C. Perez-Diaz, and P. Cossart. 1988. Expression in Escherichia coli and sequence analysis of the listeriolysin O determinant of Listeria monocytogenes. Infect. Immun. 56:766–772. 221. Mengaud, J., M. F. Vicente, and P. Cossart. 1989. Transcriptional mapping and nucleotide sequence of the Listeria monocytogenes hlyA region reveal structural features that may be involved in regulation. Infect. Immun. 57:3695–3701. 222. Michel, E., K. A. Reich, R. Favier, P. Berche, and P. Cossart. 1990. Attenuated mutants of the intracellular bacterium Listeria monocytogenes obtained by single amino acid substitutions in listeriolysin O. Mol. Microbiol. 4:2167–2178. 223. Milohanic, E., P. Glaser, J. Y. Coppee, L. Frangeul, Y. Vega, J. A. Vazquez-Boland, F. Kunst, F. P. Cossart, and C. Buchrieser. 2003. Transcriptome analysis of Listeria monocytogenes identifies three groups of genes differently regulated by PrfA. Mol. Microbiol. 47:1613–1625. 224. Milohanic, E., R. Jonquieres, P. Cossart, P. Berche, and J. L. Gaillard. 2001. The autolysin Ami contributes to the adhesion of Listeria monocytogenes to eukaryotic cells via its cell wall anchor. Mol. Microbiol. 39:1212–1224. 225. Milohanic, E., B. Pron, P. Berche, and J. L. Gaillard. 2000. Identification of new loci involved in adhesion of Listeria monocytogenes to eukaryotic cells. European Listeria Genome Consortium. Microbiology 146:731–739. 226. Moors, M. A., V. Auerbuch, and D. A. Portnoy. 1999. Stability of the Listeria monocytogenes ActA protein in mammalian cells is regulated by the N-end rule pathway. Cell. Microbiol. 1:249–257. 227. Moors, M. A., B. Levitt, P. Youngman, and D. A. Portnoy. 1999. Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes. Infect. Immun. 67:131–139. 228. Moser, J., B. Gerstel, J. E. Meyer, T. Chakraborty, J. Wehland, and D. W. Heinz. 1997. Crystal structure of the phosphatidylinositol-specific phospholipase C from the human pathogen Listeria monocytogenes. J. Mol. Biol. 273:269–282. 229. Mounier, J., A. Ryter, M. Coquis-Rondon, and P. J. Sansonetti. 1990. Intracellular and cell-to-cell spread of Listeria monocytogenes involves interaction with F-actin in the enterocytelike cell line Caco-2. Infect. Immun. 58:1048–1058. 230. Mourrain, P., I. Lasa, A. Gautreau, E. Gouin, A. Pugsley, and P. Cossart. 1997. ActA is a dimer. Proc. Natl. Acad. Sci. USA 94:10034–10039. 231. Mullins, R. D., J. A. Heuser, and T. D. Pollard. 1998. The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc. Natl. Acad. Sci. USA 95:6181–6186. 232. Nagafuchi, A. 2001. Molecular architecture of adherens junctions. Curr. Opin. Cell. Biol. 13:600–603. 233. Nair, S., I. Derre, T. Msadek, O. Gaillot, and P. Berche. 2000. CtsR controls class III heat shock gene expression in the human pathogen Listeria monocytogenes. Mol. Microbiol. 35:800–811. 234. Nair, S., C. Frehel, L. Nguyen, V. Escuyer, and P. Berche. 1999. ClpE, a novel member of the HSP100 family, is involved in cell division and virulence of Listeria monocytogenes. Mol. Microbiol. 31:185–196. 235. Nair, S., E. Milohanic, and P. Berche. 2000. ClpC ATPase is required for cell adhesion and invasion of Listeria monocytogenes. Infect. Immun. 68:7061–7068. 236. Newman, J. C., and A. Weiner. 2002. Measuring the immeasurable. Mol. Cell. 10:437–439. 237. Niebuhr, K., T. Chakraborty, M. Rohde, T. Gazlig, B. Jansen, P. Köllner, and J. Wehland. 1993. Localization of the ActA polypeptide of Listeria monocytogenes in infected tissue culture cell lines: ActA is not associated with actin comets. Infect. Immun. 61:2793–2802.

DK3089_C005.fm Page 151 Tuesday, February 20, 2007 11:40 AM

Molecular Virulence Determinants of Listeria monocytogenes

151

238. Niebuhr, K., F. Ebel, R. Frank, M. Reinhard, E. Domann, U. D. Carl, U. Walter, F. B. Gertler, J. Wehland, and T. Chakraborty. 1997. A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family. EMBO J. 16:5433–5444. 239. O’Riordan, M., M. A. Moors, and D. A. Portnoy. 2003. Listeria intracellular growth and virulence require host-derived lipoic acid. Science 302:462–464. 240. O’Riordan, M., and D. A. Portnoy. 2002. The host cytosol: front-line or home front? Trends Microbiol. 10:361–364. 241. Packer, L., E. H. Witt, and H. J. Tritschler. 1995. a-Lipoic acid as a biological antioxidant. Free Radic. Biol. Med. 19:227–250. 242. Palmer, M. 2001. The family of thiol-activated, cholesterol-binding cytolysins. Toxicon 39:1681–1689. 243. Pandiripally, V. K., D. G. Westbrook, G. R. Sunki, and A. K. Bhunia. 1999. Surface protein p104 is involved in adhesion of Listeria monocytogenes to human intestinal cell line, Caco-2. J. Med. Microbiol. 48:117–124. 244. Parida, S. K., E. Domann, M. Rohde, S. Müller, A. Darji, T. Hain, J. Wehland, and T. Chakraborty. 1998. Internalin B is essential for adhesion and mediates the invasion of Listeria monocytogenes into human endothelial cells. Mol. Microbiol. 28:81–93. 245. Park, S. F., and R. G. Kroll. 1993. Expression of listeriolysin and phosphatidylinositol-specific phospholipase C is repressed by the plant-derived molecule cellobiose in Listeria monocytogenes. Mol. Microbiol. 8:653–661. 246. Parrisius, J., S. Bhakdi, M. Roth, J. Tranum-Jensen, W. Goebel, and H. P. R. Seeliger. 1986. Production of listeriolysin by β-hemolytic strains of Listeria monocytogenes. Infect. Immun. 51:314–319. 247. Paschen, A., K. E. Dittmar, R. Grenningloh, M. Rohde, D. Schadendorf, E. Domann, T. Chakraborty, and S. Weiss. 2000. Human dendritic cells infected by Listeria monocytogenes: Induction of maturation, requirements for phagolysosomal escape and antigen presentation capacity. Eur. J. Immunol. 30:3447–3456. 248. Pfeuffer, T., W. Goebel, J. Laubinger, M. Bachmann, and M. Kuhn. 2000. LaXp180, a mammalian ActA-binding protein, identified with the yeast two-hybrid system colocalizes with intracellular Listeria monocytogenes. Cell. Microbiol. 2:101–114. 249. Pilgrim, S., A. Kolb-Mäurer, I. Gentschev, W. Goebel, and M. Kuhn. 2003. Deletion of the gene encoding p60 in Listeria monocytogenes leads to abnormal cell division and to loss of actin-based motility. Infect. Immun. 71:3473–3784. 250. Pistor, S., T. Chakraborty, K. Niebuhr, E. Domann, and J. Wehland. 1994. The ActA protein of Listeria monocytogenes acts as nucleator inducing reorganization of the actin cytoskeleton. EMBO J. 13:758–763. 251. Pistor, S., T. Chakraborty, U. Walter, and J. Wehland. 1995. The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins. Curr. Biol. 5:517–525. 252. Pistor., S., L. Grobe, A. S. Sechi, E. Domann, B. Gerstel, L. M. Machesky, T. Chakraborty, and J. Wehland. 2000. Mutations of arginine residues within the 146-KKRRK-150 motif of the ActA protein of Listeria monocytogenes abolish intracellular motility by interfering with the recruitment of the Arp2/3 complex. J. Cell Sci. 113:3277–3287. 253. Portnoy, D. A., P. S. Jacks, and D. J. Hinrichs. 1988. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J. Exp. Med. 167:1459–1471. 254. Poyart, C., E. Abachin, I. Razfimanantsoa, and P. Berche. 1993. The zinc metalloprotease of Listeria monocytogenes is required for maturation of the phosphatidylcholine phospholipase C: Direct evidence obtained by gene complementation. Infect. Immun. 61:1576–1580. 255. Pron, B., C. Boumaila, F. Jaubert, P. Berche, G. Milon, F. Geissmann, and J. L. Gaillard. 2001. Dendritic cells are early cellular targets of Listeria monocytogenes after intestinal delivery and are involved in bacterial spread in the host. Cell. Microbiol. 3:331–340. 256. Raffelsbauer, D., A. Bubert, F. Engelbrecht, J. Scheinpflug, A. Simm, J. Hess, S. H. E. Kaufmann, and W. Goebel. 1998. The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice. Mol. Gen. Genet. 260:144–158. 257. Ramnath, M., K. B. Rechinger, L. Jansch, J. W. Hastings, S. Knochel, and A. Gravesen. 2003. Development of a Listeria monocytogenes EGDe partial proteome reference map and comparison with the protein profiles of food isolates. Appl. Environ. Microbiol. 69:3368–3376.

DK3089_C005.fm Page 152 Tuesday, February 20, 2007 11:40 AM

152

Listeria, Listeriosis, and Food Safety

258. Raveneau, J., C. Geoffroy, J. L. Beretti, J. L. Gaillard, J. E. Alouf, and P. Berche. 1992. Reduced virulence of a Listeria monocytogenes phospholipase-deficient mutant obtained by transposon insertion into the zinc metalloprotease gene. Infect. Immun. 60:916–921. 259. Raybourne, R. B., and V. K. Bunning. 1994. Bacterium–host cell interaction on the cellular level: fluorescent labeling of the bacteria and analysis of short-term bacterium-phagocyte interaction by flow cytometry. Infect. Immun. 62:665–672. 260. Rechsteiner, M., and S. W. Rogers. 1996. PEST sequences and regulation by proteolysis. Trends Biochem. Sci. 21:267–271. 261. Reglier-Poupet, H., C. Frehel, I. Dubail, J. L. Beretti, P. Berche, A. Charbit, and C. Raynaud. 2003. Maturation of lipoproteins by type II signal peptidase is required for phagosomal escape of Listeria monocytogenes. J. Biol. Chem. 78:49469–49477. 262. Reglier-Poupet, H., E. Pellegrini, A. Charbit, and P. Berche. 2003. Identification of LpeA, a PsaAlike membrane protein that promotes cell entry by Listeria monocytogenes. Infect. Immun. 71:474–482. 263. Reinhard, M., K. Giehl, K. Abel, C. Haffner, T. Jarchau, V. Hoppe, B. M. Jockusch, and U. Walter. 1995. The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins. EMBO J. 14:1583–1589. 264. Reis, E., C. Sousa, A. Sher, and P. Kaye. 1999. The role of dendritic cells in the induction and regulation of immunity to microbial infection. Curr. Opin. Immunol. 11:392–399. 265. Renzoni, A., P. Cossart, and S. Dramsi. 1999. PrfA, the transcriptional activator of virulence genes, is upregulated during interaction of Listeria monocytogenes with mammalian cells and in eukaryotic cell extracts. Mol. Microbiol. 34:552–561. 266. Renzoni, A., A. Klarsfeld, S. Dramsi, and P. Cossart. 1997. Evidence that PrfA, the pleiotropic activator of virulence genes in Listeria monocytogenes, can be present but inactive. Infect. Immun. 65:1515–1518. 267. Ripio, M. T., G. Dominguez-Bernal, M. Lara, M. Suarez, and J. A. Vazquez-Boland. 1997. A Gly145Ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes. J. Bacteriol. 179:1533–1540. 268. Ripio, M. T., G. Dominguez-Bernal, M. Suarez, K. Brehm, P. Berche, and J. A. Vazquez-Boland. 1996. Transcriptional activation of virulence genes in wild-type strains of Listeria monocytogenes in response to a change in the extracellular medium composition. Res. Microbiol. 147:371–384. 269. Ripio, M. T., J. A. Vazquez-Boland, Y. Vega, S. Nair, and P. Berche. 1998. Evidence for expressional crosstalk between the central virulence regulator PrfA and the stress response mediator ClpC in Listeria monocytogenes. FEMS Microbiol. Lett. 158:45–50. 270. Robbins, J. R., A. L. Barth, H. Marquis, E. L. de Hostos, W. J. Nelson, and J. A. Theriot. 1999. Listeria monocytogenes exploits normal host cell processes to spread from cell to cell. J. Cell Biol. 146:1333–1350. 271. Robbins, J. R., and J. A. Theriot. 2003. Listeria monocytogenes rotates around its long axis during actin-based motility. Curr. Biol. 13:754–756. 272. Rosenshine, I., V. Duronio, and B. B. Finlay. 1992. Protein tyrosine kinase inhibitors block invasinpromoted bacterial uptake by epithelial cells. Infect. Immun. 60:2211–2217. 273. Rossjohn, J., S. C. Feil, W. J. McKinstry, R. K. Tweten, and M. W. Parker. 1997. Structure of a cholesterol-binding, thiol-activated cytolysin and a model of its membrane form. Cell 89:685–692. 274. Rouqette, C., J. M. Bolla, and P. Berche. 1995. An iron-dependent mutant of Listeria monocytogenes of attenuated virulence. FEMS Microbiol. Lett. 133:77–83. 275. Rouquette, C., C. de Chastellier, S. Nair, and P. Berche. 1998. The ClpC ATPase of Listeria monocytogenes is a general stress protein required for virulence and promoting early bacterial escape from the phagosome of macrophages. Mol. Microbiol. 27:1235–1245. 276. Rouquette, C., M. T. Ripio, E. Pellegrini, J. M. Bolla, R. I. Tascon, J. A. Vazquez-Boland, and P. Berche. 1996. Identification of a ClpC ATPase required for stress tolerance and in vivo survival of Listeria monocytogenes. Mol. Microbiol. 21:977–987. 277. Rowan, N. J., A. A. Candlish, A. Bubert, J. G. Anderson, K. Kramer, and J. McLauchlin. 2000. Virulent rough filaments of Listeria monocytogenes from clinical and food samples secreting wild-type levels of cell-free p60 protein. J. Clin. Microbiol. 38:2643–2648. 278. Ruhland, G. J., M. Hellwig, G. Wanner, and F. Fiedler. 1993. Cell-surface location of Listeria-specific protein p60—detection of Listeria cells by indirect immunofluorescence. J. Gen. Microbiol. 139:609–616.

DK3089_C005.fm Page 153 Tuesday, February 20, 2007 11:40 AM

Molecular Virulence Determinants of Listeria monocytogenes

153

279. Samarin, S., S. Romero, C. Kocks, D. Didry, D. Pantaloni, and M. F. Carlier. 2003. How VASP enhances actin-based motility. J. Cell. Biol. 163:131–142. 280. Sanger, J. M., J. W. Sanger, and F. S. Southwick. 1992. Host cell actin assembly is necessary and likely to provide the propulsive force for intracellular movement of Listeria monocytogenes. Infect. Immun. 60:3609–3619. 281. Sawyer, R. T., D. A. Drevets, P. A. Campbell, and T. A. Potter. 1996. Internalin A can mediate phagocytosis of Listeria monocytogenes by mouse macrophage cell lines. J. Leukoc. Biol. 60:603–610. 282. Schirmer, E. C., J. R. Glover, M. A. Singer, and S. Lindquist. 1996. HSP100/Clp proteins: A common mechanism explains diverse functions. Trends Biochem. Sci. 21:289–296. 283. Schubert, K., A. M. Bichlmaier, E. Mager, K. Wolff, G. Ruhland, and F. Fiedler. 2000. P45, an extracellular 45-kDa protein of Listeria monocytogenes with similarity to protein p60 and exhibiting peptidoglycan lytic activity. Arch. Microbiol. 173:21–28. 284. Schubert, W. D., G. Gobel, M. Diepholz, A. Darji, D. Kloer, T. Hain, T. Chakraborty, J. Wehland, E. Domann, and D. W. Heinz. 2001. Internalins from the human pathogen Listeria monocytogenes combine three distinct folds into a contiguous internalin domain. J. Mol. Biol. 312:783–794. 285. Schubert, W. D., C. Urbanke, T. Ziehm, V. Beier, M. P. Machner, E. Domann, J. Wehland, T. Chakraborty, and D. W. Heinz. 2002. Structure of internalin, a major invasion protein of Listeria monocytogenes, in complex with its human receptor E-cadherin. Cell 111:825–836. 286. Schwan, W. R., A. Demuth, M. Kuhn, and W. Goebel. 1994. Phosphatidylinositol-specific phospholipase C from Listeria monocytogenes contributes to intracellular survival and growth of Listeria innocua. Infect. Immun. 62:4795–4803. 287. Seeliger, H. P. R., and D. Jones. 1986. Genus Listeria pirie 1940. In Bergey’s manual of systematic bacteriology, vol. 2, eds. P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt. Baltimore: The Williams and Wilkins Co., pp. 1235–1245. 288. Sheehan, B., A. Klarsfeld, R. Ebright, and P. Cossart. 1996. A single substitution in the putative helixturn-helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence. Mol. Microbiol. 20:785–797. 289. Sheehan, B., A. Klarsfeld, T. Msadek, and P. Cossart. 1995. Differential activation of virulence gene expression by PrfA, the Listeria monocytogenes virulence regulator. J. Bacteriol. 177:6469–6476. 290. Shen, Y., M. Naujokas, M. Park, and K. Ireton. 2000. InIB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase. Cell 103:501–510. 291. Shetron-Rama, L. M., H. Marquis, H. G. Bouwer, and N. E. Freitag. 2002. Intracellular induction of Listeria monocytogenes actA expression. Infect. Immun. 70:1087–1096. 292. Shetron-Rama, L. M., K. Mueller, J. M. Bravo, H. G. Bouwer, S. S. Way, and N. E. Freitag. 2003. Isolation of Listeria monocytogenes mutants with high-level in vitro expression of host cytosol-induced gene products. Mol. Microbiol. 48:1537–1551. 293. Sibelius, U., T. Chakraborty, B. Krögel, J. Wolf, F. Rose, R. Schmidt, J. Wehland, W. Seeger, and F. Grimminger. 1996. The listerial exotoxins listeriolysin and phosphatidylinositol-specific phospholipase C synergize to elicit endothelial cell phosphoinositide metabolism. J. Immunol. 157:4055–4060. 294. Sibelius, U., F. Rose, T. Chakraborty, A. Darji, J. Wehland, S. Weiss, W. Seeger, and F. Grimminger. 1996. Listeriolysin is a potent inducer of the phosphatidylinositol response and lipid mediator generation in human endothelial cells. Infect. Immun. 64:674–676. 295. Sijts, A. J., I. Pilip, and E. G. Pamer. 1997. The Listeria monocytogenes-secreted p60 protein is an N-end rule substrate in the cytosol of infected cells. Implications for major histocompatibility complex class I antigen processing of bacterial proteins. J. Biol. Chem. 272:19261–19268. 296. Skoble, J., V. Auerbuch, E. D. Goley, M. D. Welch, and D. A. Portnoy. 2001. Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility. J. Cell Biol. 155:89–100. 297. Skoble, J., D. A. Portnoy, and M. D. Welch. 2000. Three regions within ActA promote Arp2/3 complexmediated actin nucleation and Listeria monocytogenes motility. J. Cell Biol. 150:527–538. 298. Slaghuis, J., M. Goetz, F. Engelbrecht, and W. Goebel. 2004. Inefficient replication of Listeria innocua in the cytosol of mammalian cells. J. Infect. Dis. 189:393–401. 299. Smith, G. A., H. Marquis, S. Jones, N. C. Johnston, D. A. Portnoy, and H. Goldfine. 1995. The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread. Infect. Immun. 63:4231–4237.

DK3089_C005.fm Page 154 Tuesday, February 20, 2007 11:40 AM

154

Listeria, Listeriosis, and Food Safety

300. Smith, G. A., D. A. Portnoy, and J. A. Theriot. 1995. Asymmetric distribution of the Listeria monocytogenes ActA protein is required and sufficient to direct actin-based motility. Mol. Microbiol. 17:945–951. 301. Smith, G. A., J. A. Theriot, and D. A. Portnoy. 1996. The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin. J. Cell Biol. 135:647–660. 302. Snyder, A., and H. Marquis. 2003. Restricted translocation across the cell wall regulates secretion of the broad-range phospholipase C of Listeria monocytogenes. J. Bacteriol. 185:5953–5958. 303. Sokolovic, Z., A. Fuchs, and W. Goebel. 1990. Synthesis of species-specific stress proteins by virulent strains of Listeria monocytogenes. Infect. Immun. 58:3582–3587. 304. Sokolovic, Z., and W. Goebel. 1989. Synthesis of listeriolysin in Listeria monocytogenes under heat shock conditions. Infect. Immun. 57:295–298. 305. Sokolovic, Z., J. Riedel, M. Wuenscher, and W. Goebel. 1993. Surface-associated, PrfA-regulated proteins of Listeria monocytogenes synthesized under stress conditions. Mol. Microbiol. 8:219–227. 306. Steffen, P., D. A. Schafer, V. David, E. Gouin, J. A. Cooper, and P. Cossart. 2000. Listeria monocytogenes ActA protein interacts with phosphatidylinositol 4,5-bisphosphate in vitro. Cell. Motil. Cytoskeleton 45:58–66. 307. Suarez, M., B. Gonzalez-Zorn, Y. Vega, I. Chico-Calero, and J. A. Vazquez-Boland. 2001. A role for ActA in epithelial cell invasion by Listeria monocytogenes. Cell. Microbiol. 3:853–864. 308. Sue, D., K. J. Boor, and M. Wiedmann. 2003. Sigma(B)-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes. Microbiology 149:3247–3256. 309. Sun, A. N., A. Camilli, and D. A. Portnoy. 1990. Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread. Infect. Immun. 58:3770–3778. 310. Tang, P., I. Rosenshine, and B. B. Finlay. 1994. Listeria monocytogenes, an invasive bacterium, stimulates MAP kinase upon attachment to epithelial cells. Mol. Biol. Cell 5:455–464. 311. Temm-Grove, C. T., B. Jokusch, M. Rohde, K. Niebuhr, T. Chakraborty, and J. Wehland. 1994. Exploitation of microfilament proteins by Listeria monocytogenes: microvillus-like composition of the comet tails and vectorial spreading in polarized epithelial sheets. J. Cell. Sci. 107:2951–2960. 312. Theriot, J. A., T. J. Mitchison, L. G. Tilney, and D. A. Portnoy. 1992. The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. Nature (London) 357:257–260. 313. Theriot, J. A., J. Rosenblatt, D. A. Portnoy, P. J. Goldschmidt-Clermont, and T. J. Mitchison. 1994. Involvement of profilin in the actin-based motility of Listeria monocytogenes in cells and cell free extracts. Cell 76:505–517. 314. Tilney, L. G., D. J. DeRosier, A. Weber, and M. S. Tilney. 1992. How Listeria exploits host cell actin to form its own cytoskeleton: II. Nucleation, actin filament polarity, filament assembly, and evidence for a poited end capper. J. Cell Biol. 118:83–93. 315. Tilney, L. G., and D. A. Portnoy. 1989. Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. J. Cell Biol. 109:1597–1608. 316. Van Dissel, J. T., J. J. M. Stikkelbroeck, and R. van Furth. 1993. Differences in the rate of intracellular killing of catalase-negative and catalase-positive Listeria monocytogenes by normal and interferon-γ activated macrophages. Scand. J. Immunol. 37:443–446. 317. Vasconcelos, J. A., and H. G. Deneer. 1994. Expression of superoxide dismutase in Listeria monocytogenes. Appl. Environ. Microbiol. 60:2360–2366. 318. Vazquez-Boland, J. A. 2002. Bacterial growth in the cytosol: lessons from Listeria. Trends Microbiol. 10:493–495. 319. Vazquez-Boland, J. A., C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart. 1992. Nucleotide sequence of the lecithinase operon in Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread. Infect. Immun. 60:219–230. 320. Vazquez-Boland, J. A., M. Kuhn, P. Berche, T. Chakraborty, G. Dominguez-Bernal, W. Goebel, B. Gonzalez-Zorn, J. Wehland, and J. Kreft. 2001. Listeria pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev. 14:584–640.

DK3089_C005.fm Page 155 Tuesday, February 20, 2007 11:40 AM

Molecular Virulence Determinants of Listeria monocytogenes

155

321. Vega, Y., C. Dickneite, M. T. Ripio, R. Böckmann, B. Gonzalez-Zorn, S. Novella, G. Dominguez-Bernal, W. Goebel, and J. A. Vazquez-Boland. 1998. Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA. J. Bacteriol. 180:6655–6660. 322. Velge, P., E. Bottreau, B. Kaeffer, N. Yurdusev, P. Pardon, and N. Van Langendonck. 1994. Protein tyrosine kinase inhibitors block the entries of Listeria monocytogenes and Listeria ivanovii into epithelial cells. Microbial Path. 17:37–50. 323. Wadsworth, S. J., and H. Goldfine. 1999. Listeria monocytogenes phospholipase C-dependent calcium signaling modulates bacterial entry into J774 macrophage-like cells. Infect. Immun. 67:1770–1778. 324. Wadsworth, S. J., and H. Goldfine. 2002. Mobilization of protein kinase C in macrophages induced by Listeria monocytogenes affects its internalization and escape from the phagosome. Infect. Immun. 70:4650–4660. 325. Webster, P. 2002. Early intracellular events during internalization of Listeria monocytogenes by J774 cells. J. Histochem. Cytochem. 50:503–518. 326. Welch, D. F. 1987. Role of catalase and superoxide dismutase in the virulence of Listeria monocytogenes. Ann. Inst. Pasteur/Microbiol. 138:265–268. 327. Welch, M. D., A. H. DePace, S. Verma, A. Iwamatsu, and T. J. Mitchison. 1997. The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly. J. Cell Biol. 138:375–384. 328. Welch, M. D., A. Iwamatsu, and T. J. Mitchison. 1997. Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes. Nature (London) 385:265–269. 329. Welch, M. D., J. Rosenblatt, J. Skoble, D. A. Portnoy, and T. J. Mitchison. 1998. Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. Science 281:105–108. 330. Williams, T., S. Bauer, D. Beier, and M. Kuhn. Construction and characterization of Listeria monocytogenes mutants with in-frame deletions in the response-regulator genes identified in the genome sequence. Infect. Immun. 73:3152–3159. 331. Williams, J. R., C. Thayyullathil, and N. E. Freitag. 2000. Sequence variations within PrfA DNA binding sites and effects on Listeria monocytogenes virulence gene expression. J. Bacteriol. 182:837–841. 332. Wood, S., N. Maroushek, and C. J. Czuprynski. 1993. Multiplication of Listeria monocytogenes in a murine hepatocyte cell line. Infect. Immun. 61:3068–3072. 333. Wuenscher, M. D., S. Köhler, A. Bubert, U. Gerike, and W. Goebel. 1993. The iap gene of Listeria monocytogenes is essential for cell viability and its gene product, p60, has bacteriolytic activity. J. Bacteriol. 175:3491–3501. 334. Zalevsky, J., I. Grigorova, and R. D. Mullins. 2001. Activation of the Arp2/3 complex by the Listeria ActA protein. ActA binds two actin monomers and three subunits of the Arp2/3 complex. J. Biol. Chem. 276:3468–3475.

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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.

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. Al Issa, M., D. R. Fowler, A. Seaman, and M. Woodbine. 1984. Role of lipid in butylated hydroxyanisole resistance of Listeria monocytogenes. Zentralbl. Bakteriol. Mikrobiol. Hyg. [A] 258:42–50. 3. Al Makhlafi, H., J. McGuire, and M. Daeschel. 1994. Influence of pre-adsorbed milk proteins on adhesion of Listeria monocytogenes to hydrophobic and hydrophilic silica surfaces. Appl. Environ. Microbiol. 60:3560–3565. 4. Allende, A., L. Jacxsens, F. Devlieghere, J. Debevere, and F. Artes. 2002. Effect of superatmospheric oxygen packaging on sensorial quality, spoilage, and Listeria monocytogenes and Aeromonas caviae growth in fresh processed mixed salads. J. Food Prot. 65:1565–1573. 5. Alpas, H., and F. Bozoglu. 2003. Efficiency of high pressure treatment for destruction of Listeria monocytogenes in fruit juices. FEMS Immunol. Med. Microbiol. 35:269–273. 6. Amezaga, M. R., I. Davidson, D. McLaggan, A. Verheul, T. Abee, and I. R. Booth. 1995. The role of peptide metabolism in the growth of Listeria monocytogenes ATCC 23074 at high osmolarity. Microbiology 141:41–49.

DK3089_C006.fm Page 199 Tuesday, February 20, 2007 11:47 AM

Characteristics of Listeria monocytogenes Important to Food Processors

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7. Ananth, V., J. S. Dickson, D. G. Olson, and E. A. Murano. 1998. Shelf life extension, safety, and quality of fresh pork loin treated with high hydrostatic pressure. J. Food Prot. 61:1649–1656. 8. Anderson, W. A., N. D. Hedges, M. V. Jones, and M. B. Cole. 1991. Thermal inactivation of Listeria monocytogenes studied by differential scanning calorimetry. J. Gen. Microbiol. 137 (Pt 6):1419–1424. 9. Andrews, L. S., D. L. Marshall, and R. M. Grodner. 1995. Radiosensitivity of Listeria monocytogenes at various temperatures and cell concentrations. J. Food Prot. 58:748–751. 10. Angelidis, A. S., and G. M. Smith. 2003. Three transporters mediate uptake of glycine betaine and carnitine by Listeria monocytogenes in response to hyperosmotic stress. Appl. Environ. Microbiol. 69:1013–1022. 11. Angelidis, A. S., L. T. Smith, and G. M. Smith. 2002. Elevated carnitine accumulation by Listeria monocytogenes impaired in glycine betaine transport is insufficient to restore wild-type cryotolerance in milk whey. Int. J. Food Microbiol. 75:1–9. 12. Annous, B. A., L. A. Becker, D. O. Bayles, D. P. Labeda, and B. J. Wilkinson. 1997. Critical role of anteiso-C15:0 fatty acid in the growth of Listeria monocytogenes at low temperatures. Appl. Environ. Microbiol. 63:3887–3894. 13. Anonymous. 1988. Update—Listeriosis and pasteurized milk. Morbid. Mortal. Wkly. Rep. 37:764–766. 14. Aureli, P., A. Costantini, and S. Zolea. 1992. Antimicrobial activity of some plant essential oils against Listeria monocytogenes. J. Food Prot. 55:344–348. 15. Barbosa, W. B., L. Cabedo, H. J. Wederquist, J. N. Sofos, and G. R. Schmidt. 1994. Growth variation among species and strains of Listeria monocytogenes. J. Food Prot. 57:765–769, 775. 16. Bari, M. L., Y. Inatsu, S. Kawasaki, E. Nazuka, and K. Isshiki. 2002. Calcinated calcium killing of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on the surface of tomatoes. J. Food Prot. 65:1706–1711. 17. Becker, L. A., M. S. Cetin, R. W. Hutkins, and A. K. Benson. 1998. Identification of the gene encoding the alternative sigma factor σB from Listeria monocytogenes and its role in osmotolerance. J. Bacteriol. 180:4547–4554. 18. Beckers, H. J., P. S. S. Soentoro, and E. H. M. Delfgour-van Asch. 1987. The occurrence of Listeria monocytogenes in soft cheeses and raw milk and its resistance to heat. Int. J. Food Microbiol. 4:249–256. 19. Bellamy, W., M. Takase, H. Wakabayashi, K. Kawase, and M. Tomita. 1992. Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin. J. Appl. Bacteriol. 73:472–479. 20. Bellamy, W., M. Takase, K. Yamauchi, H. Wakabayashi, K. Kawase, and M. Tomita. 1992. Identification of the bactericidal domain of lactoferrin. Biochim. Biophys. Acta 1121:130–136. 21. Benkerroum, N., and W. E. Sandine. 1988. Inhibitory action of nisin against Listeria monocytogenes. J. Dairy Sci. 71:3237–3245. 22. Bereksi, N., F. Gavini, T. Benezech, and C. Faille. 2002. Growth, morphology and surface properties of Listeria monocytogenes Scott A and LO28 under saline and acid environments. J. Appl. Microbiol. 92:556–565. 23. Berry, E. D., W. J. Dorsa, G. R. Siragusa, and M. Koohmaraie. 1998. Bacterial cross-contamination of meat during liquid nitrogen immersion freezing. J. Food Prot. 61:1103–1108. 24. Best, M., M. E. Kennedy, and F. Coates. 1990. Efficacy of a variety of disinfectants against Listeria spp. Appl. Environ. Microbiol. 56:377–380. 25. Beuchat, L. R., L. J. Harris, T. E. Ward, and T. M. Kajs. 2001. Development of a proposed standard method for assessing the efficacy of fresh produce sanitizers. J. Food Prot. 64:1103–1109. 26. Beumer, R. R., M. C. Te Giffel, L. J. Cox, F. M. Rombouts, and T. Abee. 1994. Effect of exogenous proline, betaine, and carnitine on growth of Listeria monocytogenes in a minimal medium. Appl. Environ. Microbiol. 60:1359–1363. 27. Blatchley, E. R. I., and M. M. Peel. 2001. Disinfection by ultraviolet irradiation. In Disinfection, sterilization and preservation, eds. S. S. Block. New York: Lippincott Williams & Wilkins, pp. 823–852. 28. Blom, H., E. Nerbrink, R. Dainty, T. Hagtvedt, E. Borch, H. Nissen, and T. Nesbakken. 1997. Addition of 2.5% lactate and 0.25% acetate controls growth of Listeria monocytogenes in vacuum-packed, sensory-acceptable cervelat sausage and cooked ham stored at 4°C. Int. J. Food Microbiol. 38:71–76.

DK3089_C006.fm Page 200 Tuesday, February 20, 2007 11:47 AM

200

Listeria, Listeriosis, and Food Safety

29. Borezee, E., E. Pellegrini, and P. Berche. 2000. OppA of Listeria monocytogenes, an oligopeptidebinding protein required for bacterial growth at low temperature and involved in intracellular survival. Infect. Immun. 68:7069–7077. 30. Boussouel, N., F. Mathieu, V. Benoit, M. Linder, A. M. Revol-Junelles, and J. B. Milliere. 1999. Response surface methodology, an approach to predict the effects of a lactoperoxidase system, nisin, alone or in combination, on Listeria monocytogenes in skim milk. J. Appl. Microbiol. 86:642–652. 31. Bower, C. K., M. A. Daeschel, and J. McGuire. 1998. Protein antimicrobial barriers to bacterial adhesion. J. Dairy Sci. 81:2771–2778. 32. Bradshaw, J. G., J. T. Peeler, J. J. Corwin, J. M. Hunt, J. T. Tierney, E. P. Larkin, and R. M. Twedt. 1985. Thermal resistance of Listeria monocytogenes in milk. J. Food Prot. 48:743–745. 33. Bradshaw, J. G., J. T. Peeler, J. J. Corwin, J. M. Hunt, and R. M. Twedt. 1987. Thermal resistance of Listeria monocytogenes in dairy products. J. Food Prot. 50:543–546. 34. Bradshaw, J. G., J. T. Peeler, and J. Lovett. 1991. Thermal resistance of Listeria species in whole milk. J. Food Prot. 54:12–14. 35. Bremer, P. J., I. Monk, and C. M. Osborne. 2001. Survival of Listeria monocytogenes attached to stainless steel surfaces in the presence or absence of Flavobacterium spp. J. Food Prot. 64:1369–1376. 36. Brody, A. L., E. R. Strupinski, and L. R. Kline. 2001. Antimicrobial packaging. In Active packaging for food applications. Lancaster, Pa.: Technomic Pub. Co., Lancaster, pp. 131–194. 37. Brondsted, L., B. H. Kallipolitis, H. Ingmer, and S. Knochel. 2003. KdpE and a putative RsbQ homologue contribute to growth of Listeria monocytogenes at high osmolarity and low temperature. FEMS Microbiol. Lett. 219:233–239. 38. Brzin, B. 1973. The effect of NaCl on the morphology of Listeria monocytogenes. Zentralbl. Bakteriol. I. Abt. Orig. A 232:287–293. 39. Buazzi, M. M., M. E. Johnson, and E. H. Marth. 1992. Fate of Listeria monocytogenes during the manufacture and ripening of swiss cheese. J. Food Prot. 55:80–83. 40. Buazzi, M. M., and E. H. Marth. 1992. Characteristics of sodium benzoate injury of Listeria monocytogenes. Microbios 70:199–207. 41. Buchanan, R. L., and M. H. Golden. 1994. Interaction of citric acid concentration and pH on the kinetics of Listeria monocytogenes inactivation. J. Food Prot. 57:567–570. 42. Buchanan, R. L., M. H. Golden, R. C. Whiting, J. G. Philips, and J. L. Smith. 1994. Nonthermal inactivation models for Listeria monocytogenes. J. Food Sci. 59:179–188. 43. Buchanan, R. L., H. G. Stahl, and R. C. Whiting. 1989. Effects and interactions of temperature, pH, atmosphere, sodium chloride and sodium nitrite on growth of Listeria monocytogenes. J. Food Prot. 52:844–851. 44. Budavari, S. 1989. The Merck index. Rahway, NJ: Merck & Co. 45. Budde, B. B., T. Hornbaek, T. Jacobsen, V. Barkholt, and A. G. Koch. 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. 46. Buncic, S., S. M. Avery, J. Rocourt, and M. Dimitrijevic. 2001. Can food-related environmental factors induce different behavior in two key serovars, 4b and 1/2a, of Listeria monocytogenes? Int. J. Food Microbiol. 65:201–212. 47. Buncic, S., S. M. Avery, and A. R. Rogers. 1996. Listeriolysin O production and pathogenicity of nongrowing Listeria monocytogenes stored at refrigeration temperature. Int. J. Food Microbiol. 31:133–147. 48. Buncic, S., C. M. Fitzgerald, R. G. Bell, and J. A. Hudson. 1995. Individual and combined listericidal effects of sodium lactate, potassium sorbate, nisin and curing salts at refrigeration temperature. J. Food Safety 15:247–264. 49. Bunduki, M. M. C., K. J. Flanders, and C. W. Donnelly. 1995. Metabolic and structural sites of damage in heat- and sanitizer-injured populations of Listeria monocytogenes. J. Food Prot. 58:410–415. 50. Bunning, V. K., R. G. Crawford, J. T. Tierney, and J. T. Peeler. 1990. Thermotolerance of Listeria monocytogenes and Salmonella typhimurium after sublethal heat shock. Appl. Environ. Microbiol. 56:3216–3219. 51. Bunning, V. K., R. G. Crawford, J. T. Tierney, and J. T. Peeler. 1992. Thermotolerance of heat-shocked Listeria monocytogenes in milk exposed to high-temperature, short time pasteurization. Appl. Environ. Microbiol. 58:2096–2098.

DK3089_C006.fm Page 201 Tuesday, February 20, 2007 11:47 AM

Characteristics of Listeria monocytogenes Important to Food Processors

201

52. Bunning, V. K., C. W. Donnelly, J. T. Peeler, E. H. Briggs, J. G. Bradshaw, R. G. Crawford, C. M. Beliveau, and J. T. Tierney. 1988. Thermal inactivation of Listeria monocytogenes within bovine milk phagocytes. Appl. Environ. Microbiol. 54:364–370. 53. Calderon-Miranda, M. L., G. V. Barbosa-Canovas, and B. G. Swanson. 1999. Inactivation of Listeria innocua in liquid whole egg by pulsed electric fields and nisin. Int. J. Food Microbiol. 51:7–17. 54. Calderon-Miranda, M. L., G. V. Barbosa-Canovas, and B. G. Swanson. 1999. Transmission electron microscopy of Listeria innocua treated by pulsed electric fields and nisin in skimmed milk. Int. J. Food Microbiol. 51:31–38. 55. Cammack, R., C. L. Joannou, X. Y. Cui, M. C. Torres, S. R. Maraj, and M. N. Hughes. 1999. Nitrite and nitrosyl compounds in food preservation. Biochim. Biophys. Acta 1411:475–488. 56. Chae, M. S., and H. Schraft. 2000. Comparative evaluation of adhesion and biofilm formation of different Listeria monocytogenes strains. Int. J. Food Microbiol. 62:103–111. 57. Charbonneau, D. L., J. M. Ponte, and B. A. Kochanowski. 2000. A method of assessing the efficacy of hand sanitizers: Use of real soil encountered in the food service industry. J. Food Prot. 63:495–501. 58. Chavant, P., B. Martinie, T. Meylheuc, M. N. Bellon-Fontaine, and M. Hebraud. 2002. Listeria monocytogenes LO28: Surface physicochemical properties and ability to form biofilms at different temperatures and growth phases. Appl. Environ. Microbiol. 68:728–737. 59. Chen, N., and L. A. Shelef. 1992. Relationship between water activity, salts of lactic acids, and growth of Listeria monocytogenes in a meat system. J. Food Prot. 55:574–578. 60. Chhabra, A. T., W. H. Carter, R. H. Linton, and M. A. Cousin. 1999. A predictive model to determine the effects of pH, milk fat, and temperature on thermal inactivation of Listeria monocytogenes. J. Food Prot. 62:1143–1149. 61. Chihib, N. E., D. S. Ribeiro, G. Delattre, M. Laroche, and M. Federighi. 2003. Different cellular fatty acid pattern behaviors of two strains of Listeria monocytogenes Scott A and CNL 895807 under different temperature and salinity conditions. FEMS Microbiol. Lett. 218:155–160. 62. Choi, H. K., M. M. Schaack, and E. H. Marth. 1988. Survival of Listeria monocytogenes in cultured buttermilk and yogurt. Milchwissenschaft 43:790–792. 63. Chung, K.-T., S. E. J. Stevens, W.-F. Lin, and C. I. Wei. 1993. Growth inhibition of selected foodborne bacteria by tannic acid, propyl gallate, and related compounds. Lett. Appl. Microbiol. 17:29–32. 64. Chung, Y. K. 2001. Control of Clostridium botulinum by bacteriocins and characterization of nisin action during spore-to-cell transformation. Ph.D. dissertation, The Ohio State University. 65. Church, I. J., and A. L. Parsons. 1995. Modified atmosphere packaging technology—a review. J. Sci. Food Agric. 67:143–152. 66. Clardy, S., D. M. Foley, F. Caporaso, M. L. Calicchia, and A. Prakash. 2002. Effect of gamma irradiation on Listeria monocytogenes in frozen, artificially contaminated sandwiches. J. Food Prot. 65:1740–1744. 67. Coma, V., I. Sebti, P. Pardon, A. Deschamps, and F. H. Pichavant. 2001. Antimicrobial edible packaging based on cellulosic ethers, fatty acids, and nisin incorporation to inhibit Listeria innocua and Staphylococcus aureus. J. Food Prot. 64:470–475. 68. Conner, D. E., R. E. Brackett, and L. R. Beuchat. 1986. Effect of temperature, sodium chloride, and pH on growth of Listeria monocytogenes in cabbage juice. Appl. Environ. Microbiol. 52:59–63. 69. Conte, M. P., G. Petrone, A. M. Di Biase, M. G. Ammendolia, F. Superti, and L. Seganti. 2000. Acid tolerance in Listeria monocytogenes influences invasiveness of enterocyte-like cells and macrophagelike cells. Microb. Pathog. 29:137–144. 70. Cotter, P. D., K. O’Reilly, and C. Hill. 2001. Role of the glutamate decarboxylase acid resistance system in the survival of Listeria monocytogenes LO28 in low-pH foods. J. Food Prot. 64:1362–1368. 71. Cowan, M. M., K. Z. Abshire, S. L. Houk, and S. M. Evans. 2003. Antimicrobial efficacy of a silver–zeolite matrix coating on stainless steel. J. Ind. Microbiol. Biotechnol. 30:102–106. 72. Dallmier, A. W., and S. E. Martin. 1988. Catalase and superoxide dismutase activities after heat injury of Listeria monocytogenes. Appl. Environ. Microbiol. 54:581–582. 73. Dawson, P. L., G. D. Carl, J. C. Acton, and I. Y. Han. 2002. Effect of lauric acid and nisin-impregnated soy-based films on the growth of Listeria monocytogenes on turkey bologna. Poult. Sci. 81:721–726. 74. Degnan, A. J., N. Buyong, and J. B. Luchansky. 1993. Antilisterial activity of pediocin AcH in model systems in the presence of an emulsifier or encapsulated within liposomes. Int. J. Food Microbiol. 18:127–138.

DK3089_C006.fm Page 202 Tuesday, February 20, 2007 11:47 AM

202

Listeria, Listeriosis, and Food Safety

75. Denis, F., and J.-P. Ramet. 1989. Antibacterial activity of the lactoperoxidase system on Listeria monocytogenes in trypticase soy broth, UHT milk and French soft cheese. J. Food Prot. 52:706–711. 76. Dionysius, D. A., and J. M. Milne. 1997. Antibacterial peptides of bovine lactoferrin: Purification and characterization. J. Dairy Sci. 80:667–674. 77. Dominguez, L., J. F. F. Garayzabal, E. R. Ferri, J. A. Vazquez, C. Ambrosio, and G. Suarez. 1987. Viability of Listeria monocytogenes in milk treated with hydrogen peroxide. J. Food Prot. 50:636–639. 78. Donnelly, C. W., E. H. Briggs, and L. S. Donnelly. 1987. Comparison of heat resistance of Listeria monocytogenes in milk as determined in two methods. J. Food Prot. 50:14–17. 79. Doyle, M. E., A. S. Mazzotta, T. Wang, D. W. Wiseman, and V. N. Scott. 2001. Heat resistance of Listeria monocytogenes. J. Food Prot. 64:410–429. 80. Doyle, M. P., K. A. Glass, J. T. Beery, G. A. Garcia, D. J. Pollard, and R. D. Schultz. 1987. Survival of Listeria monocytogenes in milk during high-temperature, short-time pasteurization. Appl. Environ. Microbiol. 53:1433–1438. 81. Duh, Y.-H., and D. W. Schaffner. 1993. Modeling the effect of temperature on the growth rate and lag time of Listeria innocua and Listeria monocytogenes. J. Food Prot. 56:205–210. 82. Dunn, J. 2001. Pulsed electric field processing: An overview. In Pulsed electric fields in food processing: Fundamental aspects and applications, eds. G. V. Barbosa–Canovas and Q. H. Zhang. Lancaster, Pa.: Technomic Pub. Co., pp. 1–30. 83. Dunn, J., T. Ott, and W. Clark. 1995. Pulsed-light treatment of food and packaging. Food Technol. 49:95–98. 84. Durst, J. 1975. The role of temperature factors in the epidemiology of listeriosis. Zentralbl. Bakteriol. [Orig. A] 233:72–74. 85. Durst, J., and A. Sawinski. 1972. Beitraege zur Untersuchung der Ausbreitungsmoeglichkeit der Listeria monocytogenes. Z. Gesamte Hyg. Grenzgeb. 18:117–118. 86. Dychdala, G. R. 2001. Chlorine and chlorine compounds. In Disinfection, sterilization and preservation, ed. S. S. Block. New York: Lippincott Williams & Wilkins, pp. 135–158. 87. Earnshaw, R. G., and J. G. Banks. 1989. A note on the inhibition of Listeria monocytogenes NTCC 11994 in milk by an activated lactoperoxidase system. Lett. Appl. Microbiol. 8:203–205. 88. Eastern Regional Research Center. 2003. USDA. Pathogen Modeling Program (version 7.0). http://www.ars.usda.gov/services/docs.htm?docid=6786. 89. El-Kest, S. E., and E. H. Marth. 1988. Inactivation of Listeria monocytogenes by chlorine. J. Food Prot. 51:520–524. 90. El-Kest, S. E., and E. H. Marth. 1988. Temperature, pH, and strain of pathogen as factors affecting inactivation of Listeria monocytogenes by chlorine. J. Food Prot. 51:622–625. 91. El-Kest, S. E., and E. H. Marth. 1991. Injury and death of frozen Listeria monocytogenes as affected by glycerol and milk components. J. Dairy Sci. 74:1201–1208. 92. El-Kest, S. E., and E. H. Marth. 1992. Lysozyme and lipase alter unfrozen and frozen/thawed cells of Listeria monocytogenes. J. Food Prot. 55:777–781. 93. El-Kest, S. E., and E. H. Marth. 1992. Transmission electron microscopy of unfrozen and frozen/thawed cells of Listeria monocytogenes treated with lipase and lysozyme. J. Food Prot. 55:687–696. 94. El-Kest, S. E., A. E. Yousef, and E. H. Marth. 1991. Fate of Listeria monocytogenes during freezing and frozen storage. J. Food Sci. 56:1068–1071. 95. El-Shenawy, M. A., and E. H. Marth. 1989. 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. 96. El-Shenawy, M. A., and E. H. Marth. 1989. Behavior of Listeria monocytogenes in the presence of sodium propionate. Int. J. Food Microbiol. 8:85–94. 97. El-Shenawy, M. A., and E. H. Marth. 1991. Organic acids enhance the antilisterial activity of potassium sorbate. J. Food Prot. 54:593–597. 98. El-Shenawy, M. A., and E. H. Marth. 1992. Behavior of Listeria monocytogenes in the presence of sodium propionate together with food acids. J. Food Prot. 55:241–245. 99. El-Shenawy, M. A., A. E. Yousef, and E. H. Marth. 1989. Thermal injury and inactivation of Listeria monocytogenes in reconstituted nonfat dry milk. Milchwissenschaft 44:741–745.

DK3089_C006.fm Page 203 Tuesday, February 20, 2007 11:47 AM

Characteristics of Listeria monocytogenes Important to Food Processors

203

100. El-Shenawy, M. A., A. E. Yousef, and E. H. Marth. 1989. Radiation sensitivity of Listeria monocytogenes in broth or in raw ground beef. Lebensm. Wiss. Technol. 22:387–390. 101. Erickson, J. P., and P. Jenkins. 1991. Comparative Salmonella spp. and Listeria monocytogenes inactivation rates in four commercial mayonnaise products. J. Food Prot. 54:913–916. 102. Fairchild, T. M., and P. M. Foegeding. 1993. A proposed non-pathogenic biological indicator for thermal inactivation of Listeria monocytogenes. Appl. Environ. Microbiol. 59:1247–1250. 103. Faith, N. G., A. E. Yousef, and J. B. Luchansky. 1992. Inhibition of Listeria monocytogenes by liquid smoke and isoeugenol, a phenolic compound found in smoke. J. Food Safety 12:303–314. 104. Fang, T. J., and L.-W. Lin. 1994. Growth of Listeria monocytogenes and Pseudomonas fragi on cooked pork in modified atmosphere packaging/nisin combination system. J. Food Prot. 57:479–485. 105. Farber, J. M., and B. E. Brown. 1990. Effect of prior heat shock on heat resistance of Listeria monocytogenes in meat. Appl. Environ. Microbiol. 56:1584–1587. 106. Farber, J. M., Y. Cai, and W. H. Ross. 1996. Predictive modeling of the growth of Listeria monocytogenes in CO2 environments. Int. J. Food Microbiol. 32:133–144. 107. Farber, J. M., J. Y. D’Aoust, M. Diotte, A. Sewell, and E. Daley. 1998. Survival of Listeria spp. on raw whole chickens cooked in microwave ovens. J. Food Prot. 61:1465–1469. 108. Fatemi, P., and J. F. Frank. 1999. Inactivation of Listeria monocytogenes/Pseudomonas biofilms by peracid sanitizers. J. Food Prot. 62:761–765. 109. Ferreira, A., C. P. O’Byrne, and K. J. Boor. 2001. Role of σB in heat, ethanol, acid, and oxidative stress resistance and during carbon starvation in Listeria monocytogenes. Appl. Environ. Microbiol. 67:4454–4457. 110. Fields, F. O. 1996. Use of bacteriocins in food: regulatory considerations. J. Food Prot. 59 (Suppl.):72–77. 111. Fisher, C. W., D. Lee, B. A. Dodge, K. M. Hamman, J. B. Robbins, and S. E. Martin. 2000. Influence of catalase and superoxide dismutase on ozone inactivation of Listeria monocytogenes. Appl. Environ. Microbiol. 66:1405–1409. 112. Foegeding, P. M., and N. W. Stanley. 1991. Listeria innocua transformed with an antibiotic resistance plasmid as a thermal-resistance indicator for Listeria monocytogenes. J. Food Prot. 54:519–523. 113. Food and Drug Administration. 1998. Lysozyme (tentative final rule; 21CFR184; Docket No. 89G-0393). Code of Federal Regulations 63:12421–12426. 114. Food and Drug Administration. 2003. Cheeses and related cheese products. Code of Federal Regulations Title 21, Chapter I, Part 133.3:309. 115. Food and Drug Administration. 2003. Direct food substances affirmed as generally recognized as safe. Code of Federal Regulations Title 21, Chapter I, Part 184:547, 515, 532. 116. Food and Drug Administration. 2003. Food additives permitted for direct addition to food for human consumption. Code of Federal Regulations Title 21, Chapter I, Part 172:32–33, 39. 117. Food and Drug Administration. 2003. Indirect food additives: adjuvants, production aids, and sanitizers. Code of Federal Regulations Title 21, Chapter I, Part 178:349–350. 118. Food and Drug Administration. 2003. Irradiation in the production, processing, and handling of food. Code of Federal Regulations Title 21, Chapter I, Part 179:439–445. 119. Food and Drug Administration. 2003. Secondary direct food additives permitted in food for human consumption. Code of Federal Regulations Title 21, Chapter I, Part 173:144–145. 120. Fraser, K. R., D. Harvie, P. J. Coote, and C. P. O’Byrne. 2000. Identification and characterization of an ATP binding cassette L-carnitine transporter in Listeria monocytogenes. Appl. Environ. Microbiol. 66:4696–4704. 121. Friedman, M., P. R. Henika, and R. E. Mandrell. 2002. Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, and Salmonella enterica. J. Food Prot. 65:1545–1560. 122. Fyfe, L., F. Armstrong, and J. Stewart. 1997. Inhibition of Listeria monocytogenes and Salmonella enteritidis by combinations of plant oils and derivatives of benzoic acid: the development of synergistic antimicrobial combinations. Int. J. Antimicrob. Agents 9:195–199. 123. Gahan, C. G., B. O’Driscoll, and C. Hill. 1996. Acid adaptation of Listeria monocytogenes can enhance survival in acidic foods and during milk fermentation. Appl. Environ. Microbiol. 62:3128–3132. 124. Gahan, C. G., J. O’Mahony, and C. Hill. 2001. Characterization of the groESL operon in Listeria monocytogenes: utilization of two reporter systems (gfp and hly) for evaluating in vivo expression. Infect. Immun. 69:3924–3932.

DK3089_C006.fm Page 204 Tuesday, February 20, 2007 11:47 AM

204

Listeria, Listeriosis, and Food Safety

125. Garcia-Graells, C., C. Valckx, and C. W. Michiels. 2000. Inactivation of Escherichia coli and Listeria innocua in milk by combined treatment with high hydrostatic pressure and the lactoperoxidase system. Appl. Environ. Microbiol. 66:4173–4179. 126. Gardan, R., P. Cossart, and J. Labadie. 2003. Identification of Listeria monocytogenes genes involved in salt and alkaline-pH tolerance. Appl. Environ. Microbiol. 69:3137–3143. 127. Gervilla, R., V. Ferragut, and B. Guamis. 2000. High pressure inactivation of microorganisms inoculated into ovine milk of different fat contents. J. Dairy Sci. 83:674–682. 128. Gianfranceschi, M., and P. Aureli. 1996. Freezing and frozen storage on the survival of Listeria monocytogenes in different foods. Ital. J. Food Sci. 8:303–309. 129. Gill, A. O., P. Delaquis, P. Russo, and R. A. Holley. 2002. Evaluation of antilisterial action of cilantro oil on vacuum packed ham. Int. J. Food Microbiol. 73:83–92. 130. Gill, A. O., and R. A. Holley. 2000. Surface application of lysozyme, nisin, and EDTA to inhibit spoilage and pathogenic bacteria on ham and bologna. J. Food Prot. 63:1338–1346. 131. Gill, A. O., and R. A. Holley. 2003. Interactive inhibition of meat spoilage and pathogenic bacteria by lysozyme, nisin and EDTA in the presence of nitrite and sodium chloride at 24°C. Int. J. Food Microbiol. 80:251–259. 132. Glass, K. A., and M. P. Doyle. 1989. Fate and thermal inactivation of Listeria monocytogenes in beaker sausage and pepperoni. J. Food Prot. 52:226–231, 235. 133. Glass, K. A., D. A. Granberg, A. L. Smith, A. M. McNamara, M. Hardin, J. Mattias, K. Ladwig, and E. A. Johnsoni. 2002. Inhibition of Listeria monocytogenes by sodium diacetate and sodium lactate on weiners and cooked bratwurst. J. Food Prot. 65:116–123. 134. Goff, J. F., A. K. Bhunia, and M. G. Johnson. 1996. Complete inhibition of low levels of Listeria monocytogenes on refrigerated chicken meat with pediocin AcH bound to heat-killed Pediococcus acidilactici cells. J. Food Prot. 59:1187–1192. 135. Gottardi, W. 2001. Iodine and iodine compounds. In Disinfection, sterilization and preservation, ed. S. S. Block. New York: Lippincott Williams & Wilkins, pp. 159–184. 136. Hanawa, T., M. Kai, S. Kamiya, and T. Yamamoto. 2000. Cloning, sequencing, and transcriptional analysis of the dnaK heat shock operon of Listeria monocytogenes. Cell Stress Chaperones 5:21–29. 137. Hansen, T. B., and S. Knochel. 2003. Image analysis method for evaluation of specific and nonspecific hand contamination. J. Appl. Microbiol. 94:483–494. 138. Harp, E., and S. E. Gilliland. 2003. Evaluation of a select strain of Lactobacillus delbrueckii subsp. lactis as a biological control agent for pathogens on fresh-cut vegetables stored at 7°C. J. Food Prot. 66:1013–1018. 139. Harris, L. J., H. P. Fleming, and T. R. Klaenhammer. 1991. Sensitivity and resistance of Listeria monocytogenes ATCC 19115, Scott A, and UAL500 to nisin. J. Food Prot. 54:836–840. 140. Harrison, M. A., Y.-W. Huang, C.-H. Chao, and T. Shineman. 1991. Fate of Listeria monocytogenes on packaged, refrigerated, and frozen seafood. J. Food Prot. 54:524–527. 141. Hefnawy, Y. A., S. I. Moustafa, and E. H. Marth. 1993. Sensitivity of Listeria monocytogenes to selected spices. J. Food Prot. 56:876–878. 142. Heldman, D. R., and R. L. Newsome. 2003. Kinetic models for microbial survival during processing. Food Technol. 57:40–46, 100. 143. Helke, D. M., and A. C. L. Wong. 1994. Survival and growth characteristics of Listeria monocytogenes and Salmonella typhimurium on stainless steel and buna-n-rubber. J. Food Prot. 57:963–968. 144. Hibma, A. M., S. A. Jassim, and M. W. Griffiths. 1997. Infection and removal of L-forms of Listeria monocytogenes with bred bacteriophage. Int. J. Food Microbiol. 34:197–207. 145. Hoffman, K. L., I. Y. Han, and P. L. Dawson. 2001. Antimicrobial effects of corn zein films impregnated with nisin, lauric acid, and EDTA. J. Food Prot. 64:885–889. 146. Holsinger, V. H., P. W. Smith, J. L. Smith, and S. A. Palumbo. 1992. Thermal destruction of Listeria monocytogenes in ice cream mix. J. Food Prot. 55:234–237. 147. Hudson, J. A. 1992. Efficacy of high sodium chloride concentrations for the destruction of Listeria monocytogenes. Lett. Appl. Microbiol. 14:178–180. 148. 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. 149. Hughey, V. L., and E. A. Johnson. 1987. Antimicrobial activity of lysozyme against bacteria involved in food spoilage and foodborne disease. Appl. Environ. Microbiol. 53:2165–2170.

DK3089_C006.fm Page 205 Tuesday, February 20, 2007 11:47 AM

Characteristics of Listeria monocytogenes Important to Food Processors

205

150. Hughey, V. L., P. A. Wilger, and E. A. Johnson. 1989. Antibacterial activity of hen egg white lysozyme against Listeria monocytogenes Scott A in foods. Appl. Environ. Microbiol. 55:631–638. 151. Islam, M., J. Chen, M. P. Doyle, and M. Chinnan. 2002. Control of Listeria monocytogenes on turkey frankfurters by generally-recognized-as-safe preservatives. J. Food Prot. 65:1411–1416. 152. Isom, L. L., K. S. Khambatta, J. L. Moluf, D. F. Akers, and S. E. Martin. 1995. Filament formation of Listeria monocytogenes. J. Food Prot. 58:1031–1033. 153. Iturriaga, M. H., S. M. Arvizu-Medrano, and E. F. Escartin. 2002. Behavior of Listeria monocytogenes in avocado pulp and processed guacamole. J. Food Prot. 65:1745–1749. 154. Jemmi, T., and A. Keusch. 1992. Behavior of Listeria monocytogenes during processing and storage of experimentally contaminated hot-smoked trout. Int. J. Food Microbiol. 15:339–346. 155. Johansen, C., L. Gram, and A. S. Meyer. 1994. The combined inhibitory effect of lysozyme and low pH on the growth of Listeria monocytogenes. J. Food Prot. 57:561–566. 156. Johnson, J. L., M. P. Doyle, R. G. Cassens, and J. L. Schoeni. 1988. Fate of Listeria monocytogenes in tissues of experimentally infected cattle and in hard salami. Appl. Environ. Microbiol. 54:497–501. 157. Jorgensen, F., B. Panaretou, P. J. Stephens, and S. Knochel. 1996. Effect of pre- and postheat shock temperature on the persistence of thermotolerance and heat shock-induced proteins in Listeria monocytogenes. J. Appl. Bacteriol. 80:216–224. 158. Jorgensen, F., P. J. Stephens, and S. Knochel. 1995. The effect of osmotic shock and subsequent adaptation on the thermotolerance and cell morphology of Listeria monocytogenes. J. Appl. Bacteriol. 79:274–281. 159. 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. 160. Juneja, V. K., and B. S. Eblen. 1999. Predictive thermal inactivation model for Listeria monocytogenes with temperature, pH, NaCl, and sodium pyrophosphate as controlling factors. J. Food Prot. 62:986–993. 161. Junttila, J. R., S. I. Niemela, and J. Hirn. 1988. Minimum growth temperatures of Listeria monocytogenes and nonhemolytic Listeria. J. Appl. Bacteriol. 65:321–327. 162. Kalchayanand, N., A. Sikes, C. P. Dunne, and B. Ray. 1998. Interaction of hydrostatic pressure, time and temperature of pressurization and pediocin AcH on inactivation of foodborne bacteria. J. Food Prot. 61:425–431. 163. Kalchayanand, N., T. Sikes, and C. P. Dunne. 1994. Hydrostatic pressure and electroporation have increased bactericidal efficiency in combination with bacteriocins. Appl. Environ. Microbiol. 60:4174–4177. 164. Kalmokoff, M. L., J. W. Austin, X. D. Wan, G. Sanders, S. Banerjee, and J. M. Farber. 2001. Adsorption, attachment and biofilm formation among isolates of Listeria monocytogenes using model conditions. J. Appl. Microbiol. 91:725–734. 165. Kamat, A., R. Warke, M. Kamat, and P. Thomas. 2000. Low-dose irradiation as a measure to improve microbial quality of ice cream. Int. J. Food Microbiol. 62:27–35. 166. Kamau, D. N., S. Doores, and K. M. Pruitt. 1990. Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system. Appl. Environ. Microbiol. 56:2711–2716. 167. Karatzas, A. K., E. P. Kets, E. J. Smid, and M. H. Bennik. 2001. The combined action of carvacrol and high hydrostatic pressure on Listeria monocytogenes Scott A. J. Appl. Microbiol. 90:463–469. 168. Kerr, K. G., D. Birkenhead, K. Seale, J. Major, and P. M. Hawkey. 1993. Prevalence of Listeria spp. on the hands of food workers. J. Food Prot. 56:525–527. 169. Kihm, D. J., G. J. Leyer, G. H. An, and E. A. Johnson. 1994. Sensitization of heat-treated Listeria monocytogenes to added lysozyme in milk. Appl. Environ. Microbiol. 60:3854–3861. 170. Kim, B., D. Kim, D. Cho, and S. Cho. 2003. Bactericidal effect of TiO2 photocatalyst on selected foodborne pathogenic bacteria. Chemosphere 52:277–281. 171. Kim, J. G., A. E. Yousef, and S. Dave. 1999. Application of ozone for enhancing the microbiological safety and quality of foods: A review. J. Food Prot. 62:1071–1087. 172. Kim, J. G., A. E. Yousef, and M. A. Khadre. 2003. Ozone and its current and future application in the food industry. Adv. Food Nutr. Res. 45:167–218. 173. Kim, J.-G., and A. E. Yousef. 2000. Inactivation kinetics of foodborne spoilage and pathogenic bacteria by ozone. J. Food Sci. 65:521–528. 174. Kim, K.-T., E. A. Murano, and D. G. Olson. 1994. Effect of heat shock on production of listeriolysin O by Listeria monocytogenes. J. Food Safety 14:273–279.

DK3089_C006.fm Page 206 Tuesday, February 20, 2007 11:47 AM

206

Listeria, Listeriosis, and Food Safety

175. Kim, K.-T., E. A. Murano, and D. G. Olson. 1994. Heating and storage conditions affect the survival and recovery of Listeria monocytogenes in ground pork. J. Food Sci. 59:30–32, 59. 176. Kim, T., J. L. Silva, and T. C. Chen. 2002. Effects of UV irradiation on selected pathogens in peptone water and on stainless steel and chicken meat. J. Food Prot. 65:1142–1145. 177. Knabel, S. J., and S. A. Thielen. 1995. Enhanced recovery of severely heat-injured, thermotolerant Listeria monocytogenes from USDA and FDA primary enrichment media using a novel, simple, strictly anaerobic method. J. Food Prot. 58:29–34. 178. Knight, K. P., F. M. Bartlett, R. C. McKellar, and L. J. Harris. 1999. Nisin reduces the thermal resistance of Listeria monocytogenes Scott A in liquid whole egg. J. Food Prot. 62:999–1003. 179. Knowles, J., and S. Roller. 2001. Efficacy of chitosan, carvacrol, and a hydrogen peroxide-based biocide against foodborne microorganisms in suspension and adhered to stainless steel. J. Food Prot. 64:1542–1548. 180. Ko, R., and L. T. Smith. 1999. Identification of an ATP-driven, osmoregulated glycine betaine transport system in Listeria monocytogenes. Appl. Environ. Microbiol. 65:4040–4048. 181. Ko, R., L. T. Smith, and G. M. Smith. 1994. Glycine betaine confers enhanced osmotolerance and cryotolerance on Listeria monocytogenes. J. Bacteriol. 176:426–431. 182. Kouassi, Y., and L. A. Shelef. 1995. Listeriolysin O secretion by Listeria monocytogenes in broth containing salts of organic acids. J. Food Prot. 58:1314–1319. 183. Kouassi, Y., and L. A. Shelef. 1995. Listeriolysin O secretion by Listeria monocytogenes in the presence of cysteine and sorbate. Lett. Appl. Microbiol. 20:295–299. 184. Kraemer, K. H., and J. Baumgart. 1993. Sliced frankfurter-type sausage. Inhibiting Listeria monocytogenes by means of a modified atmosphere. Fleischwirtschaft 73:1279–1280. 185. Krysinski, E. P., L. J. Brown, and T. J. Marchisello. 1992. Effect of cleaners and sanitizers on Listeria monocytogenes attached to product contact surfaces. J. Food Prot. 55:246–251. 186. Kuleasan, H., and M. L. Cakmakci. 2002. Effect of reuterin produced by Lactobacillus reuteri on the surface of sausages to inhibit the growth of Listeria monocytogenes and Salmonella spp. Nahrung 46:408–410. 187. Kussendrager, K. D., and A. C. van Hooijdonk. 2000. Lactoperoxidase: physicochemical properties, occurrence, mechanism of action and applications. Br. J. Nutr. 84 (Suppl. 1):S19–S25. 188. Lado, B. H. 2003. Characteristics of Listeria monocytogenes important for pulsed electric field process optimization. Ph.D. dissertation, The Ohio State University. 189. Lado, B. H., and A. E. Yousef. 2002. Alternative food-preservation technologies: Efficacy and mechanisms. Microbes Infect. 4:433–440. 190. Lado, B. H., and A. E. Yousef. 2003. Selection and identification of a Listeria monocytogenes target strain for pulsed electric field process optimization. Appl. Environ. Microbiol. 69:2223–2229. 191. Larson, A. E., E. A. Johnson, and J. H. Nelson. 1999. Survival of Listeria monocytogenes in commercial cheese brines. J. Dairy Sci. 82:1860–1868. 192. Leimeister-Wachter, M., E. Domann, and T. Chakraborty. 1992. The expression of virulence genes in Listeria monocytogenes is thermoregulated. J. Bacteriol. 174:947–952. 193. Leistner, L. 2000. Basic aspects of food preservation by hurdle technology. Int. J. Food Microbiol. 55:181–186. 194. Lemaitre, J. P., H. Echchannaoui, G. Michaut, C. Divies, and A. Rousset. 1998. Plasmid-mediated resistance to antimicrobial agents among listeriae. J. Food Prot. 61:1459–1464. 195. Leriche, V., and B. Carpentier. 2000. Limitation of adhesion and growth of Listeria monocytogenes on stainless steel surfaces by Staphylococcus sciuri biofilms. J. Appl. Microbiol. 88:594–605. 196. Leriche, V., D. Chassaing, and B. Carpentier. 1999. Behavior of Listeria monocytogenes in an artificially made biofilm of a nisin-producing strain of Lactococcus lactis. Int. J. Food Microbiol. 51:169–182. 197. Liao, C.-C., A. E. Yousef, E. R. Richter, and G. W. Chism. 1993. Pediococcus acidilactici PO2 bacteriocin production in whey permeate and inhibition of Listeria monocytogenes in foods. J. Food Sci. 58:430–434. 198. Liberti, R., G. Franciosa, M. Gianfranceschi, and P. Aureli. 1996. Effect of combined lysozyme and lipase treatment on the survival of Listeria monocytogenes. Int. J. Food Microbiol. 32:235–242. 199. Linton, R. H., M. D. Pierson, and J. R. Bishop. 1990. Increase in heat resistance of Listeria monocytogenes during pasteurization. J. Food Prot. 53:370–376.

DK3089_C006.fm Page 207 Tuesday, February 20, 2007 11:47 AM

Characteristics of Listeria monocytogenes Important to Food Processors

207

200. Lis-Balchin, M., G. Buchbauer, T. Hirtenlehner, and M. Resch. 1998. Antimicrobial activity of Pelargonium essential oils added to a quiche filling as a model food system. Lett. Appl. Microbiol. 27:207–210. 201. Liu, S., J. E. Graham, L. Bigelow, P. D. Morse, and B. J. Wilkinson. 2002. Identification of Listeria monocytogenes genes expressed in response to growth at low temperature. Appl. Environ. Microbiol. 68:1697–1705. 202. Loessner, M., S. Guenther, S. Steffan, and S. Scherer. 2003. A pediocin-producing Lactobacillus plantarum strain inhibits Listeria monocytogenes in a multispecies cheese surface microbial ripening consortium. Appl. Environ. Microbiol. 69:1854–1857. 203. Lopez, J. A. 1986. Evaluation of dairy and food-plant sanitizers against Salmonella typhimurium and Listeria monocytogenes. J. Dairy Sci. 69:2791–2796. 204. Lou, Y. 1997. Environmental stress adaptation and stress protection in Listeria monocytogenes. Ph.D. dissertation, The Ohio State University. 205. Lou, Y., and A. E. Yousef. 1996. Resistance of Listeria monocytogenes to heat after adaptation to environmental stresses. J. Food Prot. 59:465–471. 206. Lou, Y., and A. E. Yousef. 1997. Adaptation to sublethal environmental stresses protects Listeria monocytogenes against lethal preservation factors. Appl. Environ. Microbiol. 63:1252–1255. 207. Lucore, L. A., T. H. Shellhammer, and A. E. Yousef. 2000. Inactivation of Listeria monocytogenes Scott A on artificially contaminated frankfurters by high-pressure processing. J. Food Prot. 63:662–664. 208. Lunden, J., T. Autio, A. Markkula, S. Hellstrom, and H. Korkeala. 2003. Adaptive and cross-adaptive responses of persistent and nonpersistent Listeria monocytogenes strains to disinfectants. Int. J. Food Microbiol. 82:265–272. 209. Lyon, W. J., J. K. Sethi, and B. A. Glatz. 1993. Inhibition of psychrotrophic organisms by propionicin PLG-1, a bacteriocin produced by Propionibacterium thoenii. J. Dairy Sci. 76:1506–1513. 210. MacDonald, F., and A. D. Sutherland. 1993. Effect of heat treatment on Listeria monocytogenes and Gram-negative bacteria in sheep, cow and goat milks. J. Appl. Bacteriol. 75:336–343. 211. Mackey, B. M., C. Pritchet, A. Norris, and G. C. Mead. 1990. Heat resistance of Listeria: Strain differences and effects of meat type and curing salts. Lett. Appl. Microbiol. 10:251–255. 212. Mafu, A. A., D. Roy, J. Goulet, and P. Magny. 1990. Attachment of Listeria monocytogenes to stainless steel, glass, propylene, and rubber surfaces after short contact times. J. Food Prot. 53:742–746. 213. Mafu, A. A., D. Roy, J. Goulet, and L. Savoie. 1991. Characterization of physicochemical forces involved in adhesion of Listeria monocytogenes to surfaces. Appl. Environ. Microbiol. 57:1969–1973. 214. Mafu, A. A., D. Roy, J. Goulet, L. Savoie, and R. Roy. 1990. Efficiency of sanitizing agents for destroying Listeria monocytogenes on contaminated surfaces. J. Dairy Sci. 73:3428–3432. 215. Mainsnier-Patin, S., S. R. Tatini, and J. Richard. 1995. Combined effect of nisin and moderate heat on destruction of Listeria monocytogenes in milk. Lait 75:81–91. 216. Mak, P. P., B. H. Ingham, and S. C. Ingham. 2001. Validation of apple cider pasteurization treatments against Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes. J. Food Prot. 64:1679–1689. 217. Marsh, E. J., H. Luo, and H. Wang. 2004. A three-tiered approach to differentiate Listeria monocytogenes biofilm-forming abilities. FEMS Microbiol. Lett. 228:203–210. 218. Masschalck, B., D. Deckers, and C. W. Michiels. 2002. Lytic and nonlytic mechanism of inactivation of Gram-positive bacteria by lysozyme under atmospheric and high hydrostatic pressure. J. Food Prot. 65:1916–1923. 219. Matsumura, Y., K. Yoshikata, S. Kunisaki, and T. Tsuchido. 2003. Mode of bactericidal action of silver zeolite and its comparison with that of silver nitrate. Appl. Environ. Microbiol. 69:4278–4281. 220. Mazzotta, A. S. 2001. Thermal inactivation of stationary-phase and acid-adapted Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in fruit juices. J. Food Prot. 64:315–320. 221. McCarthy, S. A. 1996. Effect of sanitizers on Listeria monocytogenes attached to latex gloves. J. Food Safety 16:231–237. 222. McClements, J. M., M. F. Patterson, and M. Linton. 2001. The effect of growth stage and growth temperature on high hydrostatic pressure inactivation of some psychrotrophic bacteria in milk. J. Food Prot. 64:514–522.

DK3089_C006.fm Page 208 Tuesday, February 20, 2007 11:47 AM

208

Listeria, Listeriosis, and Food Safety

223. McClure, P. J., T. M. Kelly, and T. A. Roberts. 1991. The effects of temperature, pH, sodium chloride and sodium nitrite on the growth of Listeria monocytogenes. Int. J. Food Microbiol. 14:77–91. 224. McKellar, R. C. 1992. Effect of reduced pH on secretion, stability and activity of Listeria monocytogenes listeriolysin O. J. Food Safety 12:283–293. 225. Membre, J. M., J. Thurette, and M. Catteau. 1997. Modelling the growth, survival and death of Listeria monocytogenes. J. Appl. Microbiol. 82:345–350. 226. Mendum, M. L., and L. T. Smith. 2002. Characterization of glycine betaine porter I from Listeria monocytogenes and its roles in salt and chill tolerance. Appl. Environ. Microbiol. 68:813–819. 227. Mendum, M. L., and L. T. Smith. 2002. Gbu glycine betaine porter and carnitine uptake in osmotically stressed Listeria monocytogenes cells. Appl. Environ. Microbiol. 68:5647–5655. 228. Mereghetti, L., R. Quentin, N. Marquet-Van Der Mee, and A. Audurier. 2000. Low sensitivity of Listeria monocytogenes to quaternary ammonium compounds. Appl. Environ. Microbiol. 66:5083–5086. 229. Meylheuc, T., C. J. van Oss, and M. N. Bellon-Fontaine. 2001. Adsorption of biosurfactant on solid surfaces and consequences regarding the bioadhesion of Listeria monocytogenes LO28. J. Appl. Microbiol. 91:822–832. 230. Michalski, C. B., R. E. Brackett, Y. C. Hung, and G. O. Ezeike. 2000. Use of capillary tubes and plate heat exchanger to validate U.S. Department of Agriculture pasteurization protocols for elimination of Listeria monocytogenes in liquid egg products. J. Food Prot. 63:921–925. 231. Midelet, G., and B. Carpentier. 2002. Transfer of microorganisms, including Listeria monocytogenes, from various materials to beef. Appl. Environ. Microbiol. 68:4015–4024. 232. Miller, A. J. 1992. Combined water activity and solute effects on growth and survival of Listeria monocytogenes Scott A. J. Food Prot. 55:414–418. 233. Mohacsi-Farkas, C., J. Farkas, L. Meszaros, O. Reichart, and E. Andrassy. 1999. Thermal denaturation of bacterial cells examined by differential scanning calorimetry. J. Thermal Anal. Calorim. 57:409–414. 234. Moir, C. J., and M. J. Eyles. 1992. Inhibition, injury, and inactivation of four psychrotrophic foodborne bacteria by the preservatives methyl-p-hydroxybenzoate and potassium sorbate. J. Food Prot. 55:360–366. 235. Monk, J. D., L. R. Beuchat, and A. K. Hathcox. 1996. Inhibitory effects of sucrose monolaurate, alone and in combination with organic acids, on Listeria monocytogenes and Staphylococcus aureus. J. Appl. Bacteriol. 81:7–18. 236. Mosteller, T. M., and J. R. Bishop. 1993. Sanitizer efficacy against attached bacteria in a milk biofilm. J. Food Prot. 56:34–41. 237. Murdock, C. A., and K. R. Matthews. 2002. Antibacterial activity of pepsin-digested lactoferrin on foodborne pathogens in buffered broth systems and ultrahigh temperature milk with EDTA. J. Appl. Microbiol. 93:850–856. 238. Muriana, P. M. 1996. Bacteriocins for control of Listeria spp. in food. J. Food Prot. 59 (Suppl.):54–63. 239. Murphy, R. Y., L. K. Duncan, E. R. Johnson, M. D. Davis, and J. N. Smith. 2002. Thermal inactivation D- and z-values of Salmonella serotypes and Listeria innocua in chicken patties, chicken tenders, franks, beef patties, and blended beef and turkey patties. J. Food Prot. 65:53–60. 240. Niemira, B. A., X. Fan, and C. H. Sommers. 2002. Irradiation temperature influences product quality factors of frozen vegetables and radiation sensitivity of inoculated Listeria monocytogenes. J. Food Prot. 65:1406–1410. 241. Nilsson, L., Y. Chen, M. L. Chikindas, H. H. Huss, L. Gram, and T. J. Montville. 2000. Carbon dioxide and nisin act synergistically on Listeria monocytogenes. Appl. Environ. Microbiol. 66:769–774. 242. Nolan, D. A., D. C. Chamblin, and J. A. Troller. 1992. Minimal water activity levels for growth and survival of Listeria monocytogenes and Listeria innocua. Int. J. Food Microbiol. 16:323–335. 243. Norwood, D. E., and A. Gilmour. 2000. The growth and resistance to sodium hypochlorite of Listeria monocytogenes in a steady-state multispecies biofilm. J. Appl. Microbiol. 88:512–520. 244. Novak, J. S., and J. T. Yuan. 2003. Viability of Clostridium perfringens, Escherichia coli, and Listeria monocytogenes surviving mild heat or aqueous ozone treatment on beef followed by heat, alkali, or salt stress. J. Food Prot. 66:382–389. 245. Nykanen, A., K. Weckman, and A. Lapvetelainen. 2000. Synergistic inhibition of Listeria monocytogenes on cold-smoked rainbow trout by nisin and sodium lactate. Int. J. Food Microbiol. 61:63–72. 246. O’Driscoll, B., C. G. Gahan, and C. Hill. 1996. Adaptive acid tolerance response in Listeria monocytogenes: Isolation of an acid-tolerant mutant which demonstrates increased virulence. Appl. Environ. Microbiol. 62:1693–1698.

DK3089_C006.fm Page 209 Tuesday, February 20, 2007 11:47 AM

Characteristics of Listeria monocytogenes Important to Food Processors

209

247. Oh, D. H., and D. L. Marshall. 1996. Monolaurin and acetic acid inactivation of Listeria monocytogenes attached to stainless steel. J. Food Prot. 59:249–252. 248. Pagan, R., P. Manas, J. Raso, and S. Condon. 1999. Bacterial resistance to ultrasonic waves under pressure at nonlethal (manosonication) and lethal (manothermosonication) temperatures. Appl. Environ. Microbiol. 65:297–300. 249. Palumbo, S., and A. C. Williams. 1990. Effect of temperature, relative humidity, and suspending menstrua on the resistance of Listeria monocytogenes to drying. J. Food Prot. 53:377–381. 250. Palumbo, S., and A. C. Williams. 1991. Resistance of Listeria monocytogenes to freezing in foods. Food Microbiol. 8:63–68. 251. Pandit, V. A., and L. A. Shelef. 1994. Sensitivity of Listeria monocytogenes to rosemary (Rosmarius officinalis L.). Food Microbiol. 11:57–63. 252. Parish, M. E., and D. P. Higgins. 1989. Survival of Listeria monocytogenes in low pH model broth systems. J. Food Prot. 52:144–147. 253. Patchett, R. A., A. F. Kelly, and R. G. Kroll. 1994. Transport of glycine-betaine by Listeria monocytogenes. Arch. Microbiol. 162:205–210. 254. Patel, J. R., C.-A. Hwang, L. R. Beuchat, M. P. Doyle, and R. E. Brackett. 1995. Comparison of oxygen scavengers for their ability to enhance resuscitation of heat-injured Listeria monocytogenes. J. Food Prot. 58:244–250. 255. Payne, K. D., P. M. Davidson, S. P. Oliver, and G. L. Christen. 1990. Influence of bovine lactoferrin on the growth of Listeria monocytogenes. J. Food Prot. 53:468–472. 256. Payne, K. D., S. P. Oliver, and P. M. Davidson. 1994. Comparison of EDTA and apo-lactoferrin with lysozyme on the growth of foodborne pathogenic and spoilage bacteria. J. Food Prot. 57:62–65. 257. Payne, K. D., E. Rico-Munoz, and P. M. Davidson. 1989. The antimicrobial activity of phenolic compounds against Listeria monocytogenes and their effectiveness in a model milk system. J. Food Prot. 52:151–153. 258. 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. 259. 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. 260. Petran, R. L., and K. M. J. Swanson. 1993. Simultaneous growth of Listeria monocytogenes and Listeria innocua. J. Food Prot. 56:616–618. 261. Petran, R. L., and E. A. Zottola. 1989. A study of factors affecting growth and recovery of Listeria monocytogenes Scott A. J. Food Sci. 54:458–460. 262. Petrone, G., M. P. Conte, C. Longhi, S. di Santo, F. Superti, M. G. Ammendolia, P. Valenti, and L. Seganti. 1998. Natural milk fatty acids affect survival and invasiveness of Listeria monocytogenes. Lett. Appl. Microbiol. 27:362–368. 263. Phan-Thanh, L. 1998. Physiological and biochemical aspects of the acid survival of Listeria monocytogenes. J. Gen. Appl. Microbiol. 44:183–191. 264. Phan-Thanh, L., F. Mahouin, and S. Alige. 2000. Acid responses of Listeria monocytogenes. Int. J. Food Microbiol. 55:121–126. 265. Piccinin, D. M., and L. A. Shelef. 1995. Survival of Listeria monocytogenes in cottage cheese. J. Food Prot. 58:128–131. 266. Piyasena, P., and R. C. McKellar. 1999. Influence of guar gum on the thermal stability of Listeria innocua, Listeria monocytogenes, and gamma–glutamyl transpeptidase during high-temperature shorttime pasteurization of bovine milk. J. Food Prot. 62:861–866. 267. Poland, A. L., and B. W. Sheldon. 2001. Altering the thermal resistance of foodborne bacterial pathogens with an eggshell membrane waste by-product. J. Food Prot. 64:486–492. 268. Ponce, E., R. Pla, E. Sendra, B. Guamis, and M. Mor-Mur. 1998. Combined effect of nisin and high hydrostatic pressure on destruction of Listeria innocua and Escherichia coli in liquid whole egg. Int. J. Food Microbiol. 43:15–19. 269. Prestamo, G., P. D. Sanz, M. Fonberg-Broczek, and G. Arroyo. 1999. High-pressure response of fruit jams contaminated with Listeria monocytogenes. Lett. Appl. Microbiol. 28:313–316. 270. Rasanayagam, V., V. M. Balasubramaniam, E. Ting, C. E. Sizer, C. Bush, and C. Anderson. 2003. Compression heating of selected fatty food materials during high-pressure processing. J. Food Sci. 68:254–259.

DK3089_C006.fm Page 210 Tuesday, February 20, 2007 11:47 AM

210

Listeria, Listeriosis, and Food Safety

271. Ravishankar, S., and V. K. Juneja. 2003. Adaptation or resistance responses of microorganisms to stresses in the food processing environment, In Microbial stress adaptation and food safety, eds. A. E. Yousef and V. K. Juneja. Boca Raton, Fla: CRC Press, pp. 105–158. 272. Razavi-Rohani, S. M., and M. W. Griffiths. 1994. The effect of mono- and polyglycerol laurate on spoilage and pathogenic bacteria associated with foods. J. Food Safety 14:131–151. 273. Reina, L. D., Z. T. Jin, Q. H. Zhang, and A. E. Yousef. 1998. Inactivation of Listeria monocytogenes in milk by pulsed electric field. J. Food Prot. 61:1203–1206. 274. Reiter, B. 1985. Lactoperoxidase system in bovine milk. In The lactoperoxidase system: Chemistry and biological significance, eds. K. M. Pruitt and J. O. Tenovuo. New York: Marcel Dekker, Inc., pp. 123–141. 275. Restaino, L., E. W. Frampton, J. B. Hemphill, and P. Palinikar. 1995. Efficacy of ozonated water against various food-related microorganisms. Appl. Environ. Microbiol. 61:3471–3475. 276. Richards, R. M., D. K. Xing, and T. P. King. 1995. Activity of p-aminobenzoic acid compared with other organic acids against selected bacteria. J. Appl. Bacteriol. 78:209–215. 277. Robinson, T. P., M. J. Ocio, A. Kaloti, and B. M. Mackey. 1998. The effect of the growth environment on the lag phase of Listeria monocytogenes. Int. J. Food Microbiol. 44:83–92. 278. Romanova, N., S. Favrin, and M. W. Griffiths. 2002. Sensitivity of Listeria monocytogenes to sanitizers used in the meat-processing industry. Appl. Environ. Microbiol. 68:6405–6409. 279. Rørvik, L. M. 2000. Listeria monocytogenes in the smoked salmon industry. Int. J. Food Microbiol. 62:183–190. 280. 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. 50:452–457. 281. Rowan, N. J., S. J. MacGregor, J. G. Anderson, R. A. Fouracre, L. McIlvaney, and O. Farish. 1999. Pulsed-light inactivation of food-related microorganisms. Appl. Environ. Microbiol. 65:1312–1315. 282. 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. 283. 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. 284. Ryser, E. T., and E. H. Marth. 1988. Growth of Listeria monocytogenes at different pH values in uncultured whey or whey cultured with Penicillium camembertii. Can. J. Microbiol. 34:730–735. 285. 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. 286. Ryser, E. T., and E. H. Marth. 1989. Behavior of Listeria monocytogenes during manufacture and ripening of brick cheese. J. Dairy Sci. 72:838–853. 287. Sabanadesan, S., A. M. Lammerding, and M. W. Griffiths. 2000. Survival of Listeria innocua in salmon following cold-smoke application. J. Food Prot. 63:715–720. 288. Scannell, A. G., C. Hill, R. P. Ross, S. Marx, W. Hartmeier, and E. K. Arendt. 2000. Development of bioactive food packaging materials using immobilized bacteriocins lacticin 3147 and nisaplin. Int. J. Food Microbiol. 60:241–249. 289. Schlyter, J. H., K. A. Glass, J. Loeffelholz, A. J. Degnan, and J. B. Luchansky. 1993. The effect of diacetate with nitrite, lactate, or pediocin on the viability of Listeria monocytogenes in turkey slurries. Int. J. Food Microbiol. 19:271–281. 290. Schobitz, R., T. Zaror, O. Leon, and M. Costa. 1999. A bacteriocin from Carnobacterium piscicola for the control of Listeria monocytogenes in vacuum-packaged meat. Food Microbiol. 16:249–255. 291. Schuman, J. D., and B. W. Sheldon. 2003. Inhibition of Listeria monocytogenes in pH-adjusted pasteurized liquid whole egg. J. Food Prot. 66:999–1006. 292. Seman, D. L., A. C. Borger, J. D. Meyer, P. A. Hall, and A. L. Milkowski. 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. 293. Shelef, L. A., and L. Addala. 1994. Inhibition of Listeria monocytogenes and other bacteria by sodium diacetate. J. Food Safety 14:103–115. 294. Simpson, R. K., and A. Gilmour. 1997. The effect of high hydrostatic pressure on Listeria monocytogenes in phosphate-buffered saline and model food systems. J. Appl. Microbiol. 83:181–188.

DK3089_C006.fm Page 211 Tuesday, February 20, 2007 11:47 AM

Characteristics of Listeria monocytogenes Important to Food Processors

211

295. Siragusa, G. R., and M. G. Johnson. 1989. Inhibition of Listeria monocytogenes growth by the lactoperoxidase-thiocyanate-H2O2 antimicrobial system. Appl. Environ. Microbiol. 55:2802–2805. 296. Smith, J. L., B. S. Marmer, and R. C. Benedict. 1991. Influence of growth temperature on injury and death of Listeria monocytogenes Scott A during a mild heat treatment. J. Food Prot. 54:166–169. 297. Smith, L. T. 1996. Role of osmolytes in adaptation of osmotically stressed and chill-stressed Listeria monocytogenes grown in liquid media and on processed meat surfaces. Appl. Environ. Microbiol. 62:3088–3093. 298. Sommers, C., and X. Fan. 2003. Gamma irradiation of fine-emulsion sausage containing sodium diacetate. J. Food Prot. 66:819–824. 299. Sorrells, K. M., and D. C. Enigl. 1990. Effect of pH, acidulant, sodium chloride, and temperature on the growth of Listeria monocytogenes. J. Food Safety 11:31–37. 300. Spurlock, A. T., and E. A. Zottola. 1991. Growth and attachment of Listeria monocytogenes to cast iron. J. Food Prot. 925–929. 301. 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. 302. Stephens, P. J., and M. V. Jones. 1993. Reduced ribosomal thermal denaturation in Listeria monocytogenes following osmotic and heat shocks. FEMS Microbiol. Lett. 106:177–182. 303. Stiles, M. E. 1996. Biopreservation by lactic acid bacteria. Antonie van Leeuwenhoek 70:331–345. 304. Stratford, M., and P. A. Anslow. 1998. Evidence that sorbic acid does not inhibit yeast as a classic “weak acid preservative.” Lett. Appl. Microbiol. 27:203–206. 305. Sunen, E. 1998. Minimum inhibitory concentration of smoke wood extracts against spoilage and pathogenic microorganisms associated with foods. Lett. Appl. Microbiol. 27:45–48. 306. Szabo, E. A., and M. E. Cahill. 1999. Nisin and ALTA 2341 inhibit the growth of Listeria monocytogenes on smoked salmon packaged under vacuum or 100% CO2. Lett. Appl. Microbiol. 28:373–377. 307. Szabo, E. A., L. Simons, M. J. Coventry, and M. B. Cole. 2003. Assessment of control measures to achieve a food safety objective of less than 100 CFU of Listeria monocytogenes per gram at the point of consumption for fresh precut iceberg lettuce. J. Food Prot. 66:256–264. 308. Taormina, P. J., and L. R. Beuchat. 2002. Survival of Listeria monocytogenes in commercial foodprocessing equipment cleaning solutions and subsequent sensitivity to sanitizers and heat. J. Appl. Microbiol. 92:71–80. 309. Tarte, R. R., E. A. Murano, and D. G. Olson. 1996. Survival and injury of Listeria monocytogenes, Listeria innocua and Listeria ivanovii in ground pork following electron beam irradiation. J. Food Prot. 59:596–600. 310. Tay, A., T. H. Shellhammer, A. E. Yousef, and G. W. Chism. 2003. Pressure death and tailing behavior of Listeria monocytogenes strains having different barotolerances. J. Food Prot. 66:2057–2061. 311. Taylor, C. M., M. Beresford, H. A. Epton, D. C. Sigee, G. Shama, P. W. Andrew, and I. S. Roberts. 2002. Listeria monocytogenes relA and hpt mutants are impaired in surface-attached growth and virulence. J. Bacteriol. 184:621–628. 312. Teratanavat, R., and N. H. Hooker. 2004. Understanding the characteristics of United States meat and poultry recalls: 1994–2002. Food Control. 15:359–367. 313. 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. 314. Thimothe, J., J. Walker, V. Suvanich, K. L. Gall, M. W. Moody, and M. Wiedmann. 2002. Detection of Listeria in crawfish processing plants and in raw, whole crawfish and processed crawfish (Procambarus spp.). J. Food Prot. 65:1735–1739. 315. Thomas, L. V., E. A. Davies, J. Delves-Broughton, and J. W. Wimpenny. 1998. Synergist effect of sucrose fatty acid esters on nisin inhibition of gram-positive bacteria. J. Appl. Microbiol. 85:1013–1022. 316. Tienungoon, S., D. A. Ratkowsky, T. A. McMeekin, and T. Ross. 2000. Growth limits of Listeria monocytogenes as a function of temperature, pH, NaCl, and lactic acid. Appl. Environ. Microbiol. 66:4979–4987. 317. Ting, W. T. E., and K. E. Deibel. 1992. Sensitivity of Listeria monocytogenes to spices at two temperatures. J. Food Safety 12:129–137.

DK3089_C006.fm Page 212 Tuesday, February 20, 2007 11:47 AM

212

Listeria, Listeriosis, and Food Safety

318. To, M. S., S. Favrin, N. Romanova, and M. W. Griffiths. 2002. Postadaptational resistance to benzalkonium chloride and subsequent physicochemical modifications of Listeria monocytogenes. Appl. Environ. Microbiol. 68:5258–5264. 319. Tremoulet, F., O. Duche, A. Namane, B. Martinie, and J. C. Labadie. 2002. Comparison of protein patterns of Listeria monocytogenes grown in biofilm or in planktonic mode by proteomic analysis. FEMS Microbiol. Lett. 210:25–31. 320. Tsigarida, E., P. Skandamis, and G. J. Nychas. 2000. Behavior 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. 321. Turcotte, P., and S. A. Saheb. 1978. Antimicrobial activity of phenolic antioxidants. Can. J. Microbiol. 24:1306–1320. 322. Ultee, A., E. P. Kets, and E. J. Smid. 1999. Mechanisms of action of carvacrol on the foodborne pathogen Bacillus cereus. Appl. Environ. Microbiol. 65:4606–4610. 323. United States Department of Agriculture. 2003. Verification procedure for the Listeria monocytogenes regulation and microbial sampling of ready-to-eat products for the Food Safety and Inspection Services verification testing program. FSIS Directive #10,240.4. 324. Van Gerwen, S. J., and M. H. Zwietering. 1998. Growth and inactivation models to be used in quantitative risk assessments. J. Food Prot. 61:1541–1549. 325. Vasseur, C., N. Rigaud, M. Hebraud, and J. Labadie. 2001. Combined effects of NaCl, NaOH, and biocides (monolaurin or lauric acid) on inactivation of Listeria monocytogenes and Pseudomonas spp. J. Food Prot. 64:1442–1445. 326. Vatanyoopaisarn, S., A. Nazli, C. E. Dodd, C. E. Rees, and W. M. Waites. 2000. Effect of flagella on initial attachment of Listeria monocytogenes to stainless steel. Appl. Environ. Microbiol. 66:860–863. 327. 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 with hydrogen peroxide. J. Food Prot. 65:100–105. 328. Villani, F., O. Pepe, G. Mauriello, G. Moschetti, L. Sannino, and S. Coppola. 1996. Behavior of Listeria monocytogenes during the traditional manufacture of water-buffalo mozzarella cheese. Lett. Appl. Microbiol. 22:357–360. 329. Wade, W. N., A. J. Scouten, K. H. McWatters, R. L. Wick, A. Demirci, W. F. Fett, and L. R. Beuchat. 2003. Efficacy of ozone in killing Listeria monocytogenes on alfalfa seeds and sprouts and effects on sensory quality of sprouts. J. Food Prot. 66:44–51. 330. Walker, S. J., P. Archer, and J. G. Banks. 1990. Growth of Listeria monocytogenes at refrigeration temperatures. J. Appl. Bacteriol. 68:157–162. 331. Walsh, S. E., J. Y. Maillard, A. D. Russell, C. E. Catrenich, D. L. Charbonneau, and R. G. Bartolo. 2003. Activity and mechanisms of action of selected biocidal agents on Gram-positive and -negative bacteria. J. Appl. Microbiol. 94:240–247. 332. Wang, L. L., and E. A. Johnson. 1992. Inhibition of Listeria monocytogenes by fatty acids and monoglycerides. Appl. Environ. Microbiol. 58:624–629. 333. Wang, L. L., and E. A. Johnson. 1997. Control of Listeria monocytogenes by monoglycerides in foods. J. Food Prot. 60:131–138. 334. Wang, L. L., B. K. Yang, K. L. Parkin, and E. A. Johnson. 1993. Inhibition of Listeria monocytogenes by monoacylglycerols synthesized from coconut oil and milk fat by lipase-catalyzed glycerolysis. J. Agric. Food Chem. 41:1000–1005. 335. Weaver, R. A., and L. A. Shelef. 1993. Antilisterial activity of sodium, potassium or calcium lactate in pork liver sausage. J. Food Safety 13:133–146. 336. 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. 337. Wemekamp-Kamphuis, H. H., A. K. Karatzas, J. A. Wouters, and T. Abee. 2002. Enhanced levels of cold shock proteins in Listeria monocytogenes LO28 upon exposure to low temperature and high hydrostatic pressure. Appl. Environ. Microbiol. 68:456–463. 338. Whitley, E., D. Muir, and W. M. Waites. 2000. The growth of Listeria monocytogenes in cheese packed under a modified atmosphere. J. Appl. Microbiol. 88:52–57. 339. Winkowski, K., A. D. Crandall, and T. J. Montville. 1993. Inhibition of Listeria monocytogenes by Lactobacillus bavaricus MN in beef systems at refrigeration temperatures. Appl. Environ. Microbiol. 59:2552–2557.

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340. Wood, L. V., and M. Woodbine. 1979. Low temperature virulence of Listeria monocytogenes in the avian embryo. Zentralbl. Bakteriol. [Orig. A] 243:74–81. 341. World Health Organization. 1988. Foodborne listeriosis. Bull. WHO 66:421–428. 342. Wouters, P. C., A. P. Bos, and J. Ueckert. 2001. Membrane permeabilization in relation to inactivation kinetics of Lactobacillus species due to pulsed electric fields. Appl. Environ. Microbiol. 67:3092–3101. 343. Wouters, P. C., N. Dutreux, J. P. Smelt, and H. L. Lelieveld. 1999. Effects of pulsed electric fields on inactivation kinetics of Listeria innocua. Appl. Environ. Microbiol. 65:5364–5371. 344. Young, K. M., and P. M. Foegeding. 1993. Acetic, lactic and citric acids and pH inhibition of Listeria monocytogenes Scott A and the effect on intracellular pH. J. Appl. Bacteriol. 74:515–520. 345. Yousef, A. E., and P. D. Courtney. 2003. Basics of stress adaptation and implications in new-generation foods. In Microbial stress adaptation and food safety, eds. A. E. Yousef and V. K. Juneja. Boca Raton, Fla.: CRC Press, pp. 1–30. 346. Yousef, A. E., R. J. Gajewski, and E. H. Marth. 1991. Kinetics of growth and inhibition of Listeria monocytogenes in the presence of antioxidant food additives. J. Food Sci. 56:10–13. 347. Yousef, A. E., J. B. Luchansky, A. J. Degnan, and M. P. Doyle. 1991. Behavior of Listeria monocytogenes in wiener exudates in the presence of Pediococcus acidilactici H or pediocin AcH during storage at 4 and 5°C. Appl. Environ. Microbiol. 57:1461–1467. 348. Yousef, A. E., and E. H. Marth. 1988. Inactivation of Listeria monocytogenes by ultraviolet energy. J. Food Sci. 53:571–573. 349. 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. 350. Yuste, J., R. Pla, M. Capellas, E. Ponce, and M. Mor-Mur. 2000. High-pressure processing applied to cooked sausages: Bacterial populations during chilled storage. J. Food Prot. 63:1093–1099. 351. Zaika, L. L., and J. S. Fanelli. 2003. Growth kinetics and cell morphology of Listeria monocytogenes Scott A as affected by temperature, NaCl, and EDTA. J. Food Prot. 66:1208–1215. 352. Zhang, S., and A. Mustapha. 1999. Reduction of Listeria monocytogenes and Escherichia coli O157:H7 numbers on vacuum-packaged fresh beef treated with nisin or nisin combined with EDTA. J. Food Prot. 62:1123–1127.

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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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REFERENCES 1. Ahamad, N., and E. H. Marth. 1990. Acid injury in Listeria monocytogenes. J. Food Prot. 53:26–29. 2. Albritton, W. L., G. L. Williams, W. E. DeWitt, and J. C. Feeley. 1980. Listeria monocytogenes. In Manual of clinical microbiology, 3rd ed., eds. E. H. Lennette, A. Balows, W. J. Hausler, Jr., and J. P. Truant. Washington, D.C.: American Society for Microbiology, pp. 139–142. 3. Al-Ghazali, M. R., and S. K. Al-Azawi. 1988. Effects of sewage treatment on the removal of Listeria monocytogenes. J. Appl. Bacteriol. 65:203–208. 4. Annous, B. A., L. A. Becker, D. O. Bayles, D. P. Labed, and B. J. Wilkinson. 1997. Critical role of anteiso-C15:0 fatty acid in the growth of Listeria monocytogenes at low temperatures. Appl. Environ. Microbiol. 63:3887–3894. 5. AOAC Official Method 993.12. 2000. Listeria monocytogenes in milk and dairy products, selective enrichment and isolation method (IDF method). In Official methods of analysis of AOAC international, 17th ed., vol. 1, Agricultural chemicals, contaminants, and drugs. ed. W. Horwitz. Gaithersburg, Md.: AOAC International, Chapter 17.10.01, pp. 138–139. 6. Asperger, H., H. Heistinger, M. Wagner, A. Lehner, and E. Brandl. 1999. A contribution of Listeria enrichment methodology—growth of Listeria monocytogenes under varying conditions concerning enrichment broth composition, cheese matrices and competing microflora. Microbiology 16: 419–431. 7. Association of Official Analytical Chemists. 1996. AOAC official method 993.12. Listeria monocytogenes in milk and dairy products. In Official methods of analysis of the association of official analytical chemists. Gaithersburg, Md.: AOAC International, 17.10.01. 8. Bailey, J. S., D. L. Fletcher, and N. A. Cox. 1989. Recovery and serotype distribution of Listeria monocytogenes from broiler chickens in the southeastern United States. J. Food Prot. 52:148–150. 9. Bailey, J. S., and N. A. Cox. 1992. Universal preenrichment broth for the simultaneous detection of Salmonella and Listeria in foods. J. Food Prot. 55:256–259. 10. Bannerman, E. S., and J. Bille. 1988. A selective medium for isolating Listeria spp. from heavily contaminated material. Appl. Environ. Microbiol. 54:165–167. 11. Bayles, D. O., B. A. Annous, and B. J. Wilkinson. 1996. Cold stress proteins induced in Listeria monocytogenes in response to temperature downshock and growth at low temperatures. Appl. Environ. Microbiol. 62:1116–1119. 12. Bearns, R. E., and K. F. Girard. 1959. On the isolation of Listeria monocytogenes from biological specimens. Am. J. Med. Technol. 25:120–126. 13. Beckers, H. J., P. S. S. Soentoro, and E. H. M. Delfgou-van Asch. 1987. The occurrence of Listeria monocytogenes in soft cheeses and raw milk and its resistance to heat. Int. J. Food Microbiol. 4:249–256. 14. Beerens, H., and M. M. Tahon-Castel. 1966. Milieu a l’acide nalidixique pour l’isolement des Streptocoques, D. pneumoniae, Listeria, Erysipelothrix. Ann. Inst. Pasteur 111:90–93. 15. 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. 16. Beumer, R. R., and W. C. Hazeleger. 2003. Listeria monocytogenes: diagnostic problems. FEMS Immunol. Med. Microbiol. 35:191–197. 17. Biester, H. E., and L. H. Schwarte. 1939. Studies on Listerella infection in sheep. J. Infect. Dis. 64:135–144. 18. Blanco, M., J. F. Fernandez-Garayzabel, L. Dominguez, V. Briones, J. A. Vazquez-Boland, J. L. Blanco, J. A. Garcia, and G. Suarez. 1989. A technique for the identification of hemolytic-pathogenic Listeria on selective plating media. Lett. Appl. Microbiol. 9:125–128. 19. Bockemühl, J., H. P. R. Seeliger, and R. Kathke. 1971. Acridinfarbstoffe in Selektivnährböden zur Isolierung von L. monocytogenes. Med. Microbiol. Immunol. 157:84–95. 20. Bockemühl, J., E. Feindt, K. Höhne, and H. P. R. Seeliger. 1974. Acridinfarbstoffe in Selectivnährböden zur Isolierung von L. monocytogenes. II. Modifiziertes Stuart Medium: Ein neues ListeriaTransport-Anreicherungsmedium. Med. Microbiol. Immunol. 59:289–299. 21. Bojsen-Møller, J. 1972. Human listeriosis—diagnostic, epidemiological and clinical studies. Acta Pathol. Microbiol. Scand. Section B, 229(suppl):1–157. 22. Breuer, J., and O. Prändl. 1988. Nachweis von Listerien und deren Vorkommen in Hackfleisch und Mettwürsten in Österreich. Arch. Lebensmittelhyg. 39:28–30.

DK3089_C007.fm Page 250 Monday, February 19, 2007 12:31 PM

250

Listeria, Listeriosis, and Food Safety

22a. Brackett, R. E., and L. R. Beuchat. 1989. Methods and media for the isolation and cultivation of Listeria monocytogenes from various foods. Int. J. Food Microbiol. 8:219–223. 23. Buchanan, R. L. 1988. Advances in cultural methods for the detection of Listeria monocytogenes. Society for Industrial Microbiology Comprehensive Conference on Listeria monocytogenes, Rohnert Park, California, Oct. 2–5, Abstr. I-14. 24. Buchanan, R. L., H. G. Stahl, and D. L. Archer. 1987. Improved plating media for simplified, quantitative detection of Listeria monocytogenes in foods. Food Microbiol. 4:269–275. 25. Buchanan, R. L., H. G. Stahl, and M. M. Bencivengo. 1988. Recovery of Listeria from fresh retaillevel foods of animal origin. Proc. Annu. Meeting Am. Soc. Microbiol., Miami Beach, Florida, May 8–13, Abstr. P-46. 26. Buchanan, R. L., J. L. Smith, H. G. Stahl, and D. L. Archer. 1988. Listeria methods development research at the Eastern Regional Research Center, U.S. Department of Agriculture. J. Assoc. Off. Anal. Chem. 71:651–654. 27. Buchanan, R. L., H. G. Stahl, M. M. Bencivengo, and F. Del Corral. 1989. Comparison of lithium chloride-phenylethanol-moxalactam and modified Vogel–Johnson agars for detection of Listeria spp. in retail-level meats, poultry and seafood. Appl. Environ. Microbiol. 55:599–603. 28. Buchanan, R. L., H. G. Stahl, and R. C. Whiting. 1989. Effects and interactions of temperature, pH, atmosphere, sodium chloride, and sodium nitrite on the growth of Listeria monocytogenes. J. Food Prot. 52:844–851. 29. Buchanan, R. L., and M. L. Cygnarowicz. 1990. A mathematical approach toward defining and calculating the duration of the lag phase. Food Microbiol. 7:237–240. 30. Bunduki, M. M.-C., K. J. Flanders, and C. W. Donnelly. 1994. Metabolic and structural sites of damage in heat- and sanitizer-injured populations of Listeria monocytogenes. J. Food Prot. 58:410–415. 31. Bunning, V. K., C. W. Donnelly, J. T. Peeler, E. H. Briggs, J. G. Bradshaw, R. G. Crawford, C. M. Beliveau, and J. T. Tierney. 1988. Thermal inactivation of Listeria monocytogenes within bovine milk phagocytes. Appl. Environ. Microbiol. 54:364–370. 32. Busch, S. V., and C. W. Donnelly. 1992. Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua. Appl. Environ. Microbiol. 58:14–20. 33. Carnevale, R. A., and R. W. Johnston. 1989. Method for the isolation and identification of Listeria monocytogenes from meat and poultry products. U.S. Department of Agriculture Food Safety and Inspection Service, Laboratory Communication No. 57, Revised May 24. USDA, Washington, D.C. 34. Cassiday, P. K., R. E. Brackett, and L. R. Beuchat. 1989. Evaluation of ten selective direct plating media for enumeration of Listeria monocytogenes in ham and oysters. Food Microbiol. 55:113–125. 35. Cassiday, P. K., R. E. Brackett, and L. R. Beuchat. 1989. Evaluation of three newly developed direct plating media to enumerate Listeria monocytogenes in foods. Appl. Environ. Microbiol. 55:1645–1648. 36. Cassiday, P. K., and R. E. Brackett. 1989. Methods and media to isolate and enumerate Listeria monocytogenes: A review. Lancet 1:207–214. 37. Chawla, C. S., H. Chen, and C. W. Donnelly. 1996. Mathematically modeling the repair of heatinjured Listeria monocytogenes as affected by temperature, pH, and salt concentration. Int. J. Food Microbiol. 30:231–242. 38. Cowart, R. E., and B. G. Foster. 1985. Differential effects of iron on the growth of Listeria monocytogenes: Minimum requirements and mechanism of acquisition. J. Infect. Dis. 151:721–730. 39. Crawford, R. G., C. M. Beliveau, J. T. Peeler, C. W. Donnelly, and V. K. Bunning. 1989. Comparative recovery of uninjured and heat-injured Listeria monocytogenes cells from bovine milk. Appl. Environ. Microbiol. 55:1490–1494. 40. Curiale, M. S., and C. Lewus. 1994. Detection of Listeria monocytogenes in samples containing Listeria innocua. J. Food Prot. 57:1048–1051. 41. Curtis, G. D. W., W. W. Nichols, and T. J. Falla. 1989. Selective agents for Listeria can inhibit their growth. Lett. Appl. Microbiol. 8:169–172. 42. Curtis, G. D. W., R. G. Mitchell, A. F. King, and E. J. Griffen. 1989. A selective differential medium for the isolation of Listeria monocytogenes. Lett. Appl. Microbiol. 8:95–98. 43. Despierres, M. 1971. Isolement de Listeria monocytogenes dans un milieu défavorable a Streptococcus faecalis. Ann. Inst. Pasteur 121:493–501.

DK3089_C007.fm Page 251 Monday, February 19, 2007 12:31 PM

Conventional Methods to Detect and Isolate Listeria monocytogenes

251

44. Dijkstra, R. G. 1976. Listeria—encephalitis in cows through litter from a broiler farm. Zentralbl. Bakteriol. Hyg. I Abt. Orig. B 161:383–385. 45. Donnelly, C. W., and G. J. Baigent. 1986. Method for flow cytometric detection of Listeria monocytogenes in milk. Appl. Environ. Microbiol. 52:689–695. 46. Donnelly, C. W. 2002. Detection and isolation of Listeria monocytogenes from food samples: implications of sublethal injury. J. AOAC Int. 85:495–500. 47. Doyle, M. P., and J. L. Schoeni. 1986. Selective-enrichment procedure for isolation of Listeria monocytogenes from fecal and biologic specimens. Appl. Environ. Microbiol. 51:1127–1129. 48. Doyle, M. P., and J. L. Schoeni. 1987. Comparison of procedures for isolating Listeria monocytogenes in soft, surface-ripened cheese. J. Food Prot. 50:4–6. 49. Doyle, M. P., K. A. Glass, J. T. Beery, G. A. Garcia, D. J. Pollard, and R. D. Schultz. 1987. Survival of Listeria monocytogenes in milk during high-temperature, short-time pasteurization. Appl. Environ. Microbiol. 53:1433–1438. 50. Duarte, G., M. Vaz-Velho, C. Capell, and P. Gibbs. 1999. Efficiency of four secondary enrichment protocols in differentiation and isolation of Listeria spp. and Listeria monocytogenes from smoked fish processing chains. Int. J. Food Microbiol. 52:163–8. 51. Durst, J., and G. Berencsi. 1976. Contributions to further serovariants of L. monocytogenes. Zentralbl. Bakteriol. Hyg. I Abt. Orig. A 236:531–532. 52. Elischerova, K., and S. Stupalova. 1972. Listeriosis in professionally exposed persons. Acta Microbiol. Hung. 19:379–384. 53. El-Kest, S. E., and E. H. Marth. 1992. Freezing of Listeria monocytogenes and other microorganisms: A review. J. Food Prot. 55:639–648. 54. Farber, J. M., G. W. Sanders, and S. A. Malcolm. 1988. The presence of Listeria spp. in raw milk in Ontario. Can. J. Microbiol. 34:95–100. 55. Farber, J. M., G. W. Sanders, and J. I. Speirs. 1988. Methodology for isolation of Listeria from foods—a Canadian perspective. J. Assoc. Off. Anal. Chem. 71:675–678. 56. Fenlon, D. R. 1985. Wild birds and silage as reservoirs of Listeria in the agricultural environment. J. Appl. Bacteriol. 59:537–543. 57. Fenlon, D. R. 1986. Rapid quantitative assessment of the distribution of Listeria in silage implicated in a suspected outbreak of listeriosis in calves. Vet. Rec. 118:240–242. 58. Ferron, P., and J. Michard. 1993. Distribution of Listeria spp. in confectioners’ pastries from Western France: Comparison of enrichment methods. Int. J. Food Microbiol. 18:289–303. 59. Filice, G. A., H. F. Cantrell, A. B. Smith, P. S. Hayes, J. C. Feeley, and D. W. Fraser. 1978. Listeria monocytogenes infection in neonates: Investigation of an epidemic. J. Infect. Dis. 138:17–23. 60. Flanders, K. J. 1991. Injury, resuscitation and detection of Listeria spp. from frozen environments. M.S. thesis, University of Vermont, Burlington, Vt. 61. Flanders, K. J., C. M. Beliveau, T. J. Pritchard, and C. W. Donnelly. 1994. Enhanced recovery of Listeria from dairy plant environments using modified selective enrichment media. IFT Annual Meeting Technical Program: Book of Abstr. 59C, p. 166. 62. Flanders, K. J., T. J. Pritchard, and C. W. Donnelly. 1995. Enhanced recovery of Listeria from dairy plant processing environments through combined use of repair, enrichment and selective enrichment/detection procedures. J. Food Prot. 58:404–409. 63. Fleming, D. W., S. L. Cochi, K. L. MacDonald, J. Brondum, P. S. Hayes, B. D. Plikaytis, M. B. Holmes, A. Audurier, C. V. Broome, and A. L. Reingold. 1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. New Engl. J. Med. 312:404–407. 64. Fraser, J. A., and W. H. Sperber. 1988. Rapid detection of Listeria spp. in food and environmental samples by esculin hydrolysis. J. Food Prot. 51:762–765. 65. Fuzi, M., and I. Pillis. 1961. Selektive Züchtung von L. monocytogenes. Vortrag 3, Kongress Ung. Mikrobiol. Gesellsch., Budapest. 66. Garayzabal, J. F., and C. Genigeorgis. 1990. Quantitative evaluation of three selective enrichment broths and agars used in recovering Listeria microorganisms. J. Food Prot. 53:105–110. 67. Gibson, A. M., N. Bratchell, and T. A. Roberts. 1988. Predicting microbial growth: growth responses of Salmonella in a laboratory medium as affected by pH, sodium chloride and storage temperature. Int. J. Food Microbiol. 6:155–178.

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252

Listeria, Listeriosis, and Food Safety

68. Golden, D. A., L. R. Beuchat, and R. E. Brackett. 1988. Direct plating technique for enumeration of Listeria monocytogenes in foods. J. Assoc. Off. Anal. Chem. 71:647–650. 69. 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. 70. Golden, D. A., L. R. Beuchat, and R. E. Brackett. 1988. Inactivation and injury of Listeria monocytogenes as affected by heating and freezing. Food Microbiol. 5:17–23. 71. Goldstein, E. J. C. 1988. Structure of the fluoroquinolone group of antibacterials—introduction. Suppl. Urol. 32:4–8. 72. 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. 66:559–69. 73. Gray, M. L. 1960. Isolation of Listeria monocytogenes from oat silage. Science 132:1767–1768. 74. Gray, M. L., and A. H. Killinger. 1966. Listeria monocytogenes and listeric infections. Bacteriol. Rev. 30:309–382. 75. Gray, M. L., H. J. Stafseth, and F. Thorp, Jr. 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. 76. Gray, M. L., H. J. Stafseth, F. Thorp, Jr., L. B. Sholl, and W. F. Riley, Jr. 1948. A new technique for isolating listerellae from the bovine brain. J. Bacteriol. 55:471–476. 77. Gregorio, S. B., W. C. Eveland, and H. F. Maassab. 1986. Efficiency of various solid media for the isolation of Listeria monocytogenes. Proc. Annu. Meeting Am. Soc. Microbiol., New Orleans, Louisiana, Abstr. p. 27. 78. Hao, D. Y.-Y., L. R. Beuchat, and R. E. Brackett. 1989. Comparison of media and methods for detecting and enumerating Listeria monocytogenes in refrigerated cabbage. Appl. Environ. Microbiol. 53:955–957. 79. Hartmann, V., K. Friedrich, F. Beyer, and G. Terplan. 1988. Verbesserung des Listeriennachweises durch einen Moxalactam-enthaltenden Nährboden. Deutsche Molkerei-Zeitung 38:1164–1166. 80. 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. 81. Hayes, P. S., L. M. Graves, G. W. Ajello, B. Swaminathan, R. E. Weaver, J. D. Wenger, A. Schuchat, C. V. Broome, and the Listeria Study Group. 1991. Comparison of cold enrichment and the U.S. Department of Agriculture methods for isolating Listeria monocytogenes from naturally contaminated foods. Appl. Environ. Microbiol. 57:2109–2113. 82. Hayes, P. S., L. M. Graves, B. Swaminathan, G. W. Ajello, G. B. Malcolm, R. E. Weaver, R. Ransom, K. Deaver, B. D. Plikaytis, A. Schuchat, J. D. Wenger, R. W. Pinner, C. V. Broome, and the Listeria Study Group. 1992. Comparison of three selective enrichment methods for the isolation of Listeria monocytogenes from naturally contaminated foods. J. Food Prot. 55:952–959. 83. Heisick, J. E., D. E. Wagner, M. L. Nierman, and J. T. Peeler. 1989. Listeria spp. found on fresh market produce. Appl. Environ. Microbiol. 55:1925–1927. 84. Heisick, J. E., F. M. Harrell, E. H. Peterson, S. McLaughlin, D. E. Wagner, I.V. Wesley, and J. Bryner. 1989. Comparison of four procedures to detect Listeria spp. in foods. J. Food Prot. 52:154–157. 85. Henry, B. S. 1933. Dissociation of the genus Brucella. J. Infect. Dis. 52:374–402. 86. Hitchins, A. D. 1995. Listeria monocytogenes. In Food and Drug Administration bacteriological analytical manual, 8th ed. Gaithersburg, Md.: AOAC International, pp. 10.01–10.13. 87. Hitchins, A. D., and R. E. Duvall. 2000. Feasibility of a defined microflora challenge method for evaluating the efficacy of foodborne Listeria monocytogenes selective enrichments. J. Food Prot. 63:1064–1070. 88. Hitchins, A. D. 2003. Listeria monocytogenes. In Food and Drug Administration bacteriological analytical manual, 8th ed. AOAC International, pp. 10.01–10.13. 89. Hofer, E. 1974. Study of the occurrence of L. monocytogenes in human feces. Rev. Soc. Bras. Med. Trop. 8:109–116. 89a. Hoffman, A. D., and M. Wiedmann. 2001. Comparative evaluation of culture- and BAX polymerase chain reaction-based detection methods for Listeria spp. and Listeria monocytogenes in environmental and raw fish samples. J. Food Prot. 64:1521–6. 90. Johansson, T., H. Ahola-Luttila, T. Pirhonen, A. Taimisto, H. Haario, M. Laine, and M. SalkinojaSalonen. 2000. Improved detection of Listeria monocytogenes in soft mould-ripened cheese. J. Appl. Microbiol. 88:870–6.

DK3089_C007.fm Page 253 Monday, February 19, 2007 12:31 PM

Conventional Methods to Detect and Isolate Listeria monocytogenes

253

91. Johnson, J. L. 1998. Isolation and identification of Listeria monocytogenes from meat, poultry and egg products. In USDA–FSIS microbiology laboratory guidebook. 3rd ed., vol. 1. 92. Kampelmacher, E. H., and L. M. van Noorle Jansen. 1969. Isolation of Listeria monocytogenes from feces of clinically healthy humans and animals. Zentralbl. Bakteriol. I Abt. Orig. A 211:353–359. 93. Kampelmacher, E. H., and L. M. van Noorle Jansen. 1972. Further studies on the isolation of Listeria monocytogenes in clinically healthy individuals. Zentralbl. Bakteriol. I Abt. Orig. A 221:70–77. 94. Kampelmacher, E. H., D. E. Maas, and L. M. van Noorle Jansen. 1972. Isolierung von L. monocytogenes mittels Nalidixinsäuretrypaflavin. Zentralbl. Bakteriol. Parasit. Abt. I Orig. A 221:139–140. 95. Kells, J., and A. Gilmour. 2004. Incidence of Listeria monocytogenes in two milk processing environments, and assessment of Listeria monocytogenes blood agar for isolation. Int. J. Food Microbiol. 91:167–74. 96. Khan, M. A., A. Seaman, and M. Woodbine. 1972. Differential media for the isolation of Listeria monocytogenes. Acta Microbiol. Acad. Sci. Hung. 19:371–372. 97. Knabel, S. J. 2002. Optimized, one-step, recovery-enrichment broth for enhanced detection of Listeria monocytogenes in pasteurized milk and hot dogs. J. AOAC Int. 85:501–4. 98. Ko, R., L. T. Smith, and G. M. Smith. 1994. Glycine betaine confers enhanced osmotolerance and cryotolerance on Listeria monocytogenes. J. Bacteriol. 176:426–431. 99. Kornacki, J. L., D. J. Evanson, W. Reid, K. Rowe, and R. S. Flowers. 1993. Evaluation of the USDA protocol for detection of Listeria monocytogenes. J. Food Prot. 56:441–443. 100. Kramer, P. A., and D. Jones. 1969. Media selective for Listeria monocytogenes. J. Appl. Bacteriol. 32:381–394. 101. Lachica, R. V. 1989. Modified Henry technique for the initial recognition of Listeria colonies. Annu. Meeting, Soc. Ind. Microbiol., Seattle, Washington, August 13–18, Abstr. P-44. 102. Lammerding, A. M., and M. P. Doyle. 1989. Evaluation of enrichment procedures for recovery of Listeria monocytogenes from dairy products. Proc. Annu. Meeting Instit. Food Technologists, Chicago, June 25–29, Abstr. 460. 103. Lammerding, A. M., and M. P. Doyle. 1989. Evaluation of enrichment procedures for recovery of Listeria monocytogenes from dairy products. J. Food Microbiol. 9:249–268. 104. Larsen, H. E. 1996. Listeria monocytogenes: Studies on isolation techniques and epidemiology. Copenhagen: Carl Fr. Mortensen. 105. Leasor, S. B., C. A. Abbas, and R. Firstenberg–Eden. 1990. Evaluation of UVM as a growth medium for Listeria monocytogenes. Proc. Annu. Meeting Am. Soc. Microbiologists, Anaheim, California, May 13–19, Abstr. P-39. 106. Lee, W. H., and D. McClain. 1986. Improved Listeria monocytogenes selective agar. Appl. Environ. Microbiol. 52:1215–1217. 107. Lehnert, C. 1964. Bakteriologische, serologische und tierexperimentelle Untersuchungen zur Pathogenese, Epizootologie und Prophylaxe der Listeriose. Arch. Exp. Vet. Med. 18:981–1027, 1247–1301. 108. Leighton, I. 1979. Use of selective agents for the isolation of Listeria monocytogenes. Med. Lab. Sci. 36:283–288. 109. Lewis, S. J., and J. E. Corry. 1991. Comparison of a cold enrichment and the FDA method for isolating Listeria monocytogenes and other Listeria spp. from ready-to-eat food on retail sale in the U.K. Int. J. Food Microbiol. 12:281–286. 110. Loessner, M. J., R. H. Bell, J. M. Jay, and L. A. Shelef. 1988. Comparison of seven plating media for enumeration of Listeria spp. Appl. Environ. Microbiol. 54:3003–3007. 111. Loessner, M. J., M. Rudolf, and S. Scherer. 1997. Evaluation of a luciferase reporter bacteriophage A511::luxAB for detection of Listeria monocytogenes in contaminated foods. Appl. Environ. Microbiol. 63:2961–2965. 112. Lou, Y., A. E. Yousef, and S. K. Sastry. 1996. Stress adaptation and cross-protection in Listeria monocytogenes. In 1996 Institute of food technologists book of abstracts. Abstract 35-2. Chicago: Institute of Food Technologists, p. 75. 113. Lovett, J. 1988. Isolation and enumeration of Listeria monocytogenes. Food Technol. 42(4):172–175. 114. Lovett, J. 1988. Isolation and identification of Listeria monocytogenes in dairy products. J. Assoc. Off. Anal. Chem. 71:658–660. 115. Lovett, J., and A. D. Hitchins. 1988. Listeria isolation; revised method of analysis. Fed. Reg. 53:44148–44153.

DK3089_C007.fm Page 254 Monday, February 19, 2007 12:31 PM

254

Listeria, Listeriosis, and Food Safety

116. Lovett, J., and A. D. Hitchins. 1989. Listeria isolation. In FDA bacteriological analytical manual, 6th ed. Supplement, September 1987, Association of Official Analytical Chemists, Arlington, VA, chapter 29, p. 29.01. 117. 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. 118. Lund, A. M., E. A. Zottola, and D. J. Pusch. 1991. Comparison of methods for the isolation of Listeria from raw milk. J. Food Prot. 54:602–606. 119. Martin, R. S., R. K. Sumarah, and M. A. MacDonald. 1984. A synthetic-based medium for the isolation of Listeria monocytogenes. Clin. Invest. Med. 7:233–237. 120. Mavrothalassitis, P. 1977. A method for rapid isolation of Listeria monocytogenes from infected material. J. Appl. Bacteriol. 43:47–52. 121. McBride, M. E., and K. F. Girard. 1960. A selective method for the isolation of Listeria monocytogenes from mixed bacterial populations. J. Lab. Clin. Med. 55:153–157. 122. McCarthy, S. A., M. L. Motes, and R. M. McPhearson. 1990. Recovery of heat-stressed Listeria monocytogenes from experimentally and naturally contaminated shrimp. J. Food Prot. 53:22–25. 123. McClain, D., and W. H. Lee. 1987. A method to recover Listeria monocytogenes from meats. Proc. Annu. Meeting, Am. Soc. Microbiol., Atlanta, May 1–6, Abstr. P-23. 124. McClain, D., and W. H. Lee. 1988. Development of a USDA–FSIS method for isolation of Listeria monocytogenes from raw meat and poultry. J. Assoc. Off. Anal. Chem. 71:660–664. 125. McLauchlin, J., A. Audurier, and A. G. Taylor. 1986. Aspects of epidemiology of human Listeria monocytogenes infections in Britain: 1967–1984; the use of serotyping and phage typing. J. Med. Microbiol. 22:367–377. 126. Meyer, D. H., and C. W. Donnelly. 1992. Effect of incubation temperature on repair of heat-injured Listeria in milk. J. Food Prot. 55:579–582. 127. Murray, E. G. D., R. A. Webb, and M. B. R. Swann. 1926. A disease of rabbits characterized by a large mononuclear leucocytosis, caused by a hitherto undescribed bacillus, Bacterium monocytogenes (n. sp.). J. Pathol. Bacteriol. 29:407–439. 128. Netten, P. van, I. Perales, and D. A. A. Mossel. 1988. An improved selective and diagnostic medium for isolation and counting of Listeria spp. in heavily contaminated foods. Lett. Appl. Microbiol. 7:17–21. 129. Netten, P. van, A. Van de Ven, I. Perales, and D. A. A. Mossel. 1988. A selective and diagnostic medium for use in the enumeration of Listeria spp. in foods. Int. J. Food Microbiol. 6:187–198. 130. Netten, P. van, 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. 131. Ngutter, C., and C. Donnelly. 2003. Nitrite-induced injury of Listeria monocytogenes and the effect of selective versus nonselective recovery procedures on its isolation from frankfurters. J. Food Prot. 66:2252–7. 132. Noah, C. W., J. C. Perez, N. C. Ramos, C. R. McKee, and M. V. Gibson. 1991. Detection of Listeria species in naturally contaminated seafood using four enrichment procedures. J. Food Prot. 54:174–177. 133. Olson, C., Jr., L. A. Dunn, and C. L. Rollins. 1953. Methods for isolation of Listeria monocytogenes from sheep. Am. J. Vet. Res. 14:82–85. 134. Ortel, S. 1971. Ausscheidung von Listeria monocytogenes im Stuhl gesunder Personen. Zentralbl. Bakteriol. I Abt. Orig. 217:41–46. 135. Ortel, S. 1972. Experience with nalidixic acid-trypaflavine agar. Acta Microbiol. Acad. Sci. Hung. 19:363–365. 136. Palumbo, S. A., and A. C. Williams. 1991. Resistance of Listeria monocytogenes to freezing in foods. Food Microbiol. 8:63–68. 137. 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. 138. 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. 139. Patterson, M. 1989. Sensitivity of Listeria monocytogenes to irradiation on poultry meat and in phosphate-buffered saline. Lett. Appl. Microbiol. 8:181–184. 140. Peng, H., and L. A. Shelef. 2000. Rapid detection of low levels of Listeria in foods and next-day confirmation of L. monocytogenes. J. Microbiol. Methods 41:113–20.

DK3089_C007.fm Page 255 Monday, February 19, 2007 12:31 PM

Conventional Methods to Detect and Isolate Listeria monocytogenes

255

141. Pini, P. N., and R. J. Gilbert. 1988. A comparison of two procedures for the isolation of Listeria monocytogenes from raw chickens and soft cheeses. Int. J. Food Microbiol. 7:331–337. 142. Petran, R. I., and K. M. J. Swanson. 1993. Simultaneous growth of Listeria monocytogenes and Listeria innocua. J. Food Prot. 56:616–618. 143. Pritchard, T. J., and C. W. Donnelly. 1995. Combined secondary enrichment of UVM and LRB primary enrichment broths increases the sensitivity of Listeria detection. IFT Annu. Meeting: Book of Abstracts. Abstr. 34-2, p. 96. 144. Pritchard, T. J., K. J. Flanders, and C. W. Donnelly. 1995. Comparison of the incidence of Listeria on equipment versus environmental sites within dairy processing plants. Int. J. Food Microbiol. 26:375–384. 145. Ralovich, B. S. 1975. Selective and enrichment media to isolate Listeria. In Problems of listeriosis. Proceedings of the Sixth International Symposium, ed. M. Woodbine. Leicester, U.K.: Leicester University Press, pp. 286–294. 146. Ralovich, B. 1984. Listeriosis research—present situation and perspective. Budapest: Akademiai Kiado. 147. Ralovich, B., A. Forray, E. Mero, and H. Malovics. 1970. Additional data on diagnosis and epidemiology of Listeria infections. Zentralbl. Bakteriol. I Abt. Orig. 214:231–235. 148. Ralovich, B., L. Emody, I. Malovics, E. Mero, and A. Forray. 1972. Methods to isolate Listeria monocytogenes from different materials. Acta Microbiol. Acad. Sci. Hung. 19:367–369. 149. Ralovich, B., A. Forray, E. Mero, H. Malovics, and I. Szazados. 1971. New selective medium for isolation of L. monocytogenes. Zentralbl. Bakteriol. I Abt. Orig. 216:88–91. 150. Restaino, L., E. W. Frampton, R. M. Irbe, G. Schabert, and H. Spitz. 1999. Isolation and detection of Listeria monocytogenes using fluorogenic and chromogenic substrates for phosphatidylinositolspecific phospholipase C. J. Food Prot. 62:244–251. 151. Rijpens, N., and L. Herman. 2004. Comparison of selective and nonselective primary enrichments for the detection of Listeria monocytogenes in cheese. Int. J. Food Microbiol. 94:15–22. 152. Rodriguez, D. L., G. S. Fernandez, J. F. F. Garayzabal, and E. R. Ferri. 1984. New methodology for the isolation of Listeria microorganisms from heavily contaminated environments. Appl. Environ. Microbiol. 47:1188–1190. 153. Rodriguez, D. L., J. F. Fernandez, V. Briones, J. L. Blanco, and G. Suarez. 1988. Assessment of different selective agar media for enumeration and isolation of Listeria from dairy products. J. Dairy Res. 55:579–583. 154. Rodriguez, D. L., J. F. F. Garayzabel, J. A. V. Boland, E. R. Ferri, and G. S. Fernandez. 1985. Isolation de microorganisms de listeria a partir de lait cru destine a le consommation humaine. Can. J. Microbiol. 31:938–941. 155. Roth, T. T., and C. W. Donnelly. 1995. Injury of Listeria monocytogenes by acetic and lactic acids: mechanisms of repair and sites of sublethal damage. IFT Annu. Meeting, Book of Abstracts. Abstr. 81D-1, p. 246. 156. 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. 50:7–13. 157. 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. 158. Ryser, E. T., and E. H. Marth. 1987. Fate of Listeria monocytogenes during manufacturing and ripening of Camembert cheese. J. Food Prot. 50:372–378. 159. 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. 160. Ryser, E. T., and E. H. Marth. 1989. Behavior of Listeria monocytogenes during manufacture and ripening of brick cheese. J. Dairy Sci. 72:838–853. 161. Ryser, E. T., and E. H. Marth. 1991. Listeria, listeriosis and food safety. New York: Marcel Dekker. 162. 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. 163. Sallam, S., and C. W. Donnelly. 1992. Destruction, injury and repair of Listeria species exposed to sanitizing compounds. J. Food Prot. 55:771–776. 164. Seeliger, H. P. R. 1961. Listeriosis. New York: Hafner. 165. Seeliger, H. P. R. 1972. Reviews—a new outlook on the epidemiology and epizoology of listeriosis. Acta Microbiol. Hung. 19:273–286.

DK3089_C007.fm Page 256 Monday, February 19, 2007 12:31 PM

256

Listeria, Listeriosis, and Food Safety

166. Seeliger, H. P. R., F. Sander, and J. Bockemühl. 1970. Zum kulturellen Nachweis von Listeria monocytogenes. Z. Med. Mikrobiol. Immunol. 155:352–368. 166a. Seeliger, H. P. R., and D. Jones. 1970. Listeria. In Bergey’s manual of systematic bacteriology, 9th ed., vol. 2, eds. P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt. Baltimore, Md.: Williams and Wilkins Co., pp. 1235–1245. 167. Silk, T. M., T. M. T. Roth, and C. W. Donnelly. 2002. Comparison of growth kinetics for healthy and hear-injured Listeria monocytogenes in eight enrichment broths. J. Food Prot. 65:1333–1337. 168. 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. 169. Skovgaard, N., and C.-A. Morgen. 1988. Detection of Listeria spp. in feces from animals, in feeds, and in raw foods of animal origin. Int. J. Food Microbiol. 6:229–242. 170. Slade, P. J., and D. L. Collins-Thompson. 1987. Two-stage enrichment procedures for isolating Listeria monocytogenes from raw milk. J. Food Prot. 50:904–908. 171. Smith, J. L., and D. L. Archer. 1988. Heat-induced injury in L. monocytogenes. J. Ind. Microbiol. 3:105–110. 172. Sokolovic, Z., A. Fuchs, and W. Goebel. 1990. Synthesis of species-specific stress proteins by virulent strains of Listeria monocytogenes. Infect. Immun. 58:3582–3587. 173. Terplan, G. 1988. Provisional IDF-recommended method: Milk and milk products—detection of Listeria monocytogenes. Brussels: International Dairy Federation. 174. Truscott, R. B., and W. B. McNab. 1988. Comparison of media and procedures for the isolation of Listeria monocytogenes from ground beef. J. Food Prot. 51:626–628,638. 175. Twedt, R. M., and A. D. Hitchins. 1994. Determination of the presence of Listeria monocytogenes in milk and dairy products: IDF collaborative study. J. AOAC Int. 77:395–402. 176. U.S. Department of Agriculture. 1998. Isolation and identification of Listeria monocytogenes from meat, poultry, and egg products. In Microbiology guide book, 3rd ed., eds. B. P. Dey and C. P. Lattuada. Washington, D.C.: Government Printing Office, pp. 8-1–8-18. 177. U.S. Food and Drug Administration. 1998. Listeria monocytogenes. In Bacteriological analytical manual, 8th ed. Gaithersburg, Md.: AOAC International, pp. 10.01–10.13. 178. USDA/FSIS. 2002. Isolation and identification of Listeria monocytogenes from red meat, poultry, egg and environmental samples. In Microbiology laboratory guidebook, 3rd ed., revision 3, chapter 8. 179. USDA/FSIS. 2002. FSIS Procedure for the use of Listeria monocytogenes BAX screening test. Microbiology laboratory guidebook, chapter 8A. 179a. Vlaemynck, G., V. Lafarge, and S. Scotter. 2000. Improvement of the detection of Listeria monocytogenes by the application of ALOA, a diagnostic, chromogenic isolation medium. J. Appl. Microbiol. 88:430–441. 180. Warburton, D. W., J. M. Farber, A. Armstrong, R. Caldeira, T. Hunt, S. Messier, R. Plante, N. P. Tiwari, and J. Vinet. 1991. A comparative study of the FDA and USDA methods for the detection of Listeria monocytogenes in foods. Int. J. Food Microbiol. 13:105–117. 181. Warburton, D. W., J. M. Farber, A. Armstrong, R. Caldeira, N. P. Tiwari, T. Babiuk, P. Lacasse, and S. Read. 1991. A Canadian comparative study of modified versions of the FDA and USDA methods for the detection of Listeria monocytogenes. J. Food Prot. 54:669–676. 182. Warburton, D. W., J. M. Farber, C. Powell, N. P. Tiwari, S. Read, R. Plante, T. Babiuk, P. Laffey, T. Kauri, P. Mayers, M.-J. Champagne, T. Hunt, P. LaCasse, K. Viet, R. Smando, and F. Coates. 1992. Comparison of methods for optimum detection of stressed and low levels of Listeria monocytogenes. Food Microbiol. 9:127–145. 183. Werner, B. S., and D. V. Lim. 1990. Growth of Listeria monocytogenes in different media. Abstr. Ann. Mtg, Am. Soc. Microbiol., Anaheim, California. May 13–19, Abstr. P-41. 184. 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. 185. Wiedmann, M. 2002. Molecular subtyping methods for Listeria monocytogenes. J. AOAC Int. 85:524–531. 186. Yousef, A. E., and E. H. Marth. 1988. Behavior of Listeria monocytogenes during manufacture and storage of Colby cheese. J. Food Prot. 51:12–15.

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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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REFERENCES 1. Aherne, A., C. Alexander, M.J. Payne, N. Perez, and E.N. Vulfson, 1996. Bacteria-mediated lithography of polymer surfaces. J. Am. Chem. Soc. 118: 8771–8772. 2. Amann, R. and W. Ludwig, 2000. Ribosomal RNA-targeted nucleic acid probes for studies in microbial ecology. FEMS Microbiol. Rev. 24: 555–565. 3. Amann, R., B.M. Fuchs, and S. Behrens, 2001. The identification of microorganisms by fluorescence in situ hybridization. Curr. Opin. Biotechnol. 12: 231–236. 4. Anonymous, February 27, 2001. Performance standards for the production of processed meat and poultry products. Fed. Regist. 66: 12590–12636. 5. Aznar, R. and B. Alarcón, 2002. On the specificity of PCR detection of Listeria monocytogenes in food: a comparison of published primers. Syst. Appl. Microbiol. 25: 109–119. 6. Bassler, H.A., S.J.A. Flood, K.J. Livak, J. Marmaro, R. Knorr, and C.A. Batt, 1995. Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes. Appl. Environ. Microbiol. 61: 3724–3728. 7. Benhar, I., I. Eshkenazi, T. Neufeld, J. Opatowsky, S. Shaky, and J. Rishpon, 2001. Recombinant single chain antibodies in bioelectrochemical sensors. Talanta 55: 899–907. 8. Berry, E.D. and G.R. Siragusa, 1997. Hydroxyapatite adherence as a means to concentrate bacteria. Appl. Environ. Microbiol. 63: 4069–4074. 9. Beumer, R.R. and W.C. Hazeleger, 2003. Listeria monocytogenes: diagnostic problems. FEMS Immunol. Med. Microbiol. 35: 191–197. 10. Bhunia, A.K., 1997. Antibodies to Listeria monocytogenes. Crit. Rev. Microbiol. 23: 77–107. 11. Blais, B.W., G. Turner, R. Sooknanan, and L.T. Malek, 1997. A nucleic acid sequence-based amplification system for detection of Listeria monocytogenes hlyA sequences. Appl. Environ. Microbiol. 63: 310–313. 12. Brehm-Stecher, B.F., Hyldig-Nielsen, J.J., and E.A. Johnson, 2005. Design and evaluation of 16S rRNA-targeted peptide nucleic acid probes for whole cell detection of the genus Listeria. Appl. Environ. Microbiol. 71: 5451–5457. 13. Brooks, J.L., J.P. Back, and R.G. Kroll, 1992. Direct application to dairy foods of a Listeria-specific oligonucleotide probe to 16S rRNA. Int. J. Food Microbiol. 16: 303–312. 14. Bubert, A., J. Riebe, N. Schnitzler, A. Schönberg, W. Goebel, and P. Schubert, 1997. Isolation of catalase-negative Listeria monocytogenes strains from listeriosis patients and their rapid identification by anti-p60 antibodies and/or PCR, J. Clin. Microbiol. 35: 179–183. 15. Bubert, A., I. Hein, M. Rauch, A. Lehner, B. Yoon, W. Goebel, and M. Wagner, 1999. Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR. Appl. Environ. Microbiol. 65: 4688–4692. 16. Buchrieser, C., C. Rusniok, The Listeria Consortium, F. Kunst, P. Cossart, and P. Glaser, 2003. Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua: clues for evolution and pathogenicity. FEMS. Immunol. Med. Microbiol. 35: 207–213. 17. Call, D.R., M.K. Borucki, and T.E. Besser, 2003. Mixed-genome microarrays reveal multiple serotype and lineage-specific differences among strains of Listeria monocytogenes. J. Clin. Microbiol. 41: 632–639. 18. Call, D.R., M.K. Borucki, and F.J. Loge, 2003. Detection of bacterial pathogens in environmental samples using DNA microarrays. J. Microbiol. Methods 53: 235–243. 19. Chang, H., A. Ikram, F. Kosari, G. Vasmatzis, A. Bhunia, and R. Bashir, 2002. Electrical characterization of micro-organisms using microfabricated devices. J. Vac. Sci. Technol. B 20: 2058–2064. 20. Choi, W.S. and C.-H. Hong, 2002. Rapid enumeration of Listeria monocytogenes in milk using competitive PCR. Int. J. Food Microbiol. 84: 79–85. 21. Churchill, R.L.T., J. Tanha, C.R. MacKenzie, and J.C. Hall, 2003. Single domain antibody fragments for the detection of Listeria monocytogenes, E-057. In Abstracts of the 103rd General Meeting of the American Society for Microbiology 2003. American Society for Microbiology, Washington, D.C., p. 80. 22. Cocolin, L., K. Rantsiou, L. Iacumin, C. Cantoni, and G. Comi, 2002. Direct identification in food samples of Listeria spp. and Listeria monocytogenes by molecular methods. Appl. Environ. Microbiol. 68: 6273–6282. 23. Cohen, P., M. Bouaboula, M. Bellis, V. Baron, O. Jbilo, C. Poinot-Chazel, S. Galiègue, E.-H. Hadibi, and P. Casellas, 2000. Monitoring cellular responses to Listeria monocytogenes with oligonucleotide arrays. J. Biol. Chem. 275: 11181–11190.

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276

Listeria, Listeriosis, and Food Safety

24. Cooray, K.J., T. Nishibori, H. Xiong, T. Matsuyama, M. Fujita, and M. Mitsuyama, 1994. Detection of multiple virulence-associated genes of Listeria monocytogenes by PCR in artificially contaminated milk samples. Appl. Environ. Microbiol. 60: 3023–3026. 25. Cox, T., C. Frazier, J. Tuttle, S. Flood, L. Yagi, C.T. Yamashiro, R. Behari, C. Paszko, and R.J. Cano, 1998. Rapid detection of Listeria monocytogenes in dairy samples utilizing a PCR-based fluorogenic 5 nuclease assay. J. Ind. Microbiol. Biotechnol. 21: 167–174. 26. Crowley, E.L., C.K. O’Sullivan, and G.G. Guilbault, 1999. Increasing the sensitivity of Listeria monocytogenes assays: evaluation using ELISA and amperometric detection. Analyst 124: 295–299. 27. Cummings, C.A. and D.A. Relman, 2000. Using DNA microarrays to study host-microbe interactions. Emerg. Infect. Dis. 6: 513–525. 28. De Cesare, A., J.L. Bruce, T.R. Dambaugh, M.E. Guerzoni, and M. Wiedmann, 2001. Automated ribotyping using different enzymes to improve discrimination of Listeria monocytogenes isolates, with a particular focus on serotype 4b strains. J. Clin. Microbiol. 39: 3002–3005. 29. DeLong, E.F., G.S. Wickham, and N.R. Pace, 1989. Phylogenetic stains: ribosomal RNA-based probes for the identification of single microbial cells. Science 243: 1360–1363. 30. Donnelly, C.W. and G.J. Baigent, 1986. Method for flow cytometric detection of Listeria monocytogenes in milk. Appl. Environ. Microbiol. 52: 689–695. 31. Donnelly, C.W., G.J. Baigent, and E.H. Briggs, 1988. Flow cytometry for automated analysis of milk containing Listeria monocytogenes. J. Assoc. Off. Anal. Chem. 71: 655–658. 32. Donnelly, C.W., 2002. Detection and isolation of Listeria monocytogenes from food samples: implications of sublethal injury. J. AOAC Int. 85: 495–500. 33. Duffy, G., O.M. Cloak, J.J. Sheridan, I.S. Blair, and D.A. McDowell, 1999. The development of a combined surface adhesion and polymerase chain reaction technique in the rapid detection of Listeria monocytogenes in meat and poultry. Int. J. Food Microbiol. 49: 151–159. 34. Dunbar, S.A., C.A. Vander Zee, K.G. Oliver, K.L. Karem, and J.W. Jacobson, 2003. Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system. J. Microbiol. Methods 53: 245–252. 35. Edelson, B.T. and E.R. Unanue, 2000. Immunity to Listeria infection. Curr. Opin. Immunol. 12: 425–431. 36. Ercolini, D., P.J. Hill, and C.E.R. Dodd, 2003. Development of a fluorescence in situ hybridization method for cheese using a 16S rRNA probe. J. Microbiol. Methods 52: 267–271. 37. Firstenberg-Eden, R. and L.A. Shelef, 2000. A new rapid automated method for the detection of Listeria from environmental swabs and sponges. Int. J. Food Microbiol. 56: 231–237. 38. Franciosa, G., S. Tartaro, C. Wedell-Neergaard, and P. Aureli, 2001. Characterization of Listeria monocytogenes strains involved in invasive and noninvasive listeriosis outbreaks by PCR-based fingerprinting techniques. Appl. Environ. Microbiol. 67: 1793–1799. 39. Fu, A.Y., C. Spence, A. Scherer, F.H. Arnold, and S.R. Quake, 1999. A microfabricated fluorescenceactivated cell sorter. Nat. Biotechnol. 17: 1109–1111. 40. Geng, T., K.P. Kim, R. Gómez, D.M. Sherman, R. Bashir, M.R. Ladisch, and A.K. Bhunia, 2003. Expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments. J. Appl. Microbiol. 95: 762–772. 41. Geng, T., M.T. Morgan, and A.K. Bhunia, 2004. Detection of low levels of Listeria monocytogenes cells by using a fiber-optic immunosensor. Appl. Environ. Microbiol. 70: 6138–6146. 42. Gilot, P. and J. Content, 2002. Specific identification of Listeria welshimeri and Listeria monocytogenes by PCR assays targeting a gene encoding a fibronectin-binding protein. J. Clin. Microbiol. 40: 698–703. 43. Giraffa, G. and E. Neviani, 2001. DNA-based, culture independent strategies for evaluating microbial communities in food-associated ecosystems. Int. J. Food Microbiol. 67: 19–34. 44. Glaser, P., L. Frangeul, C. Buchrieser, C. Rusniok, A. Amend, F. Baquero, P. Berche, H. Bloecker, P. Brandt., T. Chakraborty, A. Charbit, F. Chetouani, E. Couvé, A. de Daruvar, P. Dehoux, E. Domann, G. Domínguez-bernal, E. Duchaud, L. Durant, O. Dussurget, K.-D. Entian, H. Fsihi, F. Garcia-Del Portillo, P. Garrido, L Gautier, W. Goebel, N. Gómez-López, T. Hain, J. Hauf, D. Jackson, L.-M. Jones, U. Kaerst, J. Kreft, M. Kuhn, F. Kunst, G. Kurapkat, E. Madueño, A. Maitournam, J. Mata Vicente, E. Ng, H. Nedjari, G. Nordsiek, S. Novella, B. de Pablos, J.-C. Pérez-Diaz, R. Purcell, B. Remmel, M. Rose, T. Schlueter, N. Simoes, A. Tierrez, J.-A. Vázquez-Boland, H. Voss, J. Wehland, and P. Cossart, 2001. Comparative genomics of Listeria species. Science 294: 849–852.

DK3089_C008.fm Page 277 Tuesday, February 20, 2007 11:58 AM

Rapid Methods for Detection of Listeria

277

45. Gómez, R., R. Bashir, A. Sarikaya, M.R. Ladisch, J. Sturgis, J.P. Robinson, T. Geng, A.K. Bhunia, H.L. Apple, and S. Wereley, 2001. Microfluidic biochip for impedance spectroscopy of biological species. Biomed. Microdevices 3: 201–209. 46. Gómez, R., R. Bashir, and A.K. Bhunia, 2002. Microscale electronic detection of bacterial metabolism. Sens. Actuators B Chem. 86: 198–208. 47. Gray, M.L., H.J. Stafseth, F. Thorp, Jr., L.B. Sholl, and W.F. Riley, Jr., 1948. A new technique for isolating listerellae from the bovine brain. J. Appl. Bacteriol. 50: 1–9. 48. Greisen, K., M. Loeffelholz, A. Purohit, and D. Leong, 1994. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J. Clin. Microbiol. 32: 335–351. 49. Grossman, H.L., W.R. Myers, V.J. Vreeland, R. Bruehl, M.D. Alper, C.R. Bertozzi, and J. Clarke, 2004. Detection of bacteria in suspension by using a superconducting quantum interference device. Proc. Natl. Acad. Sci. USA 101: 129–134. 50. Grow, A.E., L.L. Wood, J.L. Claycomb, and P.A. Thompson, 2003. New biochip technology for labelfree detection of pathogens and their toxins. J. Microbiol. Methods 53: 221–223. 51. Hayden, O. and F.L. Dickert, 2001. Selective microorganism detection with cell surface imprinted polymers. Adv. Mater. 13: 1480–1483. 52. Hayden, O., R. Bindeus, C. Haderspöck, K.-J. Mann, B. Wirl, and F.L. Dickert, 2003. Mass-sensitive detection of cells, viruses and enzymes with artificial receptors. Sens. Actuators B Chem. 91: 316–319. 53. Hein, I., D. Klein, A. Lehner, A. Bubert, E. Brandl, and M. Wagner, 2001. Detection and quantification of the iap gene of Listeria monocytogenes and Listeria innocua by a new real-time quantitative PCR assay. Res. Microbiol. 152: 37–46. 54. Herman, L.M.F., J.H.G.E. De Block, and R.J.B. Moermans, 1995. Direct detection of Listeria monocytogenes in 25 milliliters of raw milk by a two-step PCR with nested primers. Appl. Environ. Microbiol. 61: 817–819. 55. 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. 56. Hough, A.J., S.A. Harbison, M.G. Savill, L.D. Melton, and G. Fletcher, 2002. Rapid enumeration of Listeria monocytogenes in artificially contaminated cabbage using real-time polymerase chain reaction. J. Food Prot. 65: 1329–1332. 57. Huang, Y., K.L. Ewalt, M. Tirado, R. Haigis, A. Forster, D. Ackley, M.J. Heller, J.P. O’Connell, and M. Krihak, 2001. Electric manipulation of bioparticles and macromolecules on microfabricated electrodes. Anal. Chem. 73: 1549–1559. 58. Huang, Y., E.L. Mather, J.L. Bell, and M. Madou, 2002. MEMS-based sample preparation for molecular diagnostics. Anal. Bioanal. Chem. 372: 49–65. 59. Hudson, J.A., R.J. Lake, and M.G. Savill, 2001. Rapid detection of Listeria monocytogenes in ham samples using immunomagnetic separation followed by polymerase chain reaction. J. Appl. Microbiol. 90: 614–621. 60. Iqbal, S.S., M.W. Mayo, J.G. Bruno, B.V. Bronk, C.A. Batt, and J.P. Chambers, 2000. A review of molecular recognition technologies for detection of biological threat agents. Biosens. Bioelectron. 15: 549–578. 61. Jacobsen, C.N., J. Rasmussen, and M. Jakobsen, 1997. Viability staining and flow cytometric detection of Listeria monocytogenes. J. Microbiol. Methods 28: 35–43. 62. Jinneman, K.C. and W.E. Hill, 2001. Listeria monocytogenes lineage group classification by MAMAPCR of the listeriolysin gene. Curr. Microbiol. 43: 129–133. 63. Johansson, J., P. Mandin, A. Renzoni, C. Chiaruttini, M. Springer, and P. Cossart, 2002. An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell 100: 551–561. 64. Jothikumar, N., X. Wang, and M.W. Griffiths, 2003. Real-time multiplex SYBR Green I-based PCR assay for simultaneous detection of Salmonella serovars and Listeria monocytogenes. J. Food Prot. 66: 2141–2145. 65. Jung, Y.S., J.F. Frank, R.E. Brackett, and J.R. Chen, 2003. Polymerase chain reaction detection of Listeria monocytogenes on frankfurters using oligonucleotide primers targeting the genes encoding internalin AB. J. Food Prot. 66: 237–241. 66. Kaclíková, E., T.V. Kuchta, H. Kay, and D. Gray, 2001. Separation of Listeria from cheese and enrichment media using antibody-coated microbeads and centrifugation. J. Microbiol. Methods 46: 63–67.

DK3089_C008.fm Page 278 Tuesday, February 20, 2007 11:58 AM

278

Listeria, Listeriosis, and Food Safety

67. Kerdahi, K.F. and P.F. Istafanos, 2000. Rapid determination of Listeria monocytogenes by automated enzyme-linked immunoassay and nonradioactive DNA probe. J. AOAC Int. 83: 86–88. 68. Klein, P.G. and V.K. Juneja, 1997. Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR. Appl. Environ. Microbiol. 63: 4441–4448. 69. Koo, K., and L.-A. Jaykus, 2002. Detection of single nucleotide polymorphisms within the Listeria genus using an “asymmetric” fluorogeneic probe set and fluorescence resonance energy transfer basedPCR. Lett. Appl. Microbiol. 35: 513–517. 70. 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. 71. Kress-Rogers, E., 1997. Biosensors and electronic noses for practical applications, In Handbook of Biosensors and Electronic Noses, Medicine, Food, and the Environment, Ed., E. Kress-Rogers. CRC Press, Boca Raton, FL, pp. 3–39. 72. Lampel, K.A., P.A. Orlandi, and L. Kornegay, 2000. Improved template preparation for PCR-based assays for detection of food-borne bacterial pathogens. Appl. Environ. Microbiol. 66: 4539–4542. 73. Lan, Z., F. Fiedler, and S. Kathariou, 2000. A sheep in wolf’s clothing: Listeria innocua strains with teichoic acid-associated surface antigens and genes characteristic of Listeria monocytogenes Serogroup 4. J. Bacteriol. 182: 6161–6168. 74. Lefier, D., D. Hirst, C. Holt, and A.G. Williams, 1997. Effect of sampling procedure and strain variation in Listeria monocytogenes on the discrimination of species in the genus Listeria by Fourier transform infrared spectroscopy and canonical variates analysis. FEMS Microbiol. Lett. 147: 45–50. 75. Leonard, P., S. Hearty, J. Brennan, L. Dunne, J. Quinn, T. Chakraborty, and R. O’Kennedy, 2003. Advances in biosensors for detection of pathogens in food and water. Enzyme Microb. Technol. 32: 3–13. 76. Levin, R.E., 2003. Application of the polymerase chain reaction for detection of Listeria monocytogenes in foods: a review of methodology. Food Biotechnol. 17: 99–116l. 77. Li, H., and R. Bashir, 2002. Dielectrophoretic separation and manipulation of live and heat-treated cells of Listeria on microfabricated devices with interdigitated electrodes. Sens. Actuators B Chem. 86: 215–221. 78. Lin, M., M. Al-Holy, H. Al-Qadiri, D.-H. Kang, A.G. Cavinato, Y. Huang, and B.A. Rasco, 2004. Discrimination of intact and injured Listeria monocytogenes by Fourier transform infrared spectroscopy and principal component analysis. J. Agric. Food Chem. 52: 5769–5772. 79. Loessner, M.J., C.E.D. Rees, G.S.A.B. Stewart, and S. Scherer, 1996. Construction of luciferase reporter bacteriophage A511::luxAB for rapid and sensitive detection of viable Listeria cells. Appl. Environ. Microbiol. 62: 1133–1140. 80. Loessner, M.J., M. Rudolf, and S. Scherer, 1997. Evaluation of luciferase reporter bacteriophage A511::luxAB for detection of Listeria monocytogenes in contaminated foods. Appl. Environ. Microbiol. 63: 2961–2965. 81. Lucore, L.A., M.A. Cullison, and L.-A. Jaykus, 2000. Immobilization with metal hydroxides as a means to concentrate food-borne bacteria for detection by cultural and molecular methods. Appl. Environ. Microbiol. 66: 1769–1776. 82. Lunge, V.R., B.J. Miller, K.J. Livak, and C.A. Batt, 2002. Factors affecting the performance of 5-nuclease PCR assays for Listeria monocytogenes detection. J. Microbiol. Methods 51: 361–368. 83. Mabilat, C., S. Bukwald, N. Machabert, S. Desvarenes, R. Kurfürst, and P. Cros, 1996. Automated RNA probe assay for the identification of Listeria monocytogenes. Int. J. Food Microbiol. 28: 333–340. 84. Makino, S.-I., Y. Okada, and T. Maruyama, 1995. A new method for direct detection of Listeria monocytogenes from foods by PCR. Appl. Environ. Microbiol. 61: 3745–3747. 85. Malorny, B., P.T. Tassios, P. Rådström, N. Cook, M. Wagner, and J. Hoorfar, 2003. Standardization of diagnostic PCR for the detection of foodborne pathogens. Int. J. Food Microbiol. 83: 39–48. 86. Maquelin, K., C. Kirschner, L.-P. Choo-Smith, N. van den Braak, H.Ph. Endtz, D. Naumann, and G.J. Puppels, 2002. Identification of medically relevant microorganisms by vibrational spectroscopy. J. Microbiol. Methods 51: 255–271. 87. Marks, T. and R. Sharp, 2000. Bacteriophages and biotechnology: a review. J. Chem. Technol. Biotechnol. 75: 6–17. 88. Milner, M.G., J.R. Saunders, and A.J. McCarthy, 2001. Relationship between nucleic acid ratios and growth in Listeria monocytogenes. Microbiology 147: 2689–2696.

DK3089_C008.fm Page 279 Tuesday, February 20, 2007 11:58 AM

Rapid Methods for Detection of Listeria

279

89. Minunni, M., M. Mascini, R.M. Carter, M.B. Jacobs, G.J. Lubrano, and G.G. Guilbault, 1996. A quartz crystal microbalance displacement assay for Listeria monocytogenes. Anal. Chim. Acta 325: 169–174. 90. Mitchell, B.A., J.A. Milbury, A.M. Brookins, and B.J. Jackson, 1994. Use of immunomagnetic capture on beads to recover Listeria from environmental samples. J. Food Prot. 57: 743–745. 91. Mole, R.J. and T. WO’C Maskell, 2001. Phage as a diagnostic—the use of phage in TB diagnosis. J. Chem. Technol. Biotechnol. 76: 683–688. 92. Mossoba, M.M., F.M. Khambaty, and F.S. Fry, 2002. Novel application of a disposable optical film to the analysis of bacterial strains: a chemometric classification of mid-infrared spectra. Appl. Spectrosc. 56: 732–736. 93. Nannapaneni, R., R. Story, A.K. Bhunia, and M.G. Johnson, 1998. Unstable expression and thermal instability of a species-specific cell surface epitope associated with a 66-kilodalton antigen recognized by monoclonal antibody EM-7G1 within serotypes of Listeria monocytogenes grown in nonselective and selective broths. Appl. Environ. Microbiol. 64: 3070–3074. 94. Newsome, R., 2003. Dormant microbes: research needs. Food Technol. 57(6): 38–42. 95. Nielsen, P.E., 2001. Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology. Curr. Opin. Biotechnol. 12: 16–20. 96. Nogva, H.G., K. Rudi, K. Naterstad, A. Holck, and D. Lillehaug, 2000. Application of a 5-nuclease PCR for quantitative detection of Listeria monocytogenes in pure cultures, water, skim milk, and unpasteurized whole milk. Appl. Environ. Microbiol. 66: 4266–4271. 97. Nogva, H.K., S.M. Dromtorp, H. Nissen, and K. Rudi, 2003. Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5-nuclease PCR. Biotechniques 34: 804. 98. Norton, D.M. and C.A. Batt, 1999. Detection of viable Listeria monocytogenes with a 5-nuclease PCR assay. Appl. Environ. Microbiol. 65: 2122–2127. 99. Norton, D.M., 2002. Polymerase chain reaction-based methods for detection of Listeria monocytogenes: toward real-time screening for food and environmental samples. J. AOAC Int. 85: 505–515. 100. Odumeru, J.A., M. Steele, L. Fruhner, C. Larkin, J. Jiang, E. Mann, and W.B. McNab, 1999. Evaluation of accuracy and repeatability of identification of food-borne pathogens by automated bacterial identification systems. J. Clin. Microbiol. 37: 944–949. 101. Olier, M., F. Pierre, J.-P. Lemaître, C. Divies, A. Rousset, and J. Guzzo, 2002. Assessment of the pathogenic potential of two Listeria monocytogenes human faecal carriage isolates. Microbiology 148: 1855–1862. 102. Paoli, G.C., C.-Y. Chen, and J.D. Brewster, 2004. Single-chain Fv antibody with specificity for Listeria monocytogenes. J. Immunol. Methods 289: 147–155. 103. Pedersen, L.H., P. Skouboe, L. Rossen, and O.F. Rasmussen, 1998. Separation of Listeria monocytogenes and Salmonella berta from a complex food matrix by aqueous polymer two-phase partitioning. Lett. Appl. Microbiol. 26: 47–50. 104. Peng, H. and L.A. Shelef, 2000. Rapid detection of low levels of Listeria in foods and next-day confirmation of L. monocytogenes. J. Microbiol. Methods 41: 113–120. 105. Peng, H., and L.A. Shelef, 2001. Automated simultaneous detection of low levels of listeriae and salmonellae in foods. Int. J. Food Microbiol. 63: 225–233. 106. Petrenko, V.A. and V.J. Vodyanoy, 2003. Phage display for detection of biological threat agents. J. Microbiol. Methods 53: 253–262. 107. Piletsky, S.A., S. Subrahmanyam, and A.P.F. Turner, 2001. Application of molecularly imprinted polymers in sensors for the environment and biotechnology. Sensor Rev. 21: 292–296. 108. Ratinaud, M.-H., and S. Revidon, 1996. A flow cytometric method to assess functional state of the Listeria membrane. J. Microbiol. Methods 25: 71–77. 109. Raybourne, R.B., G. Roth, P.A. Deuster, E.M. Sternberg, and A. Singh, 2001. Uptake and killing of Listeria monocytogenes by normal human peripheral blood granulocytes and monocytes as measured by flow cytometry and cell sorting. FEMS Immunol. Med. Microbiol. 31: 219–225. 110. Raybourne, R.B., 2002. Virulence testing of Listeria monocytogenes. J. AOAC Int. 85: 516–523. 111. Reissbrodt, R., 2004. New chromogenic plating media for detection and enumeration of pathogenic Listeria spp.—an overview. Int. J. Food Microbiol. 95: 1–9. 112. Rodríguez-Lázaro, D., M. Hernández, and M. Pla, 2004. Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay. FEMS Microbiol. Lett. 233: 257–267.

DK3089_C008.fm Page 280 Tuesday, February 20, 2007 11:58 AM

280

Listeria, Listeriosis, and Food Safety

113. Rodriguez-Saona, L.E., F.M. Khambaty, F.S. Fry, and E.M. Calvey, 2001. Rapid detection and identification of bacterial strains by Fourier transform near-infrared spectroscopy. J. Agric. Food Chem. 49: 574–579. 114. Rudi, K., H.K. Nogva, B. Moen, H. Nissen, S. Bredholt, T. Møretrø, K. Naterstad, and A. Holck, 2002. Development and application of new nucleic acid-based technologies for microbial community analyses in foods. Int. J. Food Microbiol. 78: 171–180. 115. Schaferkordt, S. and T. Chakraborty, 1997. Identification, cloning, and characterization of the lma operon, whose gene products are unique to Listeria monocytogenes. J. Bacteriol. 179: 2707–2716. 116. Scheu, P.M., K. Berghof, and U. Stahl, 1998. Detection of pathogenic and spoilage micro-organisms in food with the polymerase chain reaction. Food Microbiol. 15: 13–31. 117. Schlech, W.F., 2000. Epidemiology and clinical manifestations of Listeria monocytogenes infection, In Gram-Positive Infections, Ed., V.A. Fischetti et al., ASM Press, Washington, D.C., pp. 473–479. 118. Schmid, M.W., M. Walcher, A. Bubert, M. Wagner, M. Wagner, and K.-H. Schleifer, 2003. Nucleic acid-based, cultivation-independent detection of Listeria spp. and genotypes of L. monocytogenes. FEMS Immunol. Med. Microbiol. 35: 215–225. 119. Sergeev, N., M. Distler, S. Courtney, S. F. Al-Khaldi, D. Volokhov, V. Chizhikov, and A. Rasooly, 2004. Multipathogen oligonucleotide microarray for environmental and biodefense applications. Biosens. Bioelectron. 20: 684–698. 120. Sobek, H., M. Schmidt, B. Frey, and K. Kaluza, 1996. Heat-labile uracil-DNA glycosylase: purification and characterization. FEBS Lett. 388: 1–4. 121. Somer, L., and Y. Kashi, 2003. A PCR method based on 16S rRNA sequence for simultaneous detection of the genus Listeria and the species Listeria monocytogenes in food products. J. Food Prot. 66: 1658–1665. 122. Sølve, M., J. Boel, and B. Nørrung, 2000. Evaluation of a monoclonal antibody able to detect live Listeria monocytogenes and Listeria innocua. Int. J. Food Microbiol. 57: 219–224. 123. Stender, H., M. Fiandaca, J.J. Hyldig-Nielsen, and J. Coull, 2002. PNA for rapid microbiology. J. Microbiol. Methods 48: 1–17. 124. Stephan, R., S. Schumacher, and M.A. Zychowska, 2003. The VIT technology for rapid detection of Listeria monocytogenes and other Listeria spp. Int. J. Food Microbiol. 89: 287–290. 125. Stewart, G.S.A.B., 1997. Challenging food microbiology from a molecular perspective. Microbiology 143: 2099–2108. 126. Swarts, A.J., J.W. Hastings, R.F. Roberts, and A. von Holy, 1998. Flow cytometry demonstrates bacteriocin-induced injury to Listeria monocytogenes. Curr. Microbiol. 36: 266–270. 127. Teo, A.Y.L., G.R. Ziegler, and S.J. Knabel, 2001. Optimizing detection of heat-injured Listeria monocytogenes in pasteurized milk. J. Food Prot. 64: 1000–1011. 128. Tims, T.B., S.S. Dickey, D.R. DeMarco, and D.V. Lim, 2001. Detection of low levels of Listeria monocytogenes within 20 hours using an evanescent wave biosensor. Am. Clin. Lab. 20: 28–29. 129. Titball, R.W., and D.J. Squirrell, 1997. Probes for nucleic acids and biosensors. In Handbook of Biosensors and Electronic Noses, Medicine, Food, and the Environment. Ed., E. Kress-Rogers, CRC Press, Boca Raton, FL, pp. 91–109. 130. Tompkin, R.B., V.N. Scott, D.T. Bernard, W.H. Sveum, and K. Sullivan Gombas, 1999. Guidelines to prevent post-processing contamination from Listeria monocytogenes. Dairy Food Environ. Sanit. 19: 551–562. 131. Torensma, R., M.J.C. Visser, C.J.M. Aarsman, M.J.J.G. Poppelier, A.C. Fluit, and J. Verhoef, 1993. Monoclonal antibodies that react with live Listeria spp. Appl. Environ. Microbiol. 59: 2713–2716. 132. Tran, Y.L., F. Fiedler, D.A. Hodgson, and S. Kathariou, 1999. Transposon-induced mutations in two loci of Listeria monocytogenes serotype 1/2a result in phage resistance and lack of N-acetylglucosamine in the teichoic acid of the cell wall. Appl. Environ. Microbiol. 65: 4793–4798. 133. Turner, A.P.F. and J.D. Newman, 1998. An introduction to biosensors. In Biosensors for Food Analysis, Ed., A.O. Scott, The Royal Society of Chemistry, Cambridge, pp. 13–27. 134. Uyttendaele, M., I. Van Hoorde, and J. Debevere, 2000. The use of immuno-magnetic separation (IMS) as a tool in a sample preparation method for direct detection of L. monocytogenes in cheese. Int. J. Food Microbiol. 54: 205–212. 135. Vaughan, R.D., C.K. O’Sullivan, and G.G. Guilbault, 2001. Development of a quartz crystal microbalance (QCM) immunosensor for the detection of Listeria monocytogenes. Enzyme Microb. Technol. 29: 635–638.

DK3089_C008.fm Page 281 Tuesday, February 20, 2007 11:58 AM

Rapid Methods for Detection of Listeria

281

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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REFERENCES 1. Aarnisalo, K., T. Autio, A. Sjoberg, J. Lunden, H. Korkeala, and M. Suihko. 2003. Typing of Listeria monocytogenes isolates originating from the food processing industry with automated ribotyping and pulsed-field gel electrophoresis. J. Food Prot. 66: 249–255. 2. Arpin, C., C. Carlier, P. Courvalin, and C. Quentin. 1992. Analysis of an antibiotic resistance plasmid from a clinical isolate of Listeria monocytogenes. 12 Reunion Interdisc. Chimiother. Anti-Infect., Paris. 3. Asperger, H., M. Wagner, and E. Brandl. 2001. An approach towards public health and foodborne human listeriosis—The Austrian Listeria monitoring. Berl. Mnch. Tierárztl. Wochenschr. 114: 446–452. 4. 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. 5. Bannerman, E., P. Boerlin, and J. Bille. 1996. Typing of Listeria monocytogenes by monocin and phage receptors. Int. J. Food Microbiol. 31: 245–262. 6. Bibb, W.F., B.G. Gellin, R. Weaver, B. Schwartz, B.D. Plikaytis, M.W. Reeves, R.W. Pinner, and C.V. Broome. 1990. Analysis of clinical and food-borne isolates of Listeria monocytogenes in the United States by multilocus enzyme electrophoresis and application of the method to epidemiologic investigations. Appl. Environ. Microbiol. 56: 2133–2141. 7. Bille, J. 1989. Anatomy of a listeriosis outbreak, In Foodborne Listeriosis. Proceedings of a Symposium. Hamburg: B. Behr’s GmbH and Company, pp. 29–36. 8. Bille, J. and J. Rocourt. 1996. WHO International Multicenter Listeria monocytogenes Subtyping Study- rationale and set-up of the study. Int. J. Food Microbiol. 32: 251–262. 9. Bille, J., J. Rocourt, and B. Swaminathan. 2003. Listeria and Erysipelothrix. In P.R. Murray, E.J. Baron, J.H. Jorgensen, M.A. Pfaller, and R.H. Yoken, Eds., Manual of Clinical Microbiology, 8th ed., Vol. 1. ASM Press, Washington, D.C., pp. 461–471. 10. Boerlin, P., E. Bannerman, F. Ischer, J. Rocourt, and J. Bille. 1995. Typing Listeria monocytogenes: a comparison of random amplification of polymorphic DNA with 5 other methods. Res. Microbiol. 146: 35–49. 11. Boerlin, P., J. Rocourt, F. Grimont, P.A.D. Grimont, C. Jacquet, and J.C. Piffaretti. 1992. Listeria ivanovii subsp. londoniensis subsp. nov. Int. J. Syst. Bacteriol. 42: 69–73. 12. Boerlin, P., J. Rocourt, and J.C. Piffaretti. 1991. Taxonomy of the genus Listeria by using multilocus enzyme electrophoresis. Int. J. Syst. Bacteriol. 41: 59–64. 13. Borucki, M.K., M.J. Krug, W.T. Muraoka, and D.R. Call. 2003. Discrimination among Listeria monocytogenes isolates using a mixed genome DNA microarray. Vet. Microbiol. 92: 351–362. 14. Brosch, R., M. Brett, B. Catimel, J. B. Luchansky, B. Ojeniyi, and J. Rocourt. 1996. Genomic fingerprinting of 80 strains from the WHO multicentre international typing study of Listeria monocytogenes via pulsed-field gel electrophoresis (PFGE). Int. J. Food Microbiol. 32: 343–355. 15. Brosch, R., C. Buchrieser, and J. Rocourt. 1991. Subtyping of Listeria monocytogenes serovar 4b by use of low-frequency cleavage restriction endonucleases and pulsed-field gel electrophoresis. Res. Microbiol. 142: 667–675. 16. Brosch, R., J. Chen, and J. B. Luchansky. 1994. Pulsed-field fingerprinting of listeriae: identification of genomic divisions for Listeria monocytogenes and their correlation with serovar. Appl. Environ. Microbiol. 60: 2584–2592. 17. Brosius, J., A. Ullrich, M. A. Raker, A. Gray, T. J. Dull, R. R. Gutell, and H. F. Noller. 1981. Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli. Plasmid 6: 112–118. 18. Bruce, J., R. J. Hubner, E. M. Cole, C. I. McDowell, and J. A. Webster. 1995. Sets of EcoRI fragments containing ribosomal RNA sequences are conserved among different strains of Listeria monocytogenes. Proc. Natl. Acad. Sci. U.S.A. 92: 5229–5233. 19. Buchrieser, C., R. Brosch, B. Catimel, and J. Rocourt. 1993. Pulsed-field gel electrophoresis applied for comparing Listeria monocytogenes strains involved in outbreaks. Can. J. Microbiol. 39: 395–401. 20. Buchrieser, C., R. Brosch, and J. Rocourt. 1991. Use of pulsed-field gel electrophoresis to compare large DNA-restriction fragments of Listeria monocytogenes strains belonging to serogroups 1/2 and 3. Int. J. Food Microbiol. 14: 297–304.

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300

Listeria, Listeriosis, and Food Safety

21. Cai, S., D. Y. Kabuki, A. Y. Kuaye, T. G. Cargioli, M. S. Chung, R. Nielsen, and M. Wiedmann. 2002. Rational design of DNA sequence-based strategies for subtyping Listeria monocytogenes. J. Clin. Microbiol. 40: 3319–3325. 22. Call, D. R., M. K. Borucki, and T. E. Besser. 2003. Mixed-genome microarrays reveal multiple serotype and lineage-specific differences among strains of Listeria monocytogenes. J. Clin. Microbiol. 41: 632–639. 23. Carriere, C., A. Allardet-Servent, G. Bourg, A. Audurier, and M. Ramuz. 1991. DNA polymorphism in strains of Listeria monocytogenes. J. Clin. Microbiol. 29: 1351–1355. 24. Caugant, D. A., F. E. Ashton, W. F. Bibb, P. Boerlin, W. Donachie, C. Low, A. Gilmour, J. Harvey, and B. Norrung. 1996. Multilocus enzyme electrophoresis for characterization of Listeria monocytogenes isolates: results of an international comparative study. Int. J. Food Microbiol. 32: 301–311. 25. Centers for Disease Control and Prevention. 2001. Outbreak of listeriosis associated with homemade Mexican-style cheese—North Carolina, October 2000–January 2001, Morb. Mortal. Wkly Rep. 50: 560–2. 26. Centers for Disease Control and Prevention. 2002. Public Health Dispatch: outbreak of Listeriosis—Northeastern United States. Morb. Mortal. Wkly. Rep. 51: 950–951. 27. Centers for Disease Control and Prevention. 2000. Update: multistate outbreak of listeriosis—United States. Morb. Mortal. Wkly. Rep. 49: 129. 28. Charpentier, E. and P. Courvalin. 1999. Antibiotic resistance in Listeria spp. Antimicrob. Agents Chemother. 43: 2103–2108. 29. Charpentier, E., G. Gerbaud, and P. Courvalin. 1999. Conjugative mobilization of the rolling-circle plasmid pIP823 from Listeria monocytogenes BM4293 among gram-positive and gram-negative bacteria. J. Bacteriol. 181: 3368–3374. 30. Charpentier, E., G. Gerbaud, C. Jacquet, J. Rocourt, and P. Courvalin. 1995. Incidence of antibiotic resistance in Listeria spp. J. Infect. Dis. 172: 277–281. 31. Curtis, G.D.W. and R.G. Mitchell. 1992. Bacteriocin (monocin) interactions among Listeria monocytogenes strains. Int. J. Food Microbiol. 16: 283–292. 32. Czajka, J. and C.A. Batt. 1994. Verification of causal relationships between Listeria monocytogenes isolates implicated in food-borne outbreaks of listeriosis by randomly amplified polymorphic DNA patterns. J. Clin. Microbiol. 32: 1280–1287. 33. Dalton, C.B., C.C. Austin, J. Sobel, P.S. Hayes, W.F. Bibb, L.M. Graves, B. Swaminathan, M.E. Proctor, and P.M. Griffin. 1997. An outbreak of gastroenteritis and fever due to Listeria monocytogenes in milk. N. Engl. J. Med. 336: 100–105. 34. Destro, M.T., M.F.F. Leitao, and J.M. Farber. 1996. Use of molecular typing methods to trace the dissemination of Listeria monocytogenes in a shrimp processing plant. Appl. Environ. Microbiol. 62: 705–711. 35. Dramsi, S., M. Lebrun, and P. Cossart. 1996. Molecular and genetic determinants involved in invasion of mammalian cells by Listeria monocytogenes. Curr. Top. Microbiol. Immunol. 209: 61–77. 36. Ericsson, H., P. Stalhandske, M.-L. Danielsson-Tham, E. Bannerman, J. Bille, C. Jacquet, J. Rocourt, and W. Tham. 1995. Division of Listeria monocytogenes serovar 4b into two groups by PCR and restriction enzyme analysis. Appl. Environ. Microbiol. 61: 3872–3874. 37. Facinelli, B., E. Giovanetti, P.E. Varaldo, P. Casolari, and U. Fabio. 1991. Antibiotic resistance in foodborne Listeria. Lancet 338: 1272. 38. Facinelli, B., P. Varaldo, C. Casolari, and U. Fabio. 1988. Cross-infection with Listeria monocytogenes confirmed by DNA fingerprinting. Lancet 2: 1247–1248. 39. Farber, J.M. 1996. Molecular typing of Listeria. In International Journal of Food Microbiology, special issue, 3rd ed., Vol. 32. Elsevier Science B.V., London. 40. Farber, J.M., and C.J. Addison. 1994. RAPD typing for distinguishing species and strains in the genus Listeria. J. Appl. Bacteriol. 77: 242–250. 41. Filice, G.A., H.F. Cantrell, A.B. Smith, P.S. Hayes, J.C. Feeley, and D.W. Fraser. 1978. Listeria monocytogenes infection in neonates: investigation of an epidemic. J. Infect. Dis. 138: 17–23. 42. Forbes, K.J., K.D. Bruce, J.Z. Jordens, A. Ball, and T.H. Pennington. 1991. Rapid methods in bacterial DNA fingerprinting. J. Gen. Microbiol. 137: 2051–2058. 43. Gendel, S.M. and J. Ulaszek. 2000. Ribotype analysis of strain distribution in Listeria monocytogenes. J. Food Prot. 63: 179–185.

DK3089_C009.fm Page 301 Saturday, February 17, 2007 5:18 PM

Subtyping Listeria monocytogenes

301

44. Gerner-Smidt, P., P. Boerlin, F. Ischer, and J. Schmidt. 1996. High-frequency endonuclease (REA) typing: results from the WHO collaborative study group on subtyping of Listeria monocytogenes. Int. J. Food Microbiol. 32: 313–324. 45. Glaser, P., L. Frangeul, C. Buchrieser, C. Rusniok, A. Amend, F. Baquero, P. Berche, H. Bloecker, P. Brandt, T. Chakraborty, A. Charbit, F. Chetouani, E. Couve, A. de Daruvar, P. Dehoux, E. Domann, G. Dominguez-Bernal, E. Duchaud, L. Durant, O. Dussurget, K.D. Entian, H. Fsihi, F.G. Portillo, P. Garrido, L. Gautier, W. Goebel, N. Gomez-Lopez, T. Hain, J. Hauf, D. Jackson, L.M. Jones, U. Kaerst, J. Kreft, M. Kuhn, F. Kunst, G. Kurapkat, E. Madueno, A. Maitournam, J.M. Vicente, E. Ng, H. Nedjari, G. Nordsiek, S. Novella, B. de Pablos, J.C. Perez-Diaz, R. Purcell, B. Remmel, M. Rose, T. Schlueter, N. Simoes, A. Tierrez, J.A. Vazquez-Boland, H. Voss, J. Wehland, and P. Cossart. 2001. Comparative genomics of Listeria species. Science 294: 849–852. 46. Godreuil, S., M. Galimand, G. Gerbaud, C. Jacquet, and P. Courvalin. 2003. Efflux pump Lde is associated with fluoroquinolone resistance in Listeria monocytogenes. Antimicrob. Agents Chemother. 47: 704–708. 47. Graham, T., J. Golsteyn-Thomas, V.P.J. Gannon, and J.E. Thomas. 1996. Genus- and species-specific detection of Listeria monocytogenes using polymerase chain reaction assays targeting the 16S/23S intergenic spacer region of the rRNA operon. Can. J. Microbiol. 42: 1155–1162. 48. Graves, L.M. and B. Swaminathan. 2001. PulseNet standardized protocol for subtyping Listeria monocytogenes by macrorestriction and pulsed-field gel electrophoresis. Int. J. Food Microbiol. 65: 55–62. 49. Graves, L.M., B. Swaminathan, M.W. Reeves, S.B. Hunter, R.E. Weaver, B.D. Plikaytis, and A. Schuchat. 1994. Comparison of ribotyping and multilocus enzyme electrophoresis for subtyping of Listeria monocytogenes isolates. J. Clin. Microbiol. 32: 2936–2943. 50. Graves, L.M., B. Swaminathan, M.W. Reeves, and J. Wenger. 1991. Ribosomal DNA fingerprinting of Listeria monocytogenes using a digoxigenin-labeled DNA probe. Eur. J. Epidemiol. 7: 77–82. 51. Grimont, F. and P.A.D. Grimont. 1986. Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Ann. Inst. Pasteur Microbiol. 137b: 165–175. 52. Guerra, M.M., F. Bernardo, and J. McLauchlin. 2002. Amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes. Syst. Appl. Microbiol. 25: 456–461. 53. Gur-Arie, R., C.J. Cohen, Y. Eitan, L. Shelef, E.M. Hallerman, and Y. Kashi. 2000. Simple sequence repeats in Escherichia coli: abundance, distribution, composition, and polymorphism. Genome Res. 10: 62–71. 54. Hadorn, K., H. Hachler, A. Schaffner, and F.H. Kayser. 1993. Genetic characterization of plasmidencoded multiple antibiotic resistance in a strain of Listeria monocytogenes causing endocarditis. Eur. J. Clin. Microbiol. Infect. Dis. 12: 928–937. 55. Hamon, Y. and Y. Péron. 1961. Etude dupouvoir bactériocinogène dans le genre Listeria. C. R. Acad. Sci. III 253: 1883–1885. 56. Howard, P.J., K.D. Harsono, and J.B. Luchansky. 1992. Differentiation of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri by pulsed-field gel electrophoresis. Appl. Environ. Microbiol. 58: 709–712. 57. Hubner, R.J., E.M. Cole, J.L. Bruce, C.I. McDowell, and J.A. Webster. 1995. Types of Listeria monocytogenes predicted by the positions of EcoRI cleavage sites relative to ribosomal RNA sequences. Proc. Natl. Acad. Sci. U.S.A. 92: 5232–5238. 58. Jacobs, M.R., H. Stein, A. Buqwane, A. Dubb, F. Segal, L. Rabinowitz, U. Ellis, I. Freiman, M. Witcomb, and V. Vallabh. 1978. Epidemic listeriosis: report of 14 cases detected in 9 months. S. Afr. Med. J. 54: 389–392. 59. Jacquet, C., J. Bille, and J. Rocourt. 1992. Typing Listeria monocytogenes by restriction polymorphism of the ribosomal ribonucleic acid gene region. Zentralbl. Bakteriol. 276: 356–365. 60. Klevytska, A.M., L.B. Price, J.M. Schupp, P.L. Worsham, J. Wong, and P. Keim. 2001. Identification and characterization of variable-number tandem repeats in the Yersinia pestis. Genome. J. Clin. Microbiol. 39: 3179–3185. 61. Lawrence, L.M., J. Harvey, and A. Gilmour. 1993. Development of random amplification of polymorphic DNA typing method for Listeria monocytogenes. Appl. Environ. Microbiol. 59: 3117–3119.

DK3089_C009.fm Page 302 Saturday, February 17, 2007 5:18 PM

302

Listeria, Listeriosis, and Food Safety

62. Lehmann, S. and A. Schonberg. 2001. Report on the Phase III of the WHO Serotyping Study of Listeria monocytogenes. International Symposium on Problems of Listeriosis (ISOPOL XIV), Mannheim, Germany, p. 148. 63. Lemaitre, J.P., A. Delcourt, and A. Rousset. 1997. Optimization of the detection of bacteriophages induced from Listeria sp. Lett. Appl. Microbiol. 24: 51–54. 64. Loessner, M.J., L.A. Estela, R. Zink, and S. Scherer. 1994. Taxonomical classification of 20 newly isolated Listeria bacteriophages by electron microscopy and protein analysis. Intervirology 37: 31–35. 65. Lukinmaa, S., M. Miettinen, U.-M. Nakari, H. Korkeala, and A. Siitonen. 2003. Listeria monocytogenes isolates from invasive infections: variation of sero- and genotypes during an 11-year period in Finland. J. Clin. Microbiol. 41: 1694–1700. 66. MacGowan, A.P., D.S. Reeves, and J. McLauchlin. 1990. In-vitro synergy testing of nine antimicrobial combinations against Listeria monocytogenes. Lancet 336: 513–514. 67. Maiden, M.C., J.A. Bygraves, E. Feil, G. Morelli, J.E. Russell, R. Urwin, Q. Zhang, J. Zhou, K. Zurth, D.A. Caugant, I.M. Feavers, M. Achtman, and B.G. Spratt. 1998. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. U.S.A. 95: 3140–3145. 68. Marquet-van der Mee, N., M. Loessner, and A. Audurier. 1997. Evaluation of seven experimental phages for inclusion in the international phage set for the epidemiological typing of Listeria monocytogenes. Appl. Environ. Microbiol. 63: 3374–3377. 69. Mazurier, S.I., A. Audurier, N. Marquet-Van der Mee, S. Notermans, and K. Wernars. 1992. A comparative study of randomly amplified polymorphic DNA analysis and conventional phage typing for epidemiological studies of Listeria monocytogenes isolates. Res. Microbiol. 143: 507–512. 70. McLauchlin, J., A. Audurier, A. Frommelt, P. Gerner-Smidt, C. Jacquet, M.J. Loessner, N. van der Mee-Marquet, J. Rocourt, S. Shah, and D. Wilhelms. 1996. WHO study on subtyping Listeria monocytogenes: results of phage-typing. Int. J. Food Microbiol. 32: 289–299. 71. McLauchlin, J., A. Audurier, and A.G. Taylor. 1986. Aspects of the epidemiology of human Listeria monocytogenes infections in Britain 1967–1984; the use of serotyping and phage typing. J. Med. Microbiol. 22: 367–377. 72. McLauchlin, J., A. Audurier, and A.G. Taylor. 1986. The evaluation of a phage-typing system for Listeria monocytogenes for use in epidemiological studies. J. Med. Microbiol. 22: 357–365. 73. McLauchlin, J., S.M. Hill, S.K. Velani, and R.J. Gilbert. 1991. Human listeriosis and pâté: a possible association. Br. Med. J. 303: 773–775. 74. Niederhauser, C., C. Hoelfelein, M. Allman, P. Burkhalter, J. Luethy, and U. Candrian. 1994. Random amplification of polymorphic bacterial DNA: evaluation of 11 oligonucleotides and application to food contaminated with Listeria monocytogenes. J. Appl. Bacteriol. 77: 574–582. 75. Nocera, D., M. Altwegg, G. Martinetti Lucchini, E. Bannerman, F. Ischer, J. Rocourt, and J. Bille. 1993. Characterization of Listeria strains from a food borne listeriosis outbreak by rDNA gene restriction patterns compared to four other typing methods. Eur. J. Clin. Microbiol. Infect. Dis. 12: 162–169. 76. Nocera, D., E. Bannerman, J. Rocourt, K. Jaton-Ogay, and J. Bille. 1990. Characterization by DNA restriction endonuclease analysis of Listeria monocytogenes strains related to the Swiss epidemic of listeriosis. J. Clin. Microbiol. 28: 2259–2263. 77. Norrrung, B. 1992. Characterization of Danish isolates of Listeria monocytogenes by multilocus enzyme electrophoresis. Int. J. Food Microbiol. 15: 51–59. 78. Norrung, B. and P. Gerner-Smidt. 1993. Comparison of multilocus enzyme electrophoresis (MEE), ribotyping, restriction enzyme analysis (REA) and phage typing for Listeria monocytogenes. Epidemiol. Infect. 111: 71–79. 79. O’Donoghue, K., K. Bowker, J. McLauchlin, D.S. Reeves, P.M. Bennett, and A.P. MacGowan. 1995. Typing of Listeria monocytogenes by random amplified polymorphic DNA (RAPD) analysis. Int. J. Food Microbiol. 27: 245–252. 80. Ortel, S. 1989. Listeriocins (monocins). Int. J. Food Microbiol. 8: 249–250. 81. Owens, R. 1989. Chromosomal DNA fingerprinting—a new method of species and strain identification applicable to microbial pathogens. J. Med. Microbiol. 30: 89–99.

DK3089_C009.fm Page 303 Saturday, February 17, 2007 5:18 PM

Subtyping Listeria monocytogenes

303

82. Piffaretti, J.C., H. Kressebuch, M. Aeschbacher, J. Bille, E. Bannerman, J.M. Musser, R.K. Selander, and J. Rocourt. 1989. Genetic characterization of clones of the bacterium Listeria monocytogenes causing epidemic disease. Proc. Natl. Acad. Sci. U.S.A. 86: 3818–3822. 83. Pinner, R.W., A. Schuchat, B. Swaminathan, P.S. Hayes, K.D. Deaver, R.E. Weaver, B.D. Plikaytis, M. Reeves, C.V. Broome, and J.D. Wenger. 1992. Role of foods in sporadic listeriosis II. Microbiologic and epidemiologic investigation. JAMA 267: 2046–2050. 84. Poyart-Salmeron, C., P. Trieu-Cuot, C. Carlier, A. MacGowan, J. McLauchlin, and P. Courvalin. 1992. Genetic basis of tetracycline resistance in clinical isolates of Listeria monocytogenes. Antimicrob. Agents Chemother. 36: 463–466. 85. Proctor, M.E., R. Brosch, J.W. Mellen, L.A. Garrett, C.W. Kaspar, 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. 86. Rasmussen, O.F., T. Beck, J.E. Olsen, L. Dons, and L. Rossen. 1991. Listeria monocytogenes isolates can be classified into two major types according to the sequence of the listeriolysin gene. Infect. Immun. 59: 3945–3951. 87. Rasmussen, O.F., P. Skouboe, L. Dons, L. Rossen, and J.E. Olsen. 1995. Listeria monocytogenes exists in at least three evolutionary lines: evidence from flagellin, invasive associated protein and listeriolysin O genes. Microbiology 141: 2053–2061. 88. Ridley, A.M. 1995. Evaluation of a restriction fragment length polymorphism typing method for Listeria monocytogenes. Res. Microbiol. 146: 21–34. 89. Ripabelli, G., J. McLauchin, and E.J. Threlfall. 2000. Amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes. Syst. Appl. Microbiol. 23: 132–136. 90. Rocourt, J., A. Audruier, A.L. Courtieu, J. Durst, S. Ortel, A. Schrettenbrunner, and A.G. Taylor. 1985. A multi-centre study on the phage typing of Listeria monocytogenes. Zentralbl. Bakteriol. Hyg. 259: 489–497. 91. Rocourt, J., V. Goulet, and A. Lepoutre-Toulemon. 1993. Epidemie de listeriose en France en 1992. Med. Mal. Infect. 23: 481–484. 92. Rudi, K., T. Katla, and K. Naterstad. 2003. Multi locus fingerprinting of Listeria monocytogenes by sequence-specific labeling of DNA probes combined with array hybridization. FEMS Microbiol. Lett. 220: 9–14. 93. Ryser, E.T., S.M. Arimi, M.M. 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. 94. Salcedo, C., B. Arreaza, L. Alcala, L. de la Fuente, and J.A. Vazquez. 2003. Development of a multilocus sequence typing method for analysis of Listeria monocytogenes clones. J. Clin. Microbiol. 41: 757–762. 95. Sauders, B.D., E.D. Fortes, D.L. Morse, N. Dumas, J.A. Kiehlbauch, Y. Schukken, J.R. Hibbs, and M. Wiedmann. 2003. Molecular subtyping to detect human listeriosis clusters. Emerg. Infect. Dis. 9: 672–680. 96. Saunders, N.A., A.M. Ridley, and A.G. Taylor. 1989. Typing of Listeria monocytogenes for epidemiological studies using DNA probes. Acta Microbiol. Immunol. Hung. 36: 205–209. 97. Schaferkordt, S. and T. Chakraborty. 1997. Identification, cloning, and characterization of the lma operon whose gene products are unique to Listeria monocytogenes. J. Bacteriol. 179: 2707–2716. 98. Schlech, W.F., III, P. M. Lavigne, R.A. Brotolussi, A.C. Allen, E.V. Haldane, A.J. Wort, A.W. Hightower, S.E. Johnson, A.H. King, E.S. Nicholls, and C.V. Broome. 1983. Epidemic listeriosis: evidence for transmission by food. N. Engl. J. Med. 308: 203–206. 99. Schönberg, A., E. Bannerman, A.L. Courtieu, R. Kiss, J. McLauchlin, S. Shah, and D. Wilhelms. 1996. Serotyping of 80 strains from the WHO multicentre international typing study of Listeria monocytogenes. Int. J. Food Microbiol. 32: 279–287. 100. Schwartz, D.C. and C.R. Cantor. 1984. Separation of yeast chromosome-sized DNAs by pulsed-field gradient gel electrophoresis. Cell 37: 67–75. 101. Seeliger, H.P.R. and K. Hohne. 1979. Serotyping of Listeria monocytogenes and related species. Methods Microbiol. 13: 31–49.

DK3089_C009.fm Page 304 Saturday, February 17, 2007 5:18 PM

304

Listeria, Listeriosis, and Food Safety

102. Selander, R.K., D.A. Caugant, H. Ochman, J.M. Musser, M.N. Gilmour, and T.S. Whittam. 1986. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl. Environ. Microbiol. 51: 873–884. 103. Sheehan, B., C. Kocks, S. Dramsi, E. Gouin, A.D. Klarsfeld, J. Mengaud, and P. Cossart. 1994. Molecular and genetic determinants of Listeria monocytogenes infectious process. Curr. Top. Microbiol. Immunol. 192: 187–216. 104. Slade, P.J. and D.L. Collins-Thompson. 1990. Listeria, plasmids, antibiotic resistance, and food. Lancet 336: 1004. 105. Sontakke, S. and J.M. Farber. 1995. The use of PCR ribotyping for typing strains of Listeria spp. Eur. J. Epidemiol. 11: 665–673. 106. Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503–517. 107. Spratt, B.G. 1999. Multilocus sequence typing: molecular typing of bacterial pathogens in an era of rapid DNA sequencing and the Internet. Curr. Opin. Microbiol. 2: 312–316. 108. Stull, T.L., J.J. LiPuma, and T.D. Edlind. 1988. A broad-spectrum probe for molecular epidemiology of bacteria: ribosomal RNA. J. Infect. Dis. 157: 280–286. 109. 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. 110. Swaminathan, B., S.B. Hunter, P.M. Desmarchelier, P. Gerner-Smidt, L.M. Graves, S. Harlander, R. Hubner, C. Jacquet, B. Pedersen, K. Reineccius, A. Ridley, N.A. Saunders, and J.A. Webster. 1996. WHO-sponsored international collaborative study to evaluate methods for subtyping Listeria monocytogenes: restriction fragment length polymorphism (RFLP) analysis using ribotyping and Southern hybridization with two probes derived from L. monocytogenes chromosome. Int. J. Food Microbiol. 32: 263–278. 111. Sword, C.P. and M.J. Pickett. 1961. The isolation and characterization of bacteriophages from Listeria monocytogenes. J. Gen. Microbiol. 25: 241–248. 112. Unnerstad, H., H. Ericsson, A. Alderborn, W. Tham, M.L. Danielsson-Tham, and J.G. Mattsson. 2001. Pyrosequencing as a method for grouping of Listeria monocytogenes strains on the basis of singlenucleotide polymorphisms in the inlB gene. Appl. Environ. Microbiol. 67: 5339–5342. 113. Versalovic, J., T. Koeuth, and J.R. Lupski. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucl. Acid. Res. 19: 6823–6831. 114. Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. van de Lee, M. Hornes, A. Frijters, J. Pot, J. Peleman, M. Kuiper, and M. Zabeau. 1995. AFLP: a new technique for DNA fingerprinting. Nucl. Acid. Res. 23: 4407–4414. 115. Wagner, M. and F. Allerberger. 2003. Characterization of Listeria monocytogenes recovered from 41 cases of sporadic listeriosis in Austria by serotyping and pulsed-field gel electrophoresis. FEMS Immunol. Med. Microbiol. 35: 227–234. 116. Weaver, R.E. 1989. Morphological, physiological and biochemical characterization. In Isolation and identification of Listeria monocytogenes, CDC Lab Manual, 1st ed., Vol. 1. U.S. Department of Health and Human Services, Centers for Disease Control, Atlanta, GA. 117. Welsh, J. and M. McClelland. 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucl. Acid. Res. 18: 7213–7218. 118. Wernars, K., P. Boerlin, A. Audurier, E.G. Russell, G.D.W. Curtis, L. Herman, and N. van der MeeMarquet. 1996. The WHO multicentre study on Listeria monocytogenes subtyping: random amplification of polymorphic DNA (RAPD). Int. J. Food Microbiol. 32: 325–341. 119. Wesley, I.V. and F. Ashton. 1991. Restriction enzyme analysis of Listeria monocytogenes strains associated with food-borne epidemics. Appl. Environ. Microbiol. 57: 969–975. 120. 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. 121. Wilhelms, D. and D. Sandow. 1989. Preliminary studies on monocine typing of Listeria monocytogenes strains. Acta. Microbiol. Hung. 36: 235–238. 122. Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski, and S.V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Acid. Res. 18: 6531–6535. 123. Wuenscher, M.D., S. Kohler, A. Bubert, U. Gerike, and W. Goebel. 1993. The iap gene of Listeria monocytogenes is essential for cell viability, and its gene product, p60, has bacteriolytic activity. J. Bacteriol. 175: 3491–3501.

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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.

REFERENCES 1. Adak, G.K., S.M. Meakins, H. Yip, B.A. Lopman, and S.J. O’Brien 2005. Disease risk from foods, England and Wales. Emerging Infect Dis 11: 365–372. 2. Allerberger, F. and J.P. Gaggenbichler 1989. Listeriosis in Austria — report of an outbreak in 1986. Acta Microbiol Hung 36: 149–152. 3. Anonymous 1989. Fed Reg 54, 22345. 4. Anonymous 1987. Fed Reg 52, 7264. 5. Anonymous, ILSI Research Foundation Risk Science Institute 2005. Achieving continuous improvement in reductions in foodborne listeriosis: a risk-based approach. J Food Prot 68: 1932–1994. 6. Anonymous 2004. Assessing the Effectiveness of Listeria monocytogenes Interim Final Rule: Summary Report. United States Department of Agriculture, Washington, DC: Food Safety and Inspection Service. 7. Anonymous 1997. Case definitions for infectious conditions under public health surveillance. Morbidity Mortality Wkly Rep 46: 1–55. 8. Anonymous 2003. Control of Listeria monocytogenes in Ready-To-Eat Meat and Poultry Products; Final Rule, p. 9 CFR Part 30. U.S. Department of Agriculture, Washington, DC: Food Safety and Inspection Service. 9. Anonymous 2003. First documented outbreak of Listeria monocytogenes in Quebec, 2002. Canada Communicable Disease Report 29. 10. Anonymous 1985. Jalisco-Brand Mexican-Style Soft Cheese Recalled. FDA Enforcement Report. June 26. 11. Anonymous 1999. Listeria cases linked to pâté. Westchester County Department of Health. [Online] http: //www.westchestergov.com/health/PR991230.htm. 12. Anonymous 1986. Listeriosis—survey in French-speaking Switzerland. Weekly Epidemiological Record 41: 317–318. 13. Anonymous 1989. Listeriosis associated with consumption of turkey franks. Morbidity Mortality Wkly Rep 38: 267–268. 14. Anonymous 1998. Multistate outbreak of listeriosis—United States, 1998. Morbidity Mortality Wkly Rep 47: 1085–1086. 15. Anonymous 2004. Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food—10 sites, United States. Morbidity Mortality Wkly Rep 54: 352–356. 16. Anonymous 2003. Quantitative assessment of relative risk to public health from foodborne Listeria monocytogenes among selected categories of ready-to-eat foods. U.S. Department of Agriculture, Food Safety and Inspection Service/Food and Drug Administration, Washington, DC [Online] http: //www.foodsafety.gov/~dms/lmr2-toc.html. 17. Anonymous 1999. Recall Notification Report 061-99, Expanded (2nd). Food Safety and Inspection Service, U.S. Department of Agriculture, Washington, DC. 18. Anonymous August 15, 2003. TDH warns of health hazards from Mexican cheese. [Online] https: //www.tdh.state.tx.us/news/b_new489.htm. 19. Ansbacher, R., K.A. Borchardt, M.W. Hannegan, and W.A. Boryson 1966. Clinical investigation of Listeria monocytogenes as a possible cause of human fetal wastage. Am J Obstet Gynecol 94: 386–390. 20. Arnold, G.J., and J. Coble 1995. Incidence of Listeria species in foods in NSW. Food Aust 47: 71–75.

DK3089_C010.fm Page 350 Tuesday, February 20, 2007 12:01 PM

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21. Ashton, F.E., J.A. Ryan, and E.P. Ewan 1988. Enzyme electrophoretic analysis of Listeria monocytogenes serotype 4b associated with listeriosis in Canada. Presented at the Proceedings of the X International Symposium on Listeriosis, August 22–26, Hungary. 22. 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. N Engl J Med 342: 1236–1241. 23. Autio, T., S. Hielm, M. Miettinen, A. Sjöberg, K. Aarnisalo, T. Björkroth, T. Mattila-Sandholm, and H. 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. 24. Bagge-Ravn, D., K. Gardshodn, L. Gram, and B.F. Vogel 2003. Comparison of sodium hypochloritebased foam and peroxyacetic acid-based fog sanitizing procedures in a salmon smokehouse: survival of the general microflora and Listeria monocytogenes. J Food Prot 66: 592–598. 25. Barrett, T.J., H. Lior, J.H. Green, R. Khakhria, J.G. Wells, B.P. Bell, K.D. Greene, J. Lewis, and P.M. Griffin 1994. Laboratory investigation of a multistate food-borne outbreak of Escherichia coli O157: H7 by using pulsed-field gel electrophoresis and phage typing. J Clin Microbiol 32: 3013–3017. 26. Bearns, R.E. and K.F. Girard 1958. The effect of pasteurization on Listeria monocytogenes. Can J Microbiol 4: 55–61. 27. Becroft, D.M.O., K. Farmer, R.J. Seddon, R. Sowden, J.H. Stewart, A. Vines, and D.A. Wattie 1971. Epidemic listeriosis in the newborn. Br Med J 3: 747–751. 28. Bell, R.A., V.N. Hillers, and T.A. Thomas 1999. The Abuela project: safe cheese worksh