Listeria, Listeriosis, & Food Safety 2nd Edition (Food Science and Technology)

l i steriu, Listeriosis, and Food Safety FOOD SCIENCE AND TECHNOLOGY A Series of Monographs, Textbooks, and Refere...

Author: Elliot T. Ryser | Elmer H. Marth

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l i steriu,

Listeriosis,

and

Food Safety

FOOD SCIENCE AND TECHNOLOGY A Series of Monographs, Textbooks, and Reference Books EDITORIAL BOARD

Owen R. Fennema University of Wisconsin-Madison Marcus Karel Rutgers University Gary W. Sanderson Universal Foods Corporation Steven R. Tannenbaum Massachusetts Institute of Technology Pieter Walstra Wageningen Agricultural University John R. Whitaker University of California-Davis

1. Flavor Research: Principles and Techniques, R. Teranishi, I, Hornstein, P. Issenberg, and E. L. Wick 2. Principles of Enzymology for the Food Sciences, John R, Whitaker 3. Low-Temperature Preservation of Foods and Living Matter, Owen R. Fennema, William D. Powrie, and Elmer H. Marth 4. Principles of Food Science Part I: Food Chemistry, edited by Owen R, Fennema Part I I : Physical Methods of Food Preservation, Marcus Karel, Owen R. Fennema, and Daryl 8.Lund 5. Food Emulsions, edited by Stig E. Friberg 6 . Nutritional and Safety Aspects of Food Processing, edited by Steven R. Tannenbaum 7 . Flavor Research: Recent Advances, edited by R. Teranishi, Robert A. Flath, and Hiroshi Sugisaw a 8. Computer-Aided Techniques in Food Technology, edited by Israel Saguy 9. Handbook of Tropical Foods, edited by Harvey T. Chan 10. Antimicrobials in Foods, edited by Alfred Larry Branen and P, Michael Davidson 1 1. Food Constituents and Food Residues: Their Chromatographic Determination, edited by James F. Lawrence 12. Aspartame: Physiology and Biochemistry, edited by Lewis D. Steghk and L. J, Filer, Jr. 13. Handbook of Vitamins: Nutritional, Biochemical, and Clinical Aspects, edited by Lawrence J. Machlin 14. Starch Conversion Technology, edited by G. M. A. van Beynum and J. A. Roels 15. Food Chemistry: Second Edition, Revised and Expanded, edited by Owen R. Fennema 16 . Sensory Evaluation of Food: Statistical Methods and Procedures, Michael 0 'Mahony 1 7 . Alternative Sweetners, edited by Lyn O'Brien Nabors and Robert C. Gelardi 18. Citrus Fruits and Their Products: Analysis and Technology, S. V. Ting and Russell L. Rouseff

19. 20. 21, 22. 23. 24. 25. 26. 27. 28. 29.

30. 31. 32. 33. 34. 35.

36. 37. 38.

39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

49.

50. 51. 52. 53. 54.

Engineering Properties of Foods, edited by M. A. Rao and S. S. H. Rizvi Umami: A Basic Taste, edited by Yojiro Kawamura and Morley R. Kare Food Biotechnology, edited by Dietrich Knorr Food Texture: Instrumental and Sensory Measurement, edited by Howard R. Moskowitz Seafoods and Fish Oils in Human Health and Disease, John E. Kinsella Postharvest Physiology of Vegetables, edited by J. Weichmann Handbook of Dietary Fiber: An Applied Approach, Mark L. Dreher Food Toxicology, Parts A and 6, Jose M. Concon Modern Carbohydrate Chemistry, Roger W. Binkley Trace Minerals in Foods, edited by Kenneth T. Smith Protein Quality and the Effects of Processing, edited by R. Dixon Phillips and John W. Finley Adulteration of Fruit Juice Beverages, edited by Steven Nagy, John A. Attaway, and Martha E. Rhodes Foodborne Bacterial Pathogens, edited by Michael P, Doyle Legumes: Chemistry, Technology, and Human Nutrition, edited by Ruth H. Ma tth ews Industrialization of Indigenous Fermented Foods, edited by Keith H. Steinkraus International Food Regulation Handbook: Policy Science Law, edited by Roger D. Middlekauff and Philippe Shubik Food Additives, edited by A. Larry Branen, P. Michael Davidson, and Seppo Salminen Safety of Irradiated Foods, J, F. Diehl Omega-3 Fatty Acids in Health and Disease, edited by Robert S. Lees and Marcus Karel Food Emulsions: Second Edition, Revised and Expanded, edited by Ksre Larsson and Stig E. Friberg Seafood: Effects of Technology on Nutrition, George M, Pigott and Barbee W. Tucker Handbook of Vitamins: Second Edition, Revised and Expanded, edited by Lawrence J. Machlin Handbook of Cereal Science and Technology, Klaus J! Lorenz and Karel Kulp Food Processing Operations and Scale-Up, Kenneth ,J. Valentas, Leon Levine, and J. Peter Clark Fish Quality Control by Computer Vision, edited by L. F. Pau and R, Olafsson Volatile Compounds in Foods and Beverages, edited by Henk Maarse Instrumental Methods for Quality Assurance in Foods, edited by Daniel Y. C. Fung and Richard F. Matthews Listeria, Listeriosis, and Food Safety, Elliot T. Ryser and Elmer H. Marth Acesulfame-K, edited by D. G. Mayer and F. H. Kemper Alternative Sweeteners: Second Edition, Revised and Expanded, edited by Lyn O'Brien Nabors and Robert C. Gelardi Food Extrusion Science and Technology, edited by Jozef L. Kokini, Chi-Tang Ho, and Mukund V. Karwe Surimi Technology, edited by Tyre C. Lanier and Chong M. Lee Handbook of Food Engineering, edited by Dennis R. Heldman and Daryl B. Lund Food Analysis by HPLC, edited by Leo M. L. Nollet Fatty Acids in Foods and Their Health Implications, edited by Ching Kuang Chow Clostridium botulinum: Ecology and Control in Foods, edited by Andreas H. W. Hauschild and Karen L. Dodds

55. Cereals in Breadmaking: A Molecular Colloidal Approach, Ann- Charlotte Eliasson and Ksre Larsson 56. Low-Calorie Foods Handbook, edited by Aaron M. Altschul 57. Antimicrobials in Foods: Second Edition, Revised and Expanded, edited by P. Michael Davidson and Alfred Larry Branen 58. Lactic Acid Bacteria, edited by Seppo Salminen and Atte von Wright 59. Rice Science and Technology, edited by Wayne E. Marshall and James l. Wadsworth 60. Food Biosensor Analysis, edited by Gabriele Wagner and George G. Guilbault 61. Principles of Enzymology for the Food Sciences: Second Edition, John R, Whitaker 62. Carbohydrate Polyesters as Fat Substitutes, edited by Casimir C. Akoh and Barry G. Swanson 63. Engineering Properties of Foods: Second Edition, Revised and Expanded, edited by M. A. Rao and S. S. H. Rizvi 64. Handbook of Brewing, edited by William A. Hardwick 65, Analyzing Food for Nutrition Labeling and Hazardous Contaminants, edited by lke J. Jeon and William G. lkins 66. Ingredient Interactions: Effects on Food Quality, edited by Anilkumar G. Gaonkar -67. Food Polysaccharides and Their Applications, edited by Alistair M. Stephen 68. Safety of Irradiated Foods: Second Edition, Revised and Expanded, J. F, Diehl 69. Nutrition Labeling Handbook, edited by Ralph Shapiro 70. Handbook of Fruit Science and Technology: Production, Composition, Storage, and Processing, edited by D. K. Salunkhe and S. S. Kadam 7 1 . Food Antioxidants: Technological, Toxicological, and Health Perspectives, edited by D. L. Madhavi, S. S. Deshpande, and D. K. Salunkhe 72. Freezing Effects on Food Quality, edited by Lester E. Jeremiah 73. Handbook of Indigenous Fermented Foods: Second Edition, Revised and Expanded, edited by Keith H. Steinkraus 74. Carbohydrates in Food, edited by Ann-Charlotte Eliasson 75. Baked Goods Freshness: Technology, Evaluation, and Inhibition of Staling, edited by Ronald E. Hebeda and Henry F. Zobel 76. Food Chemistry: Third Edition, edited by Owen R. Fennema 77. Handbook of Food Analysis: Volumes 1 and 2, edited by Leo M. L. Nollet 78. Computerized Control Systems in the Food Industry, edited by Gauri S, Mittal 79. Techniques for Analyzing Food Aroma, edited by Ray Marsili 80. Food Proteins and Their Applications, edited by Srinivasan Damodaran and Alain Paraf 81. Food Emulsions: Third Edition, Revised and Expanded, edited by Stig E, Friberg and K&e Larsson 82. Nonthermal Preservation of Foods, Gustavo V. Barbosa-Canovas, Usha R. Pothakamury, Enrique Palou, and Barry G. Swanson 83. Milk and Dairy Product Technology, Edgar Spreer 84. Applied Dairy Microbiology, edited by Elmer H. Marth and James L. Steele 85. Lactic Acid Bacteria: Microbiology and Functional Aspects, Second Edition, Revised and Expanded, edited by Seppo Salminen and Atte von Wright 86. Handbook of Vegetable Science and Technology: Production, Composition, Storage, and Processing, edited by D. K. Salunkhe and S. S. Kadam 87. Polysaccharide Association Structures in Food, edited by Reginald H. Walter 88. Food Lipids: Chemistry, Nutrition, and Biotechnology, edited by Casimir C. Akoh and David B. Min 89. Spice Science and Technology, Kenji Hirasa and Mitsuo Takemasa

90. Dairy Technology: Principles of Milk Properties and Processes, P. Walstra, T. J. Geurts, A. Noomen, A. Jellema, and M. A. J. S. van Boekel 91. Coloring of Food, Drugs, and Cosmetics, Gisbert Otterstiitter 92. Listeria, Listeriosis, and Food Safety, edited by Elliot 7: Ryser and Elmer U. Marth

Additional Volumes in Preparation

Complex Carbohydrates in Foods, edited by Susan Sungsoo Cho, Leon Prosky, and Mark Dreher Handbook of Food Preservation, edited by M. Shafiur Rahman Food Safety: Science, International Regulation, and Control, edited by van der Heeden, Sanford Miller, Maged Younes, and Lawrence Fishbein

C. A.

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Listeriu, Listeriosis, and

Food Safety Second Edition, Revised and Expanded edited by

EIIiot T.

Ryser

Department of Food Science and Human Nutrition Michigan State University East Lansing, Michigan

EImer H. Marth Department of Food Science University of Wisconsin-Ma dison Madison, Wisconsin

M A R C E L

MARCEL DEKKER, INC. D E K K E R

NEWYORK BASEL

Library of Congress Cataloging-in-Publication Data

Listeria, listeriosis, and food safety / edited by Elliot T. Ryser, Elmer Marth. -- 2nd ed., rev. and expanded. p. cm. -- (Foodscienceand technology ; 92) Includes bibliographical references and index. ISBN: 0-8247-0235-2 (alk. paper) 1. Listeriosis. 2. Listeria monocytogenes. 3. Foodborne diseases. 4. Food-Microbiology. 1. Ryser, Elliot T. 11. Marth, Elmer H. 111. Series: Food science and technology (Marcel Dekker, Inc.) ; 92 QR201 .L7R9 1999 615.9’ 5 2 9 3 7 4 ~ 2 1

98-50963 CIP

This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York, NY 10016 tel: 212-696-9000; fax: 2 12-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 8 12, CH-4001 Basel, Switzerland tel: 44-6 1-26 1-8482; fax: 44-6 1-261-8896 World Wide Web http://www.dekker.com

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Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

Preface to the Second Edition

Listeriosis and Listeria monocytogenes continue to be of worldwide interest to the food industry and regulatory agencies, to scientists in various disciplines, and to consumers of food. Such interest is prompted by the occasional appearance of L. monocytogenes in ready-to-eat foods leading to the removal of such products from the marketplace. Furthermore, sporadic cases of listeriosis continue to occur and there have been several foodassociated outbreaks of the disease since the first edition of this book was published. Scientists in several disciplines have studied and 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 writing of 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 first, published in 1991. Whereas we were the sole 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. We feel that the contributions by the new authors have resulted in an improved book that has appeared in a timely fashion. Each of the chapters in the first edition has been retained, but each has been revised and expanded with new information added where approprhte. Two additional chapters not found in the first edition, dealing with typing methods and pathogenesis, have also been included. Thus, this book contains 17 chapters which address the following topics: (a) description of L. monocytogenes; (b) occurrence and behavior of this pathogen in various natural environments; (c) animal and human listeriosis; (d) pathogenesis of L. monocytogenes; (e) characteristics of L. monocytogenes that are kmportant to food processors; (f) conventional and rapid methods to isolate, detect, and identify L. monocytogenes; (g) strain-specific typing of L. monocytogenes; (h) foodborne listeriosis; (i) incidence of behavior of L. monocytogenes in unfermented and fermented dairy products, meat, poultry (including eggs), fish and seafood, and products of plant origin; and (j) incidence and control of this pathogen within various types of food-processing facilities. iii

iv

Preface to the Second Edition

This book is useful to advanced undergraduate students, graduate students, and practitioners in fooddairy microbiology, fooddairy science, bacteriology/microbiology, public health, dietetics, meat science, poultry science, and veterinary medicine. It also will be helpful to personnel in the fooddairy industry and in regulatory agencies and to researchers in industrial, governmental, and university laboratories. Elliot T.Ryser Elmer H. Marth

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 financ.ia1 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 Wiirzburg, published his time-honored book entitled Listeriosis. While his monograph has provided scientists, veterinarians, and the medical profession with much-needed information regarding Listeria and humadanimal listeriosis as well as pathological, bacteriological, and serological methods to diagnose this disease, 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 andor 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 V

wi

Preface t o the First Edition

together with the virtual flood of Listeria-related papers that have appeared in scientific journals, trade journals, and 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 which address the following topics: (a) L. monocytogenes as the causative agent of listeriosis; (b) occurrence and survival of this pathogen in various natural environments; (c) human and animal listeriosis; (d) characteristics of L. monocytogenes that are important to food processors; (e) conventional and rapid methods for isolating, detecting, and identifying L. monocytogenes in food; (f ) recognition of cases and outbreaks of foodborne listeriosis; (g) incidence and behavior of L. monocytogenes in fermented and unfennented dairy products, meat, poultry (including eggs), seafood and products of plant origin; and finally (h) incidence and control of this pathogen within various types of food-processing facilities. It is evident that major emphasis has been given to information that is directly applicable to food processors. Since information concerning the bacterium and the disease has been admirably reviewed by Professor Seeliger and others, 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. While 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 3year task of compiling an up-to-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 writing of the book was completed. We acknowledge with gratitude the many investigators whose findings made this book both necessary and possible. Special thanks go to those 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 those scientists who provided photographs or drawings; each person is acknowledged where 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 go to Dr. Ralston B. Read, Jr., formerly 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 WisconsinMadison 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 U.S.; 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

Preface to the First Edition

vii

University of Wisconsin. We thank all of these agencies for their interest in and support of research on L. rnonocytogenes. 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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Contents

Preface to the Second Edition Preface to the First Edition Contributors 1. The Genus Listeria and Listeria monocytogenes: Phylogenetic Position, Taxonomy, and Identification Jocelyne Rocourt

iii V

xi

1

2. Listeria monocytogenes in the Natural Environment David R. Fenlon

21

3. Listeriosis in Animals Irene V. Wesley

39

4.

Listeriosis in Humans Laurence Slutsker and Anne Schuchat

5. Pathogenesis of Listeria monocytogenes Michuel Kuhn and Werner Goebel

75 97

6. Characteristics of Listeria monocytogenes Important to Food Processors Yuqian Lou and Ahmed E. Yousef

131

7. Conventional Methods to Detect and Isolate Listeria monocytogenes Catherine W. Donnelly

225

8. Rapid Methods for Detection of Listeria Car1 A. Batt

26 1

9. Subtyping Listeria rnonocytogenes Lewis M. Graves, Bala Swaminathan, and Susan B. Hunter

279

ix

Contents

X

10. Foodborne Listeriosis Elliot T. Ryser

299

11. Incidence and Behavior of Listeria monocytogenes in Unfermented Dairy Products Elliot T. Ryser

359

12. Incidence and Behavior of Listeria monocytogenes in Cheese and Other Fermented Dairy Products Elliot T. Ryser

41 1

13. Incidence and Behavior of Listeria monocytogenes in Meat Products Jeflrey M. Farber and Pearl I. Peterkin

505

14. Incidence and Behavior of Listeria monocytogenes in Poultry and Egg Products Nelson A. Cox, J. Stan Bailey, and Elliot T. Ryser

565

15. Incidence and Behavior of Listeria monocytogenes in Fish and Seafood Products Karen C. Jinneman, Marleen M. Wekell, and Me1 W. Eklund

60 1

16. Incidence and Behavior of Listeria monocytogenes in Products of Plant Origin Robert E. Brackett

63 1

17. Incidence and Control of Listeria in Food-Processing Facilities Robert Gravani

657

Appendix

711

Index

719

Contributors

J. Stan Bailey Russell Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, Georgia

Car1 A. Batt Department of Food Science, Cornell University, Ithaca, New York Robert E. Brackett Center for Food Safety and Quality Enhancement, The University of Georgia, Griffin, Georgia Nelson A. Cox Russell Research Center, Agricultural Research Service, U.S. ,Department of Agriculture, Athens, Georgia Catherine W. Donnelly University of Vermont, Burlington, Vermont Me1 W. Eklund" U.S. National Marine Fisheries Service, Northwest Fisheries Science Center, Seattle, Washington Jeffrey M. Farber Bureau of Microbial Hazards, Food Directorate, Health Canada, Ottawa, Ontario, Canada David R. Fenlon Animal Biology Division, Scottish Agricultural College, Aberdeen, Scotland Werner Goebel Department of Biology, University of Wiirzburg, Wurzburg, Germany Robert Gravani Department of Food Science, Cornell University, Ithaca, New York Lewis M. Graves Foodborne Diseases Laboratory Section, Centers for Disease Control and Prevention, Atlanta, Georgia Susan B. Hunter Foodborne Diseases Laboratory Section, Centers for Disease Control and Prevention, Atlanta, Georgia

* Retired. xi

xii

Contributors

Karen C. Jinneman Seafood Products Research Center, U.S. Food and Drug Administration, Bothell, Washington Michael Kuhn Department of Biology, University of Wurzburg, Wurzburg, Gerrnany Yuqian Lou Bil Mar Foods, Inc., Zeeland, Michigan Pearl I. Peterkin Bureau of Microbial Hazards, Food Directorate, Health Canada, Ottawa, Ontario, Canada Jocelyne Rocourt Listeria Laboratory, Institut Pasteur, Paris, France Elliot T. Ryser Department of Food Science and Human Nutrition, Michigan State University, East Laming, Michigan Anne Schuchat Respiratory Diseases Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia Laurence Slutsker Foodborne and Diarrheal Diseases Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia Bala Swaminathan Centers for Disease Control and Prevention, Atlanta, Georgia Marleen M. Wekell Seafood Products Research Center, U.S. Food and Drug Administration, Bothell, Washington Irene V. Wesley National Animal Disease Center, U.S. Department of Agriculture, Ames, Iowa Ahmed E. Yousef Department of Food Science and Technology and Department of Microbiology, The Ohio State University, Columbus, Ohio

The Genus Listeria and Listeria monocytogenes: Bhylogenetic Position, Taxonomy, and Identification JOCELYNE ROCOURT lnstitut Pasteur, Paris, France

HISTORY The first published description of Listeria monocytogenes, which rapidly became the reference, was written by Murray et al. in 1926 [ ZOO]. A few earlier.reports may have described Listeria isolation [53,141], the most plausible of which is certainly that by Hulphers [64]. 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 et al. [loo] observed six cases of rather sudden death of young rabbits in 1924 in the animal breeding establishment of the Department of Pathology of Cambridge University, and many more such cases occurred in the succeeding 15 months. The interesting characteristics presented by the disease and the increasing mortality prompted an investigation. The authors wrote at that time [loo]: 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

Rocount

2

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 ’sManual ofDeterminative 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 cooperation 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 [ 1 161 discovered a new microorganism, agent of what he called the Tiger River disease. He named this new agent “Listerella hepatolytica”’ for the following reasons: The causative organism is a Gram-positive 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

Both discoverers, Murray and Pirie, 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. As the identity was clear, they decided to call this bacterium “Listerella monocytogenes” [99,117]. However, in 1939, the Judicial Commission of the International Committee on Systematic Bacteriology rejected the generic name “Listerella” because it had been previously used for a mycetozoan (a slime mold) in 1906 in honor of Arthur Lister (young brother of Lord Lister) and for a species of foraminifer (a marine protozoan) in 1933 in honor of Joseph Jackson Lister (father of Lord Lister). As noted by Gibbons in 1972 [48], it is certainly unique that the same name was chosen for three quite different groups of microorganisms to honor the contributions of a father and his two sons. The next year, in 1940, Pirie proposed the name Listeria [ 1171. Before, and even after this date, numerous names were used to designate L. monocytogenes: “Bacterium rnonocytogenes hominis’ ’ and later “Listerella hominis’ ’ by Nyfeldt, who considered that it was the agent of infectious mononucleosis [ 104,1051; “Corynebacterium pawulum” by Schultz et al. in 1934 [ 1401; “Listerella ovis” by Gill in 1937 [49]; “Listerella bovina,” “L. gallinaria,” “L. cunniculi,” and “L. gerbilli” by Nyfeldt [105,106]; “Erysipelothrix monocytogenes” by Wilson and Miles in 1946 [ 1731; and “Corynebacterium infantisepticurn” by Potel in 1951 during his first observations of fetal and neonatal listeriosis in Germany [ 1191. Unlike some pathogenic agents responsible for large outbreaks which have marked the history of humans for centuries, for example, Vibrio cholerae or Yersinia pestis, the history of L. monocytogenes and listeriosis is recent: It began officially in 1924. The first confirmed diagnosis in a human was that of a soldier suffering from meningitis at the end

’ Names of species within quotes are no longer valid. Were “Listerella,” and later Listeria, named in honor of Lord (Joseph) Lister, the father of antiseptic surgery, or in honor of Sir Frederick Spencer Lister, Director of the South African Institute of Medical Research from 1926 to 1939? Gibbons, in 1972, tried to elucidate this nomenclatural point and came to the conclusion, together with other authors, that Pirie chose Listerella to honor Lord Joseph Lister [48,99,142].

The Genus Listeria and Listeria monocytogenes

3

of World War I (retrospective identification of the strain [24]), and before this case, there are no validated observations. Interestingly, however, a historian has suggested that L. monocytogenes could have been the cause of Queen Am’s 17 unsuccessful pregnancies ( 17th century) [ 1371.

PHYLOGENETIC POSITION OF THE GENUS LISTERIA The relationship of Listeria to other bacteria remained obscure until the 1970s. Absent from the first three editions of Bergey ’sManual 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, whereas in the next edition (1974), Listeria was considered as a genus of uncertain affiliation and was placed with Erysipelothrix and Caryophanon after the family of Lactobacillaceae [3-61. 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), Listerin has long been associated with the coryneform group of bacteria. However, with the successive introduction and development of numerical taxonomy, chemotaxonomy, DNA/I)NA hybridation, and more recently rRNA (ribosomal RNA) sequencing, the phylogenetic position of Listeria has been far more clearly defined.

Numerical Taxonomy With development of computers for handling large amounts of data, 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 either with the corynebacteria [13,28] or in an indefinite position [16,62]. In contrast, since 1969, more natural relationships were described when Listeria was compared with various representatives of lactic acid bacteria [29,159,160]. The close relatedness with these microorganisms was clearly demonstrated in 1975 by the broader numerical taxonomic survey of Jones, who studied 173 characteristics of 233 strains of various genera, including both coryneform and lactic acid bacteria 1701. The refined position of Listeria was later investigated by Wilkinson and Jones in 1977 and Feresu and Jones in 1988 137, 1721. From these works, it became clear that Listeria is distinct from other known genera, including Erysipelothrix and Brochothrix thermosphacta (formerly Microbacterium ,thermosphactum), and that it is closely related to Lactobacillus and Streptococcus. Consequently, Wilkinson and Jones [ 1721 suggested that Listeria, Gemella, Brochothrix, Streptococcus, and Lactobacillus be classified in the family Lactobacillaceae. Despite some imprecision concerning the exact position of higher taxonomic relationships, especially of Brochothrix, certain lactobacilli, and Carnobacterium [37,172], conclusions based on numerical analysis of data for large numbers of phenotypic features were the precursors of the current phylogenetic classification of the genus Listeria.

4

Rocourt

Chemotaxonomy Several 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 as evidenced by numerical taxonomic studies. The G+C % DNA content of L. monocytogenes isolates ranges from 36 to 42% [37,125,160], indicating that Listeria belongs to the low G+C % DNA content (<55%) group of grampositive bacteria. Lipoteichoic acids (amphilic polymers of the cytoplasmic membranes) have been isolated from L. monocytogenes [59,135,164]. These acids consist of hydrophilic polyglycerophosphate 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 so may be used as a taxonomic marker. For Listeria, the presence of particular lipoteichoic acids provides further evidence that the genus is a biochemically coherent taxon [ 1351. Furthermore, lipoteichoic acids are absent from coryneform bacteria but are found in Bacillus, Staphylococcus, Streptococcus, and Lactobacillus, indicating that Listeria should be grouped with these latter microorganisms [ 1351. With the exception of one report [94], free mycolic acids, which are specific for high G+C % DNA content gram-positive bacteria, have not been detected in Listeria [37,73]. The presence of respiratory menaquinones (seven isoprene units), of predominantly methyl-branched cellular fatty acids and meso-DAP as the major peptidoglycan diamino acid support the close relatedness of Listeria and Brochothrix and the greater distance from lactobacilli [2 1,22,37,40,41,12I]. Analysis of low molecular weight RNA profiles also supports the independent identity of L. monocytogenes among other gram-positive taxa [ 1571.

rRNA Sequencing Recent 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, Ludwig et al. [9 11 unambiguously demonstrated, using the 16s rRNA cataloguing approach (partial sequencing), that Listeria forms with Brochothrix thermosphacta one of several sublines within the “Clostridium subdivision.” They did not detect any relationship with coryneform bacteria (except for universal and highly conserved 16s rRNA sequences that are common 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-Carnobacteriumsublines [22]. On the basis of these data and chemotaxonomic properties, Collins et al. 1221 considered that (a) Listeria is phylogenetically remote from Lactobacillus and should not be included in the family Lactobacillaceae and (b) the Listeria-Brochothrix subline probably merits a separate family, the Listeriaceae. This great distance between Lactobacillus and Listeria has been recently confirmed by sequencing 23s rRNA, with Listeria to be most similar to Bacillus and Staphylococcus [ 1361.

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.

The Genus Listeria and Listeria monocytogenes

5

It is not a coryneform bacterium as evidenced by numerical phenetic studies, chemotaxonomic properties (low G+C % DNA content, lack of mycolic acids, and presence of lipoteichoic acid) and various rRNA sequencing analyses. However, the exact phylogenetic position of this genus remains controversial. Although it is generally agreed that Listeria’s nearest neighbor is Brochothrix, its relationship to other low G+C % DNA content gram-positive bacteria, especially Luctobacillus, needs further clarification.

TAXONOMY OF THE GENUS LlSTERIA For many years, the genus Listeria was monospecific containing only the type species, L. monocytogenes. L. denitriJcicans(because of its ability to reduce nitrates) was added in 1948 [158], 1,. grayi (in honor of M.L. Gray, an American microbiologist) in 1966 [89], L. murrayi (in honor of E.G.D. Murray, a Canadian microbiologist) in 1971 [171], L. innocua (because of its innocuousness or harmlessness) in 1981 [143], L. ivanovii (in honor of I. Ivanov, a Bulgarian microbiologist) in 1985 [147], L. welshimeri (in honor of H.J. Welshimer, an American microbiologist) in 1983, and L. seeligeri (in honor of H.P.R. Seeliger, a German microbiologist) in 1983 [ 1251. As for the phylogenetic analysis of Listeria, introduction of molecular biology methods allowed a better appreciation of the diversity within the genus Listeria which now contains only six species: L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seeligeri, and L. grayi.

L. monocytogenes, L. ivanovii, L. welshimeri, and seeligeri

L.

Among methods used to compare strains, serotyping was the first and crucial approach to elucidate the infrageneric structure of the genus Listeria. Until 1960, Listeria was nearly exclusively isolated from pathological samples, thus isolation of virtually no species but L. monocytogenes. The first antigenic scheme was devised ’by Paterson [ 1 10,111], who described the first four serovars. This scheme was later exl~endedby Donker-Voet and Seeliger with the addition of new serovars [32,145]. In 1962, Ivanov observed atypical L. rnonocytogenes strains isolated from sheep abortions 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) [66,67]. Years later, with development of selective media, many additional strains were isolated from various environmental sources. Seeliger collected and serotyped hundreds of strains between 1965 and 1980 and observed that many 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 these strains L. innocua in 1981 [ 1431. Thus, simple phenotypic methods, serotyping and hemolysis, led to the demonstration that the species .L. monocytogenes, as defined in the eighth edition of the Bergey’s Manual of Determinative Bacteriology [6], was heterogeneous, covering a number of different species. Early DNA/DNA hybridizations (filter study), by Stuart and Welshimer in 1974, showed L. monocytogenes to be heterogeneous [161]. However, the number of DNA hybridization groups in their collection of strains could not be ascertained, as only one DNA from L. monocytogenes was labeled; moreover, serovars were not indicated. Further DNA/ DNA hybridization studies (S 1 method), aimed at resolving the genomic heterogeneity of the so-called L. monocytogenes and evaluating the validity of the new species L. bulgar-

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6

ica and L. innocua (not officially validated at that time), were undertaken in 1982 with many strains of various origins [ 1291. 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 consisted of strongly hemolytic strains, all belonging to serovar 5, confirming Ivanov’s proposal that these strains, for which he suggested the name “L. bulgarica,” deserved species rank [66]; they were named L. ivanovii in 1985 [147]. Further investigations of strains of this species using multilocus enzyme electrophoresis, DNA/DNA hybridizations, and rRNA gene restriction patterns resolved to describe two subspecies, L. ivanovii subsp. ivanovii (ribose positive) and L. ivanovii subsp. londoniensis (ribose negative) [ 101. Genomic group 3 contained strains of serovars 4ab, 6a, 6b, and undesignated serovars that were nonhemolytic and nonpathogenic for mice, including the two strains that Seeliger previously proposed as reference strains for L. innocua [ 1431. This group of strains corresponded to L. innocua, with this species being officially validated in 1983 [165]. Genomic group 4 contained nonhemolytic strains of serovar 6a and 6b. These strains produced acid from D-xylose and were nonpathogenic for mice [127,128], and therefore corresponded to a group of strains previously described by Groves and Welshimer in 1977 [56].The group was given species status and called L. welshimeri in 1983 [125]. Genomic group 5, an unexpected group, included hemolytic and nonpathogenic strains of various serovars (1/2c, 4c, 4d, 6b, and undesignated serovars) [ 127,1281 and was subsequently named L. seeligeri [ 1251. DNA/DNA homology experiments (optical method) in 1993 supported these results [58]. 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 which differ by few characteristics, with these studies also being of little help in resolving the heterogeneity [37,72]. Interestingly, this new genomic classification was tested using multilocus enzyme electrophoresis ( 18 enzyme loci analyzed). Matrix cluster analysis of the genetic distances between paired electrophoretic types revealed that L. monocytogenes, L. ivanovii, L. welshimeri, and L. seeligeri each corresponded to a single cluster with no overlaps between them [lO].

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 (a) a low DNA relatedness between L. monocytogenes and L. grayi and L. murrayi and (b) a high genomic homology between L. grayi and L. murrayi; these data were supported by numerical phenetic analyses [ 160,1611. The authors proposed to transfer L. grayi and L. murrayi to a new genus, “Murraya” with “M. grayi” as the type species, divided into two subspecies “M.grayi” subsp. “grayi” and “M. grayi” subsp. “murrayi.” Two questions arose from this proposal which was not officially validated in the Approved

The Genus Listeria and Listeria monocytogenes

7

Lists of Bacterial Names: (a) do L. grayi and L. murrayi belong to the genus Listeria?; (b) do L. grayi and L. murrayi belong to a single species? L. grayi, L. murrayi, and L. monocytogenes share several similarities: They cluster in all numerical taxonomic studies [70,159,16 I , 1721, possess lipoteichoic acid [ 1351, teichoic acid of the polyribitol phosphate type [41], peptidoglycan of the A1 gamma variation [40], nonhydrogenated menaquinones of the MK-7 type [21,37], and the same cytochromes (albdo) [37]. However, other features support the distinctness of the L. grayi and L. murrayi pair within the genus Listeria: A slight difference in G+C % DNA content (42 vs 36-38) 137,1611, several biochemical reactions [146], the nature of substitution of lipoteichoic acids [ 1351, slight differences in protein electrophoregrams [88], cellular fatty acid composition [ 1021, antigenic structure [ 1461, and low DNA homology values [ 1601. Finally, the 16s rRNA oligonucleotide cataloging 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 [ 1301. The close relationship between L. grayi and L. murrayi was evidenced by various investigations: These two species comprise a single distinct cluster in numerical taxonomic analysis [37,172], in multilocus enzyme electrophoresis analysis [ 101, and in DNA/DNA hybridization studies [ 1601. In addition, they share a number of chemotaxonomic properties which distinguish them from the other Listeria species: same DNA base composition values [37,160], same substitution of lipoteichoic acids [ 1351, same cellular fatty acid and fatty aldehyde patterns [75], and a common antigenic structure despite small differences [145,168]. They have been distinguished from each other only on the basis of nitrate reduction [171]. Finally, recent 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 to be members of a single species, L. grayi [132]. These data are consistent with 16s and 23s rRNA sequencing and cellular protein electrophoretic pattern data [22,78,136].

L, denitrificans/Jonesia denitrificans Although only a single isolate is currently known for this species, there have been an amazing number of papers dealing with its taxonomic position. As early as 1966, a numerical taxonomic study showed that L. denitrificans clustered with certain coryneform bacteria, and this was later confirmed [ 16,70,159,160]. Results of chemotaxonomic studies and DNA/DNA hybridization further emphasized the phenotypic differences between L. denitrijicans and other members of the genus Listeria [20,21,40,41,135,161]. In 1987, 16s rRNA cataloging confirmed that this species is not a member of the genus Listeria and belongs to the coryneform group of bacteria [ 13I]. Consequently, this species was transferred to the newly formed genus, Jonesia, and is now officially recognized as Jonesia denitrijicans .

Present State of the Taxonomy of the Genus Listeria 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 sequencing homology, chemotaxonomic properties, and multilocus enzyme analysis. Based on DNA/DNA hybridization, 16s rRNA cataloging and reverse transcriptase sequencing of 16s and 23s rRNA, the genus embraces two closely related but

8

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obviously distinct lines of descent: One contains L. grayi and the other L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, and L. seeligeri. The species within this line can be divided into two groups, (a) L. monocytogenes and L. innocua and (b) L. ivanovii, L. seeligeri, and L. welshimeri [22,129,130,136].

IDENTIFICATION OF BACTERIA OF THE GENUS LISTERIA One of the most practical goals of bacterial taxonomy is generation of information for construction of reliable identification schemes so that newly isolated bacteria can be identified and their role in a particular environment can be assessed.

Genus Characteristics Morphology Listeria is a small (0.5 pm in diameter and 1-2 pm in length), regular gram-positive rod with rounded ends. Cells are found singly, or in short chains, or may be arranged in V and Y forms or in palisades. Sometimes cells are coccoid, averaging about 0.5 pm 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 Haernophilus. Long, thin, filamentous cells appear in old and rough cultures and also after osmotic shock [57,74,84]. Listeria does not produce spores and capsules are not formed [144]. Listeria is motile because of its few peritrichous flagella when cultured at 20-25°C (not or very weakly motile at 37°C) [45]. Hanging-drop preparations of fresh cultures in tryptose phosphate broth incubated at 20°C show characteristic tumbling motility: cells start with twisting and wriggling movements which increase to fast, eccentric rotations before they suddenly move quickly in various directions. Stab cultures in semisolid motility medium produce a typical picture of “umbrella” or inverted ‘‘pine tree” growth about one half centimeter below the surface because of the microaerophilic nature of the organism. A recent report indicates that L. rnonocytogenes 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 [8 11.

Cu Iture On nutrient agar, colonies are 0.2-0.8 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 5-10 days, well-separated colonies may be 5 mm or more in diameter. When cultures of Listeria grown for 18-24 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 [52,85,97]. Even when the population of contaminants is rather high and that of Listeria low, Listeria can still be recognized because of this characteristic [52,90], Rough colonies may occasionally be observed [54]. Conversion of smooth colonies to rough colonies is not reversible [141]. Differences in virulence between rough and smooth colonies have been observed [61,84,87]. Petite colony formation by strains grown on esculin-containing agar has been described [ 1531. 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 which may be caused by

The Genus Listeria and Listeria monocytogenes

9

formation of carboxylic acids, hydroxy acids, and alcohols [27]. In broth, the medium becomes turbid after 8-24 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 of air [141]. The normal temperature limits for growth are + 1-2°C to 45°C [76,144]; however, some multiplication has been reported to occur in chicken broth and pasteurized milk during extended incubation at -0.1 to 0.4"C [ 1691. Growth is slow at refrigeration temperatures, with generation times of 30-40 h at +4"C in skim milk, for example [134]. This property was first used by Gray [55] for selective cold enrichment of a contaminated sample. In broth, Listeria normally grows from pH 4.4-9.6, optimally at pH 7 [ 15,47,107,112]. Growth can occur in media containing 10% (w/v) NaCl with survival at higher concentrations [ 146,1491. Survival at low pH and high salt concentration is strongly temperature-dependent [ 191. Listeria is one of the few foodborne pathogens that can grow at an a, value below 0.93 [35,112].

Nutritional Requirements According to published data, growth factors for Listeria include cystine, leucine, isoleucine, arginine, methionine, valine, cysteine, riboflavin, biotin, thiamine, and thioctic acid [ 120,151,1701. Growth is stimulated by Fe3 and phenylalanine [ 120,15I]. In some experiments, virulent strains grew faster in the presence of iron than did avirulent strains 1251. Glucose and glutamine are required as primary sources of carbon and nitrogen [ 146,1201. Chemically defined media have been described for Listeria [ 120,122,1501. +

Metabolism and Biochemical Characters Listeria is aerobic, microaerophilic, facultatively anaerobic, catalase-positive (rare catalase-negative strains have been observed) and oxidase-neg,ative. Although Feresu and Jones [37] found cytochrome a,bdo, the presence of cytochrome is controversial [ 109,1631. Listeria is homofermentative and oxidizes glycolytic intermediate compounds [27]. It possesses glucose oxidase and NADH oxidase activities [ 1091. All strains grow on glucose forming lactate, acetate, and acetoin as main endproducts under aerobic conditions [27,113,133]. Acetoin is not produced under anaerobic conditions. Anaerobically, only hexoses and pentoses support growth; aerobically, maltose and lactose support growth of some strains, but sucrose does not [ 1 131. Catabolism of glucose proceeds by the EmbdenMeyerhof pathway both aerobically and anaerobically [ 1461. L. monocytogenes imports glucose by a high-affinity phosphoenolpyruvate-dependentphosphotransferase system and a low-affinity proton motive force-mediated system [ 17,1081. 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. Phenylalanine-deaminase,ornithine, 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 can be found in references 37, 79, 126, 146, 148, and 172.

Species Identification All Listeria species are phenotypically very similar, but they can be distinguished by the following tests: hemolysis, acid production from D-xylose, L-rhamnose, alpha-methy1-D-

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FIGURE1 Identification o f

Listeria species.

mannoside, and mannitol [ 1281 (Fig. 1). The phenotypic similarities are consistent with the high genomic homologies between the different species [22,129,136]. Hemolysis is a key characteristic for speciation of isolates and clearly is the most difficult characteristic to detect. During a collaborative study on Listeria identification, Higgins and Robison [60] noted that a large percentage of errors in identification of L. seeligeri and L. ivanovii was caused by inaccurate reading of the CAMP3-testand 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 on certain media [ 118,1561. L. monocytogenes hemolyzes blood from sheep, horses, cows, guinea pigs, piglets, and humans [ 138,144,154,166l.Various methods have been developed to determine hemolytic activity, especially for weakly hemolytic strains (L. seeligeri and some L. monocytogenes isolates) and include examination of hemolysis underneath the colony, prolonged incubation (48 h), incubation for several hours at +4"C, use of thin layer blood agar plates [86], several media 1441, tube tests and microplate techniques with erythrocyte suspensions [30,31,1621, addition of an exosubstance from Rhodococcus equi, Staphylococcus aureus, or L. ivanovii to the blood agar [95,154,155],and the CAMP-test with S. aureus and R. equi [ 14,42,56,65,66,97,101,128]. 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 considering L. monocytogenes to be positive [39,139,167] and others negative [128,146]. 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. rnonocy-

The original CAMP-test was described by Christie, Atkins, and Munch-Peterson, who observed this lytic phenomenon for Srreprococcus in 1944, and the test is named after these authors [ 141.

The Genus Listeria and Listeria monocytogenes

11

togenes, the shape is that of an onion. This could reflect either different abilities of R. equi strains to interact with L. monocytogenes because of different amounts of listeriolysin 0 secreted by L. monocytogenes strains or variations in the capability of R. equi strains to secrete cholesterol oxidase [38,167]. However, whether or not it is positive, this test is not essential, as 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. uureus and R. equi is listeriolysin 0 [ 1231. The exosubstances of S. aureus and R. equi are a sphingomyelinase C and a cholesterol oxidase, respectively [38,96,123]. Hemolysin is a major virulence factor of L. monocytogenes. Three species, L. monocytogenes, L. ivanovii, and L. seeligeri, are hemolytic and possess the virulence gene cluster as recently demonstrated [50]; however, only two species, L. monocytogenes and L. ivanovii, are naturally and experimentally pathogenic [93,127], L. ivanovii being 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, with the best known example being the type strain of this species [6 1,71,801. Dissociation between hemolytic and nonhemolytic colonies is rarely observed [ 1 151. Nonhemolytic and several weakly hemolytic strains are non- or weakly pathogenic [ 12,23,30,36,61,114,1621.Despite these atypical strains, routine pathogenicity testing for L. monocytogenes is generally unnecessary [90]. Additional tests, especially to distinguish L. monocytogenes from L. iiznocua, have been proposed and include detection of phospolipase C activity [ 18,1031, hydrolysis of D-alanine-p-nitroanilide[79], and hydrolysis of a naphthylamide substrate (API Listeria [81)* Various commercial miniaturized culture or enzyme multitest assays are now used for Listeria identification, since conventional culture procedures for identification are tedious and time consuming. They include API 50 CH [83,126], API-ZYM [127], API 20 STREP [92], API Listeria [8,9,44], API Coryne [82], Micro-ID Listeria [2,8,60,124], Mast ID [83], RAPID CORYNE [46], RAPID ID 32 Strep [43], and a microtiter plate method [ 1521. 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 both genus and species identification. Phenotypic markers are used for routine identification of Listeria isolates. More sophisticated methods have been described and some can help speciate atypical isolates. These methods include 16s rRNA sequencing [26], sequence analysis of the 16s-23s internal transcribed spacer loci [33,5 11, ribotyping 1681, random amplification of polymorphic DNA [34], repetitive element sequence-based PCR 1691, multilocus enzyme electrophoresis [ 10I, cellular protein electrophoretic pattern analysis [78], enzymatic profiling using fluorogenic substrates [77], analysis of fermentation products by frequency-pulsed electon-capture gas-liquid chromatography [27], Fourier transform infrared spectroscopy analysis [63], and thermogram determination [ 11.

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. These data were confirmed by 16s rRNA cataloging. The refined location of Listeria within the low C + C % DNA

12

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content gram-positive bacteria was later determined by reverse transcriptase 16s and 23s sequencing data. Numerical taxonomic studies revealed a certain heterogeneity within this genus with the exact species content determined by DNA/DNA hybridization and rRNA sequencing. Based on this genomic dissection, the genus contains six species which are divided into two sublines of descent. The present state of Listeria taxonomy is the result of more than 20 years of work done in various laboratories in different countries, using as many methods as imaginable. Most of this work was done during the last two decades, and the number of publications in this field is now decreasing. Fortunately, the distinction between L. rnonocytogenes and nonpathogenic species was already defined when foodborne transmission of listeriosis became a public health problem with a major economic impact on the food industry. This allowed efforts to be restricted to food contaminated with L. rnonocytogenes, since food contaminated by other Listeria species is of no public health concern. Now, both introduction of molecular biology methods and the need to develop new tools to understand listeriosis epidemiology are generating renewed interest in classification of L. rnonocytogenes strains (see Chap. 9).

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. 1992. Evaluation of the Organon-Teknika MICRO-ID Listeria System. Appl. Environ. Microbiol. 58:2011-2015. 3. Bergey’s Manual of Determinative Bacteriology. 1934. 4th edition (Bergey, D.H., ed.). Williams & Wilkins c o, Baltimore. 4. Bergey’s Manual of Determinative Bacteriology. 1948.6th ed. (Breed, R.D., Murray, E.G.D., and Hitchens, A.P., eds.). Williams & Wilkins Co., Baltimore. 5. Bergey’s Manual of Determinative Bacteriology. 1957.7th ed. (Breed, R.D., Murray, E.G.D., and Smith, N.R., eds.). Williams & Wilkins Co., Baltimore. 6. Bergey’s Manual of Determinative Bacteriology. 1974. 8th ed. (Buchanan, R.E., and Gibbons, N.E., eds.). Williams & Wilkins Co., Baltimore. 7. Bergey’s Manual of Systematic Bacteriology, vol. 2, 1986. (Sneath, P.H.A., Mair, N.S., Sharpe, N.E., and Holt, J.G., eds.). Williams & Wilkins Co., Baltimore. 8. Beumer, R.R., M.C.T. Giffel, M.T.C. Kok, and F.M. Rombouts. 1996. Confirmation and identification of Listeria spp. Lett. Appl. Microbiol. 22:448-452. 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, and J.C. Piffaretti. 1991. Taxonomy of the genus Listeria by using multilocus enzyme electrophoresis. Int. J. System. Bacteriol. 41 :59-64. 11. Boerlin, P., J. Rocourt, F. Grimont, P.A.D. Grimont, Ch. Jacquet, and J.C. Piffaretti. 1992. Listeria ivanovii subsp. Londoniensis. Int. J. System. Bacteriol. 1542-46. 12. Bosgiraud, C., A. Menudier, M.J. Cornuejols, N. Hangard-Vidaud, and J.A. Nicolas. 1989. Etude de la virulence de Listeriu monocytogenes isolies d’aliments de I’homme. Microbiol. Alim. Nut. 7:4 13-420. 13. Bousfield, I. 1972. A taxonomic study of some coryneform bacteria. J. Gen. Microbiol. 71: 441-455. 14. Brzin, B., and H.P.R. Seeliger, 1975. A brief note on the CAMP phenomenon in Listeria. In: M. Woodbine, ed. Problems of Listeriosis. Leicester, UK: University of Leicester. pp. 34-37. 15. Buchanan, R.L., and L.A. Klawitter, 1990. Effects of temperature and oxygen on the growth of Listeria monocytogenes at pH 4.5. J. Food Sci. 55:1754-1756.

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species. In: T. Bergan, and J . Norris, (eds.), Methods in Microbiology New York: Academic Press, pp 33-48. Seeliger, H.P.R., and D. Jones. 1986. Genus Listeria Pirie 1940. In: P.H.A. Sneath, N.S. Mail, N.E. Sharpe, and J. G. Holt (eds.), Bergey’s Manual of Systematic Bacteriology, Vol. 2. Baltimore: Williams & Wilkins. pp. 788-795. Seeliger, H.P.R., J. Rocourt, A. Schrettenbrunner, P.A.D. Grimont, and D. Jones. 1984. Listeria ivanovii sp. nov. Int. J. Syst. Bacteriol. 34:336-337. Seiler, H., and M. Busse. 1989. Biochemische Differenzierung von Listerien aus Kase. Berl. Munch. tierarztl. Wschr. 102:166- 170. Shahamat, M., A. Seaman, and M. Woodbine. 1980. Survival of Listeria monocytogenes in high salt concentrations. Zbl. Bakteriol. Hyg., I. Abt. Orig. A. 246:506-5 1 1. Siddiqi, K.,and M.A. Khan. 1982. Vitamin and nitrogen base requirements for Listeria nzonocytogenes and haemolysin production. Zbl. Bakteriol. Hyg. I. Abt. Orig. A. 253:225-235. Siddiqi, K.,and M.A. Khan. 1989. Amino acid requirement of six strains of Listeria monocytogenes. Zbl. Bakteriol. 27 1 : 146-1 52. Siragusa, G.R., and J.W. Nielsen. 1991. A modified microtiter plate for biochemical characterization of Listeriu spp. J . Food Prot. 54: I2 I - 125. 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. Skalka, 13., and J. Smola. 1982. Hemolytic properties of exosubstance of serovar 5 Listeriu monocytogenes compared with beta toxin of Stuphylococcus aureus. Zbl. Bakteriol. Hyg., 1. Abt. Orig. A. 252:17-25. Shalka, B., J. Smola, and K. Elischerova. 1982. Routine test for in vitro differentiation of pathogenic and apathogenic Listeria monocytogenes strains. J. Clin. Microbiol. 15503-507. Skalka, 13., J. Smola, and K. Elischerova. 1983. Hemolytic phenomenon under the cultivation of Listeria innocua. Zbl. Bakteriol. Hyg., I. Abt. Orig. A. 253:559-565. Slade, P.J., and D.L. Collins-Thompson. 1991. Differentiation of the genus Listeriu from other Gram-positive species based on low molecular weight (L,MW) RNA profiles. J. Appl. Bacteriol . 70:355 -360. Sohier, I<., F. Benazet, and M. Pikhaud. 1948. Sur un germe du genre Listeriu apparemment non pathogkne. Ann. Inst. Pasteur 7454-57, Stuart, M.R., and P.E. Pease. 1972. A numerical study on the relationships of Listeria and Erysipeiothrix. J. Gen. Microbiol. 7355 1-565. Stuart, S.E., and H.J. Welshimer. 1973. Intrageneric relatedness of Listeria Pirie. Int. J. Syst. Bacteriol. 23:8- 14. Stuart, S.E., and H.J. Welshimer. 1974. Taxonomic reexamination of Listeria Pirie and tranfer of Listeria grayi and Listeria murruyi to a new genus Murruya. Int. J. Syst. Bacteriol. 24: 177-185. Tabouret, M., J. Derycke, A. Audurier, and B. Poutrel. I99 1. Pathogenicity of Listeria monocytogenes isolates in immunocompromised mice in relation to listeriolysin production. J. Med. Microbiol. 34: 13- 18. Trivett, T.L., and E.A. Meyer. 1967. Effect of erythritol on the in vitro growth and respiration of Listrria monocytogenes. J. Bacteriol. 93: 1 197- I 198. Uchikawa, K.-I., I. Sekikawa, and I. Azuma. 1986. Structural studies on lipoteichoic acids from four Listeria strains. J. Bacteriol. 168:1 15-122. Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 10. 1983. Int. J. Syst. Bacteriol. 33:438-440. Van der Kelen, D., and J.A. Lindsay. 1990. Differential hemolytic response of Listeria monocytogerres strains on various blood agars. J. Food Safety I 1 3 - 12. Vazquez-Boland, J.A., L. Dominguez, J. F. Fernandez-Garayzabal, and G. Suarez. 1992. Listeritr monocytogenes CAMP reaction. Clin. Microbiol. Rev. 5:343.

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168. 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. 169. Walker, S.J., P. Archer, and J.G. Banks. 1990. Growth of Listeria monocytogenes at refrigeration temperatures. J. Appl. Bacteriol. 68: 157- 162. 170. Welshimer, H.J. 1963. Vitamin requirements of Listeria monocytogenes. J. Bacteriol. 85: 1156-1 159. 171. Welshimer, H.J., and A.L. Meredith. 1971. Listeria murrayi: a nitrate-reducing mannitolfermenting Listeria. Int. J. Syst. Bacteriol. 21 :3-7. 172, Wilkinson, B.J., and D. Jones. 1975. Some serological studies on Listeria and possibly related bacteria. In: M. Woodbine (ed.), Problems of Listeriosis. Leicester, UK: University of Leicester. pp. 251-261. 173. Wilson, G.S., and A.A. Miles. 1946. Topley and Wilson’s principles of bacteriology and immunity, Vol. 1. 3rd ed., London: Edward Arnold.

Listeria monocytogenes in the Natural Environment DAVIDR. FENLON Scottish Agricultural College, Aberdeen, Scotland

INTRODUCTION Listeria monocytogenes is commonly found in soil and water and on plant material, particularly that undergoing decay, with these environments being re,gardedas the natural habitat of the organism [86]. Decayed vegetation, such as aerobically spoiled silage, supports development of high numbers of L. monocytogenes, and has been cited as the source of infection in numerous cases of listeriosis in farm animals, and may be the origin of contamination capable of spreading along the food chain. The organism can survive longer under adverse environmental conditions than many other non-spore-forming bacteria of importance in foodbome disease. This resistance, together with the ability to colonize, multiply, and persist on processing equipment makes L. monocytogenes a particular threat to the food industry. Table 1 shows the persistence of L. monocytogenes in various natural and farm environments and summarizes results from some of the studies mentioned in this chapter. This ability to survive for long periods may explain why the natural environment can act as a reservoir of contamination capable of spreading to animal and plant food products. It is only relatively recently, with the introduction of improve:d molecular typing methods such as multilocus enzyme electrophoresis (MEE), random fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE), that the full story of listeriosis epidemiology is emerging.

21

Fenlon

22

TABLE 1 Survival of L. monocytogenes in Various Environmental Samples Sample Soil sterile soil (I)a 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 Fecal material cattle feces (NC)' moist horse/sheep feces (I) dry horse/sheep feces (I) sheep feces liquid manure liquid manure sewage sewage sludge cake (NC) surface interior sprayed on field Water sterilized pond water (I) unsterilized pond water (I) pond water pond water pond water/ice pond/river water pond/river water water distilled water (I) Animal feed silage (NC) silage (NC) mixed feed (I) oats (I) hay (1) straw (NC/I) straw (I) straw straw Inoculated. Not given. Naturally contaminated. Source: Adapted from Refs. 3, 4, and 75. a

Storage temperature ("C) Outside-winter/spring 24-26 24-26

154 225 67

24-26 24-26

295 67

NGh NG NG NG 4-12 18-20

12 182 -497 >730 240-3 1 I 201-271

5 Outside Outside Outside Summer Winter

182-2 190 347 730 242 36 106

28-32 48-56 Outside

35 49 >56

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

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

450 180-2 190 188-275 150-300 145-189 365 47-207 23 135

Listeria monocytogenes in the Natural Environment

23

ISOLATION OF LISTERIA MONOCYTOGENES FROM ENVIRONMENTAL SAMPLES Although L. monocytogenes is widely distributed, the numbers of the organism present in most environmental habitats are very low. Isolation methods have therefore required that relatively large samples be selectively enriched to increase numbers to detectable levels and suppress the competing background microflora of thc: sample. Such procedures have ranged from the original cold enrichment at 4°C over many weeks or months [ 51] to the present widely used and relatively rapid enrichment broths of the Food and Drug Administration (FDA) [66] and UVM [70] methods with selectivity based on acriflavine and nalidixic acid. Plating media also have improved; PALCAM agar [93] is less prone to overgrowth with competitive organisms than Oxford agar [53]and is sensitive enough for direct plating of samples for enumeration of L. morzocytogenes, particularly when high numbers of Listeria are present [36]. It does, however, require incubation at 30°C for 48 h, whereas with less contaminated samples, Oxford agar gives similar results in a shorter time at 37°C. In the United States, lithium chloride phenylethanol moxolactam agar (LPM) is a popular medium [29]. In many highly contaminated environmental samples, such as feces or decayed plant material, where L. monocytogenes numbers may be low and high numbers of competing microorganisms are present, a two-stage enrichment procedure is often necessary. This is particularly so when Enterococcus spp. (941 or B a d lus spp. are present, as some strains of these species can initially have a colonial morphology similar to L. monocytogenes. These two-stage enrichment methods, such as the US Department of Agriculture (USDA) procedure, consist of a first stage with lower acriflavine concentration (12 mg/L) from which, if necessary, a subculture can be made after 24 h to a second broth containing a higher acriflavine concentration (25 mg/L). This is the standard procedure using UVM and Fraser enrichment broths; a similar technique was described for fecal and other contaminated environmental samples by Fenlon et al. [36]. A draft international standard (DIS 1 1290- 1) based on a two-stage enrichment has recently been proposed by the International Organization for Standardization [5]. Enrichment methods can be adapted to provide quantitative “most probable number’ ’ (MPN) estimates of L. monocytogenes populations in environmental samples [36,981. Since L. monocytogenes is widely distributed, the pathogen must be enumerated so that the relative risks posed by each habitat to the food chain and the circumstances that allow survival and proliferation of the organism can be determined [28].

Soil and Vegetation Soil is often referred to as the source of Listeria contamination particularly for silage [33]. Fertile agricultural soil receives decaying plant material, animal waste, and sewage sludge, all of which are well-documented sources of L. monocytogenes. The study of Weiss and Seeliger [ l o l l showed that L. monocytogenes was present in plant samples from 9.7% of cornfields, 13.3% of grainfields, 12.5% of cultivated fields, 44% of uncultivated fields, 15.5% of meadows and pastures, 21.3% of forests, and 23.1% of wildlife feeding areas examined in southern Germany. Surface soils had similar levels, but analysis of soil samples taken at a depth of 10 cm gave significantly fewer positive samples, indicating that vegetation is a principal component in Listeria contamination of the soil. Welshimer and Donker-Voet [ 1041 were unable to isolate L. monocytogenes from soil or dead vegetation in early autumn, yet soil and decayed vegetation taken in the following spring were nearly

24

Fenlon

all positive for the organism. Fenlon et al. [36] examined five 25-g samples each of grass, leaves, stems, and roots/stems from two crops of growing sward before harvesting. No L. monocytogenes were detected in any samples. Only L. innocua and L. seeligeri were isolated from 3 of 10 samples from the root/stem area. However, L. monocytogenes was detected in 9 of 10 samples of cut grass from the same crops after wilting for 24 h before ensiling. Other vegetable crops such as lettuce and carrots postharvest did not carry the organism to the same extent. Farber [27] similarly found little Listeria contamination of unprocessed vegetables. Other workers [46] have reported higher levels, with the degree of processing and packaging having a significant effect on Listeria levels [10,16]. The higher incidence of L. monocytogenes associated with harvested (processed) grass compared with other plant products has been attributed to the presence of a sheath of decaying plant material at the base of the plant which might act as an inoculum at harvest [36]. Whittenbury [ 1061 demonstrated the importance of the sheath area as a source of lactic acid bacteria, which have a similar natural habitat to Listeria in ensiling of grass. This may be a model for contamination of grass with Listeria during the ensiling process. Other potential sources of L. monocytogenes in soils are from natural deposition of feces by animals and spreading of animal waste and sewage sludge as fertilizer. Survival of L. monocytogenes in soil depends on soil type and its moisture content. Welshimer [ 1031, using cotton wool-plugged tubes containing either clay or fertile soils inoculated with L. monocytogenes and stored for 67 days at 24-26"C, showed numbers decreased in both by seven orders of magnitude as the soils dried out. Repeating the experiment with sealed tubes to prevent water loss, the decrease was similar for the clay soil but only two orders of magnitude for fertile soil. Watkins and Sleath [98] demonstrated that in soil on land to which sewage sludge had been applied, there was little decrease in Listeria spp. numbers (approximately 170 cfu per 100 g soil) over an 8-week period, whereas salmonellae, applied at a similar rate, decreased to undetectable levels in less than 4 weeks. This difference in rate of survival may be a reflection on the original habitat of each species. Fenlon et al. [36] did not find L. monocytogenes in the few soils examined that were associated with vegetable crops, but soil from fields where cattle or sheep had been kept on silage diets did contain L. monocytogenes. In a study using autoclaved soils inoculated with L. monocytogenes (approximately 5 X 102cfu/g) and stored at ambient winter temperatures ranging from -15" to +18"C, Botzler [14] found an increase to 1 X 107cfu/g over a 154-day period. However, it is not known what effect the autoclaving process had on Listeria growth by increasing availability of nutrients and eliminating competing organisms. From evidence in the literature to date, it does not appear that soil is a natural reservoir in which L. monocytogenes multiplies. The widespread presence of the organism in soil probably results from contamination by decaying plant and fecal material, with damp surface soil providing a cool, moist protective environment and decaying vegetation the substrate, which together enable L. monocytogenes to survive from season to season.

Fecal Material L. monocytogenes has been found in the feces of a wide variety of healthy animal species. Gray and Killinger [49] listed 37 mammals from whose feces the organism had been isolated. Given that vegetation is the natural habitat of the organism, and that most reports have involved cultivation of L. monocytogenes solely by enrichment techniques with few quantitative data, it is not surprising that carriage in grazing animals such as healthy sheep

Listeria monocytogenes in the Natural Environment

25

[52,68,90], goats [65,96], and cattle [36,56,96] is well documented. However, L. rnonocytogenes excretion also has been reported in pigs [25,83], chickens [7,47,77], turkeys [9,55,77], pheasants [89], gulls [30], rooks [30], pigeons [82], fish [6], and crustaceans [6]. As L. rnonocytogenes appears to be a food-related pathogen [73] with naturally occurring listeriosis being recorded in many other animals, including mice [92], voles [79], rats [90], rabbits [79,90,99], guinea pigs [84], chinchillas [38], lemmings [82], hyraxes [82], mink [63], skunks [84], horses [102], dogs, [63,92], cats [79], foxes [79], deer [26,79], buffalo [25], giraffes [ 181, bats [57], ducks [82], partridges [82], eagles [82], parrots [82], canaries [82], starlings [63], frogs [13], turtles [13J,ticks [4], and flies [4], it is reasonable to suspect these animals to also excrete the organism in their feces. Humans, both symptomatic and asymptomatic carriers, excrete L. monocytogenes . Ralovich [841 summarized data, primarily of European origin, indicating that 1.8-9.0% of healthy individuals excrete L. rnonocytogenes in their feces. The influence of diet on excretion of L. monocytogenes has recently been studied, particularly in ruminants. Low et al. [68] reported a low incidence of excretion in a flock of 100 grazing sheep; this increased significantly ( P < .OOOOl) to between 10 and 33% once silage feeding commenced, confirming the finding of Husu [58] that the tendency is for excretion rates to be lower in grazing animals. Fenlon et al. [36] monitored two groups of cattle. While grazing, none of those tested (n = 10 and 13) 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. rnonocytogenes.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. Formulated diets for intensively reared pigs and poultry also were free of the organism. Furthermore, of 9 samples of broiler poultry litter tested, only 1 was positive and all 10 swabs of feces taken at the processing plant from crates used to transport the birds were negative. Similarly, only 1 of 47 fecal samples from pigs and piglets was positive. Genigeorgis et al. [41], in a study at a poultry processing plant, found the incidence of Listeria on carcasses increased as processing progressed. L. monocytogenes was not isolated from composite feather samples (n = 16) from live hanging birds or their hind gut contents (n = 16). Husu et al. [59] noted that most 2-day-old chicks dosed orally with L. rnonocytogenes had eliminated the organism within 9 days, indicating that chickens are unlikely reservoirs of the organism with any carriage probably being transient. Dijkstra [21J examined the intestines of 2373 broilers from 146 farms, and showed that 4.1 % were contaminated with Listeria. Increased excretion because of stress, as associated with salmonellosis, has not been well documented for listeriosis. Ralovich [84] observed increased excretion of L. rnonocytogenes by sheep housed under stressful conditions. Fenlon [36] reported that in animals traveling to three abattoirs, the level of L. rnonocytogenes in feces from cattle traveling a long distance (>loo km) was greater than in those traveling to nearby abattoirs (<25 km) (P = .003). 'The highest excretion rate found for L. rnonocytogenes was 800 cfu/g feces, although L. ivanovii was found at a level of 6.4 X 104cfu/g in sheep feces. Numbers of L. monocytogenes transferred to red meat carcasses tended to be low and rarely were detected by swabbing, usually requiring enrichment of meat samples taken from the lower parts of hanging carcasses. In abattoirs with a good hygienic standard, feces are only responsible for intermittent low-level direct contamination. This may be sufficient to provide an inoculum for colonization of equipment used for further processing of the meat and therefore may be a source of indirect contamination of foods, especially if hygiene

26

Fenlon

standards are poor. Studies have shown that more highly processed meat products are more consistently contaminated with L. rnonocytogenes [36,87].

Sewage One of the earliest definitive quantitative studies on L. monocytogenes was that of Watkins and Sleath [98]. They reported levels >18,000 cfu/L in trade effluents associated with animals and sewage sludges from treatment plants in northeast England. Levels of the organism in primary tank settled raw sewage ranged from 700 to 18,000 cfu/L. Much higher levels of Listeria spp, were reported by Geuenich and Muller [42] in a West German sewage treatment plant. Both untreated waste and filtered effluent had 103- 10scfu 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 [36] showed L. monocytogenes numbers in untreated sewage to be 120 cfu/mL and in treated effluent 221 cfu/mL. Anaerobic digestion reduced numbers to 1. I cfu/g, and lime-treated sludge had no detectable Listeria. In Iraq, Al-Ghazali and Al-Azawi [2,3] studied survival of L. monocytogenes during sewage treatment and in stored sewage sludge cake. A decrease of 85-97% in viable Listeria numbers occurred during activation and digestion stages of the sewage treatment process [2], although all steps were detrimental to the organism’s survival. They recovered <3-15 L. monocytogenes cfu/mL from final effluent and <3-7 cfu/mL from sludge cake. The latter is frequently dried and used as a fertilizer in developing countries, although treated sludge is increasingly subjected to land disposal in European countries as restrictions on sea disposal come into force. These same authors [3] demonstrated that the listeriae were inactivated when sludges were stored in direct sunlight for at least 8 weeks. Inactivation was slower in the interior of the sludge piles. As reported earlier [98], 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. Since fecal contamination has been linked to one foodborne outbreak of human listeriosis associated with coleslaw [SS], potentially contaminated sludges and animal wastes should be plowed into the soil and not spread on crops which are eaten raw.

Water The ubiquitous nature of L. monocytogenes and use of surface waterways for discharge of sewage effluents will inevitably result in the presence of the organism in a wide range of lakes, rivers, and streams. Dijkstra [22] found the organism in 21% of the surface water samples in northern Netherlands, and noted that even though the lakes were used by swimmers, no human cases were linked to this activity. A study of the course of the River Don in northeastern Scotland [36] showed 42% of 19 samples (100 mL) were positive for L. monocytogenes in May and 53% 6 months later; numbers ranged from 10 to 350 cfu/L. Highest numbers were found at a sampling point below a sewage plant, but this was not consistent for both sampling occasions. No factor, seasonal or otherwise, could be related to the presence or numbers of L. monocytogenes which occurred over the entire course of the river. In another study in the United Kingdom [39], 30 samples (100 mL) from 21 sites showed 8 sites positive for L. seeligeri (27%), 1 for L. innocua,

Listeria monocytogenes in the Natural Environment

27

and 1 for L. welshirneri. Soil may be the source of contamination at these sites, since McGowan [69] reported that L. seeligeri was more frequently isolated from soils than either L. innocua or L. rnonocytogenes. No waterborne cases of human listeriosis have been reported; however, water may act as a source of contamination for certain foods. Soonthoranant and Garland [91] found L. rnonocytogenes in 35- 100%of discharges from a sewage treatment pond and fish processing factory effluents, which also contained sewage. Inshore marine waters, which eventually received these discharges, contained L. monocytogenes in 6.6% of samples with the contamination rate of Pacific oysters and blue mussels being 15.4%, which probably reflected their filter feeding habits. These findings confirm and extend the California study of Colburn et al. [17] on fresh and low-salinity waters in which 81 and 62%, respectively, harbored Listeria spp. including L. rnonocytogenes. One of three bay water samples contained L. rnonocytogenes, and L. innocua was found in one of 35 oysters sampled. Furthermore, Motes [76] isolated L. rnonocytogenes from shrimp caught off the U.S. Gulf Coast, and Destro et al. [ 191 showed that some strains associated with shrimp can persist in processing plants and enter the final product. Gray et al. [50] successfully infected two sheep, four goats, and one cow by supplying them with L. rnonocytogenes-contaminated water. Althoug,h current evidence on direct infection of humans and animals with Listeria via water remains sparse, there is a legitimate risk. A greater risk would appear to be contamination of foods such as marine and freshwater fish with polluted water, especially those going for further processing [76], as it is known that certain strains of L. rnonocytogenes can colonize equipment and contaminate the final product [72].

Animal Feeds Most formulated animal feeds have low levels of available water which restricts multiplication of L. monocytogenes. The same is true of hay and cereal grains, so although L. rnonocytogenes has been reported in such materials 1401, the numbers are unlikely to reach levels which present a serious risk to animals. Many formulated feeds are sold in a pelleted 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 I811 noted the link between “Listerella” encephalitis in ruminants and silage in 1940, and in 1960, Gray [48] reported isolating L. monocytogenes from the fetus of a pregnant mouse fed poor-quality L. rnonocytogenes-contaminated silage implicated in death and abortion in cattle. Identical serotypes of L. rnonocytogenes were isolated postmortem from the mice and cattle. Today, there are numerous reports linking the feeding of silage with listeriosis outbreaks in sheep and cows [32,37,44,45,52,68,78,100]. Much of this problem can be attributed to the high numbers of L. monocytogenes present in contaminated silage [31,321 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 e n d i n g , the onset of anaerobic Conditions stimulates the indigenous or inoculated lactic acid bacteria to multiply rapidly; generally reaching a maximum of around 10’ cfu/g within 48 h. These bacteria convert the plant sugars to lactic acid, causing a rapid decrease in pH [7 11 with well-preserved silages generally having a pH 5 4 . 5 . These acidic conditions inhibit growth of both spoilage microorganisms and Listeria as long as anaerobic conditions are maintained. Higher dry matter silages tend to have a higher pH,

28

Fenlon

but the lower level of available water compensates for any lessening in the preservative effect caused by reduced acidity. Grass silages in cooler, wetter climates tend to have lower sugar levels and higher moisture contents resulting in a poorer, slower fermentation, and so are more susceptible to L. rnonocytogenes contamination than grass and maize silages grown in countries with warmer climates. L. monocytogenes Contamination is most frequently associated with poor-quality silage. In 1979, Gr@ntsol[52] analyzed 291 grass silages from 113 farms and isolated L. rnonocytogenes from 22% of samples with a pH <4.0; 37% with a pH of 4.0-5.0, and 56% with a pH of >5.0. In a study of clamp silage implicated in an outbreak of listeriosis in cattle [32], levels of L. rnonocytogenes in excess of 12,000 cfu/g were found in the surface layer (pH 8.3-8.5), whereas no Listeria spp. were detected 15 cm into the silage mass and the pH was 4.5. Gitter [44] noted that from 1975 to 1985, the incidence of listeriosis in sheep in Great Britain increased from less than 50 incidents per annum to over 250. During the same period, the rise in cattle listeriosis was much lower. He also reported that the pattern changed from isolated single incidents to much larger flock 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 197Os, as 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 [30]. As L. monocytogenes is a surface problem (Figure 1) and big bales have a much greater surface area than clamp or silo silage, the potential for contamination to develop is much greater in bale silage if it is not made correctly and aerobic deterioration takes place. Fenlon [31] reproduced the problem in 500-g laboratory bales ensiled in plastic bags. After 2-3 weeks, L. rnonocytogenes could be detected at levels of 2 1.1 X 106cfu/g silage in moldy areas near the tie end of the bag where air infiltrated. In the center of bags, the pH remained at 3.8, silage appeared to be of good visual quality, and it was free of Listeria. The association between L. monocytogenes growth in silage and pH is shown in Figure 2. The relationship between oxygen tension, pH, and L. rnonocytogenes contamination was demonstrated by Donald et al. [24], who infused laboratory silos with gas mixtures containing from 0.1 to 5.0% oxygen and demonstrated that the greater the oxygen level, the more rapidly the pH rose with subsequent multiplication of Listeria. Listeria die in well-fermented silage; however, if the pH increases before all cells are killed, then surviving Listeria will multiply. Dijkstra [20] showed that L. rnonocytogenes can survive 4-6 years in naturally contaminated silage. Fenlon et al. [36] noted that the organism could survive over 1 year in bags which had been 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, the challenge to the animal is considerably reduced [33]. Inflammation of the iris (iritis) caused by L. rnonocytogenes has been increasingly reported [97], particularly in cattle. This condition occurs when cattle and sheep burrow their heads into bale silage in self-feeding systems. The eye 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 [67]. More obscure

Listeria monocytogenes in the Natural Environment Big Bale Silage

29

air

air air

f

tie

Bagged bale

Wrapped bale

1 1 1 - 1 1 1 1 1 1 1 1 1 1 D D 1 1 1 1 1

plastic sheet

Clamp Silage

1

air

good quality silage pH4.0

Silage showing aerobic deterioration and therefore high risk of L. monocytogenes contamination

FIGURE1 Effect of air on development of Listeria rnonocytogenes contamination in clamp and bale silage.

7.5

6

7

5

6.5 4

6

5.5 PH

3

5

2

4.5 1 0.1

4 0

5

10

15

20

25

30

35

3.5

Days Center bale:

43 Lmon

Tie end:

Q

L.mon

+ pH; + pH

FIGURE 2 Effect of aerobic deterioration o n pH and survival and growth of Listeria rnonocytogenes in laboratory bale silage. (From Ref. 33.)

30

Fenlon

causes of listeriosis have been reported, including silages made from orange peel and artichokes [95], several outbreaks in Canada and the northern United States have been caused by cattle feeding on ponderosa pine needles [ I].

TRANSMISSION Being so widely distributed in the environment, animals and humans frequently come in contact with L. monocytogenes through a variety of sources. What is also apparent is the relatively low incidence of clinical listeriosis in both animals and humans. Traditionally, the disease in both animals and humans has occurred as individual sporadic cases, and although this is probably still the predominant form of the disease in humans [73], the organism has since emerged as a serious foodborne pathogen, with well-documented outbreaks being associated with processed foods, such as coleslaw [%], soft cheese [11,61], pat6 [43], pork tongue [60], and pork rilletts [61]. These larger outbreaks have almost all been attributable to serovar 4 strains principally serovar 4b, which, when typed by several methods, have been shown to be closely related [15,60]. The origin of these outbreak strains is unclear. Boerlin and Piffaretti [ 121 used MEE and showed that the electrophoretic type (ET) which 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, thus suggesting an environmental origin. However, relying on one typing method can be misleading. MEE reportedly has good discrimination for serotype 1/2 isolates, which can be subdivided into a large number of ETs. However, it is less effective for serotype 4 strains, most of which fall into relatively few ETs. Donachie et al. [23] subjected serotype 4 isolates of the same ET from a variety of sources to analysis using PFGE and found that all but one of the human isolates could be grouped into exclusive human PFGE types. Multiple isolates from other sources, such as silage, also fell into a single type. This study confirmed the need to use a combination of typing methods to obtain maximum discrimination of strains. Further evidence for an environmental origin for outbreak-related strains was obtained by Wesley and Ashton [ 1051, who in a retrospective subtyping study subjected clinical, environmental, and factory isolates from the Mexican-style soft cheese outbreak to restriction enzyme analysis. This study showed that the causative strain was recovered from samples of curd, the pasteurizer, cooler water, a floor drain, and insects caught in the factory, with the widespread presence being related to poor hygiene in the plant. McLauchlin and Nichols [74] demonstrated a direct relationship between poor hygiene, as measured by total viable count, and the presence of Listeria spp. in 4405 samples of seafood. A similar relationship between food processing hygiene and Listeria was shown when pit6 samples were tested in the recent UK outbreak [43]. Sporadic and small outbreaks (<10 cases) tend to be caused by a much more diverse range of strains than the larger outbreaks 1721, and although serovar 4b predominates over others, serovar 1/2b is often found. In one study [72], isolates of L. monocytogenes from cases of human listeriosis and foods were collected in the United Kingdom over a 30-year period, and were sent to the Public Health Laboratory Service Food Hygiene Laboratory in London for serotyping. Isolates from human listeriosis cases were of serovars 4b (60%), 1/2a (17%), 1/2b (1 l%), and 1/2c (4%) and from foods 4b (22%), 1/2a (32%), 1/2b (15%) and 1/2c (21%), although this predominance of serovar 4b in human cases may be the result of more virulent qualities of this serovar, the greater preponderance of serovar 112 in food isolates may be explained by the ability of these strains to adapt better to ecological niches in the food processing environment. In a recent 12-month survey of raw

Listeria monocytogenes in the Natural Environment

31

milk from 160 farm bulk tanks, L. monocytogenes contamination was low, 4.3-9.3%, all were serovar 1, and the maximum level was 35 cfu/mL. Most contamination was sporadic with a diverse array of ETs present [35]. Harvey and Gilmour [54], using MEE and RFLP analysis, compared L. monocytogenes isolates from four milk processing centers and two dairy farms in Northern Ireland with food and clinical isolates and found the dairy-related isolates to be quite distinct. 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 [64] showeld that a single RAPD type was predominant in the raw processing environment over the 6-month period of the study, surviving the clean-in-place schedules. In a follow-up study 12 months later, the same RAPD type was again isolated from final cooked products. Norrung and Skovgard [SO] found the genetic diversity of ETs isolated from cooked meat products to be lower (0.439) than those from raw meat (0.903). They suggested that certain clones may be better adapted to processing. An alternative explanation might be that the diverse range of strains found on raw meat has been eliminated during heating, and that strains on cooked products represent a more restricted range of strains present in the postcooking environment. The extremely diverse range of strains on raw meat and poultry products was shown by Ryser et al. [87], who tested by ribotyping up to 10 isolates per sample from enriched samples of ground beef, pork sausage, ground turkey, and chicken. They demonstrated that the strain selected was influenced by the enrichment medium, but, more importantly, over half of all positive saniples had more than one L. monocytogeizes ribotype (RT), some with as many as three. In some instances, detection of certain clinically important ribotypes of L. monncytogenes, which were apparently overgrown by other RTs, was only possible when 10 isolates from a sample were typed. In animal listeriosis, where the change from hay to silage feeding for sheep has resulted in outbreaks involving whole flocks, a similar pattern of diversity is emerging. A temporal difficulty also exists in linking contaminated silage with cases of clinical disease, particularly the encephalitic form, because of the long incubation period 1681. Identical strains have been recovered from animals with clinical disease and silage fed to them [68,105]. However, silage may contain a diverse array of L. monocytogenes strains, resulting in flocks being exposed to and infected by [8] multiple strains. Nonetheless, within a single sheep, one strain dominates during infection. Low et al. [68] noted that 6 distinct phage types were present among 45 isolates of L. monocytogenes from baled silage. The 67 positive fecal isolates from 100 sheep being fed the silage were even more diverse with 10 phage types other than those present in the silage being identified. The authors commented that several strains present in feces were absent from silage. They noted that since sheep consumed 100- 1000 times more silage than was analyzed, Listeria strains present in excreta were more likely to be representative of the total silage population. Two of the isolates recovered from the three clinical cases during the trial period were serovar 4b and one was 1/2a. Identical phage types were isolated from silage and fecal samples taken before the onset of disease, but were not necessarily the dominant strain present, thus confirming the necessity to examine and type rriultiple isolates from individual samples. In another study 1341, the extent of Listeria contamination in silage was directly related to its hygienic quality, as measured by the numbers of Enterobacteriaeceae present, illustrating that management of postharvest processing of the grass can markedly affect numbers of Listeria present. The natural environment appears to be the initial reservoir for virulent strains of L. monocytogenes which can enter and pass along the food chain, but this contamination is

Fenlon

32

usually of a low level and sporadic. It is significant that poultry products are more contaminated than beef, yet the environment in which beef cattle are reared presents a greater risk of contact with the organism than that of the intensively reared broiler chicken. However, in processing, the latter is exposed to greater risk of contamination from other carcasses and mechanical equipment than is in the beef carcass [85].It is at the processing stage of food and feedstuffs that amplification of numbers and persistent contamination occur, which in turn present a potentially more serious challenge to human and animal health (Fig. 3). Although discriminating power of typing methods is improving, a standardized system is not yet in place, and it appears that a significant number of isolates must be typed per sample, particularly with raw meats and silages, to ensure detection of the most important strains. What is clear is that the natural environment harbors a highly diverse range of L. monocytogenes strains, including some with the potential to cause clinical listeriosis in humans and animals. This contamination is usually minimal and sporadic. However, in the absence of good manufacturing and hygiene practices in both human and animal food production, the food processing environment can become a ready source of virulent strains with the ability to colonize equipment and contaminate food products.

f 1

0

Plants Soil

Manure

Water

Sewage

0

~

Other meat producing animals and

Equipment and Environmental Sources

Areas of greatest potential risk of L. monocytogenes multiplication.

4 Consumer at risk

Ruminants

Direct consumption of minimally processed products i.e. whole fresh vegetables, cooked carcass cuts of meat and fish and effectively pasteurised milk presents a low risk.

FIGURE3 Spread of Listeria monocytogenes to the food chain from the natural environment.

Listeria monocytogenes in the Natural Environment

33

ACKNOWLEDGMENTS The author gratefully acknowledges the contribution of Professors E. T. Ryser and E. H. Marth on which this revised chapter is based, and also the assistance and critical comments of colleagues within and outside the Scottish Agricultural College (SAC). SAC receives financial support from The Scottish Office Agriculture Environment and Fisheries Department.

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82. Plagemann, O., and A. Weber. 1988. Listeria monocytogenes als Abortursache bei Klippschliefern (Procavia capensis). Kleintierpraxis 33:3 17-3 18. 83. 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. 84. Ralovich, B. 1984. Listeriosis Research-Present Situation anti Perspective. Budapest: Akademiai Kiado. 85. Richmond, M. 1990. The Microbiological Safety of Food, Parts I & 11. Report of the Committee on the Micribiological Safety of Food, London, HMSO. 86. Rocourt, J., and H.P.R. Seeliger. 1985. Distribution des especes du genre Listeria. Zentral. Bakteriol. Mikrobiol. Hyg. A. 259:3 17-330. 87. 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 with two primary enrichment media. Appl. Environ. Microbiol. 62: 1781- 1787. 88. Schlech, W.F., P.M. Lavigne, R.A. Bortolussi, A.C. Allen, C.V. Haldane, A.J. Wort, A.W. Hightower, S.E. Johnson, S.H. King, E.S. Nicholls, and C.V. Broome. 1993. Epidemic listeriosis-evidence for transmission by food. N. Engl. J. Med. 308:203-206. 89. Schwartz, J.C. 1969. Attempted isolations of Listeria monocytogenes from diagnostic accessions. Am. J. Vet. Res. 30:483-484. 90. Seeliger, H.P.R. 1961. Listeriosis. New York: Hafner Publishing. 91. Soontharanont, S., and C.D. Garland. 1995. The occurrence of Listeria in temperate aquatic habitats. Proceedings of XI1 International Symposium on Problems of Listeriosis. Perth, Western Australia, Publ. Promaco Conventions, pp. 145- 146. 92. Sturgess, C.P. 1989. Listerial abortion in the bitch. Vet. Rec. 124:177-87. 93. Van Netten, P., I. Perales, A. Van de Moosdijk, G.D.W. Curtis, and D.A.A. Mossel. 1989. Liquid and solid selective differential media for the detection and enumeration of Listeria monoc-ytogenesand other Listeria spp. Int. J. Food Microbiol. 6: 187- 198. 94. Van Renterghem, B., F. Huysman, R. Rygole, and W. Verstraete. 1991. Detection and prevalence of Listeria rnonocytogenes in the agricultural ecosystem. J. Appl. Bacteriol. 7 1:211217. 95. 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, Abst. P29. 96. Von Winkenwerder, W. 1967. Das Vorkommen von Listeria monocytogenes bei Rindern in Neidersachsen. Berl. Munch. tieriirztl. Wschr. 23:445-449. 97. Walker, J.K., and J.H. Morgan. 1993. Ovine ophthalmitis associated with Listeria monocytogenes. Vet. Rec. 132:636. 98. 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. 99. Watson, G.L., and M.G. Evans. 1985. Listeriosis in a rabbit. Vet. Pathol. 22:191-193. 100. Weidmann, M., J. Czajka, N. Bsat, M. Bodia, M.C. Smith, T.J. Divers, and C.A. Batt. 1994. Diagnosis and epidemiological association of Lis#teria monocytogenes strains in two outbreaks of listerial encephalitis in small ruminants. J. Clin. Microbiol. 32:99 1-996. 101. Weis, J., and H.P.R. Seeliger. 1975. Incidence of Listeria monocytogenes in nature. Appl. Microbiol. 30:29-32. 102. Welsh, R.D. 1983. Equine abortion caused by Listeria monocytogenes serotype 4. J. Am. Vet. Med. Assoc. 182:291. 103. Welshimer, H.J. 1960. Survival of Listeria monocytogenes in soil. J. Bacteriol. 80:3 16-320. 104. Welshimer, H.J., and J. Donker-Voet. 1971. Listeria rnonocytogenes in nature. Appl. Microbiol. 21516-519. 105. 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. 106. Wittenbury, R. 1968. Microbiology of grass silage. Process. Biochem. 3:27-3 1.

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Listeriosis in Animals IRENE V. WESLEY National Animal Disease Center, U.S. Department of Agriculture, Ames, Iowa

INTRODUCTION Numerous animal species are susceptible to listerial 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 either sporadically or as epidemics and often leads to fatal forms of encephalitis. Virtually all domestic animals are susceptible to listeriosis [ 1781, with sheep [7,109,205,207,239,285,302], cattle [ 109, 205,209,2 17,226,255,2991, goats [ 169,170,2431, and less frequently chickens [ 106,200, 205,2231 succumbing to infection. Several comprehensive reviews have detailed the distribution and pathology of L. mnnocytrogenes in food animals [7,108,127,160,176,2191.

INCIDENCE Listeriosis in domestic livestock is being recognized with increasing frequency around the world [107,279]. Since listeriosis is not a reportable disease in animals, the exact incidence of listerial infections in domestic livestock remains unknown. According to Ralovich [221], the annual number of cases in animals has increased substantially since 1966 with about 2200, 1000, and 900 cases being reported in Bulgaria, eastern Germany, and Hungary in 1976, 1972, and 1980, respectively. Reports of listeriosis in domestic animals also have increased in New Zealand, Germany, Greece, and England. This may reflect a natural emergence, increased awareness, and/or improved detection methods. 39

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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 I million and 2000-2500 sheep, respectively, from listerial infection. Before a vaccine became available and lowered the incidence of listeriosis, infections in Norwegian livestock remained relatively constant with approximately 19002300 sheep herds, 90-160 goat herds, and 3-17 cattle herds being affected during the 10-year period between 1977 and 1986 [301]. In the United Kingdom, approximately 30,000 ovine abortions occur annually with 0.05-0.13% attributed to listeriosis [S]. 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% (average of 3.2%), which represented 234-928 cases [70]. 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]. Although the humoral immune response may not have a major role in acquired resistance against listeriosis, serum antibody levels are useful for serodiagnosis of Listeria carriage in healthy animals [29]. Sindoni et al. [247] conducted a serological survey in Italy on the frequency of Listeria antibodies in apparently healthy sheep, cows, goats, and swine. Based on an agglutination test, sheep ( I U%), cows ( 1 0.4%), goats (17.3%), and swine ( 1 3.3%) tested positive, suggesting previous exposure to L. monocytogenes. However, these data should be interpreted with some caution, since antibodies to several gram-positive bacteria can cross react with Listeria antigens. In a 1995 German survey of domestic and companion animals [288], L. monocytogenes was found in fecal samples from healthy bovines (33%), sheep (8%), birds (8%), pigs (5.9%), horses (4.8%), and dogs (0.9%). 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]. Seasonal variation in the number of cases of animal listeriosis has often been observed. 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 [ 1081. Numbers of 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 [70] indicate that most cases of listerial abortion in cattle occurred between December and May. Approximately 40% of these cases were linked to consumption of contaminated silage. Although in Norway listeriosis can be diagnosed year-round in sheep and goats, the illness is far more prevalent from October to June and also appears to be influenced by housing and feeding conditions [221]. Recent changes in production methods have reduced levels of L. monocytogenes in silage, which in turn has led to a considerable decrease in the incidence of listeriosis in silage-fed animals. Yet an increase in ovine listeriosis with conversion to big bale silage production has been reported in the United Kingdom [297]. The quality of the silage, including pH and micronutrient composition, may predispose livestock to infection. To illustrate, sheep fed silage had a lower number of lymphocytes, lower total serum protein levels, and elevated serum iron compared with sheep fed hay [7]. Transmission of L. monocytogenes from livestock to humans occurs by (a) direct contact with infected animals, especially during calving or lambing, and (b) consumption of contaminated raw milk. Primary cutaneous listeriosis is regarded as an occupational

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disease of veterinarians and farmers who have attended deliveries of stillborn or aborting bovine fetuses [4,44,191,207,211,2751. 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 [199]. Apart from human infections acquired from contact with infected animals or from food directly contaminated by 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 food and animal feed is usually environmental [ 176,1901. The rise in cases of human listeriosis is probably the result, in part, of changes in food manufacturing and postprocessing contamination [ 1761. The World Health Organization (WHO) [294] concluded that foodborne listeriosis is predominantly transmitted by nonzoonotic means, and that L. rnonocytogenes is an environmental organism whose primary route of transmission to humans is via foods contaminated during production. Several strain-specific typing methods such as multilocus enzyme electrophoresis and pulsed field gel electrophoresis have shown that L. rnonocytogenes strains isolated from meat or raw milk mainly originated from the processing environment rather than from animals [32,119]. Although an animal origin of contamination was inferred for the three major epidemics of listeriosis occurring in North America, in the absence of documented evidence, the role of direct animal involvement in human foodborne outbreaks of the disease remains speculative [290].

Sheep Ovine listeriosis is commonly caused by L. rnonocytogenes serotypes 1/2,3, and 4 as well as by L. ivanovii [ 1741. Although “listeric-like” infections were previously observed in sheep [239], Gill is credited with the first isolation of L. rnonocytogenes from domestic farm animals [loo]. In 1929, he observed an illness in sheep in New Zealand which he called circling disease. This name is still used today to describe listerial encephalitis, encephalomyelitis, and meningioencephalitis [239]. Clinical manifestations of ovine listeriosis are (a) encephalitis, (b) placentitis with abortions occurring in the last trimester [ 109,I8 1,2801, and (c) gastrointestinal septicemia with hepatitis, splenitis, and pneumonitis [ 1541. Encephalitis is the most common form diagnosed in sheep [154]. Lambs as young as 5 weeks of age may develop septicemia with older feedlot lambs (4-8 months) manifesting encephalitis. All sheep are probably exposed to the same contaminated feed, indicating a high natural resistance with 5-10% of exposed animals exhibiting clinical signs [ 1541. L. rnonocytogenes usually enters the animal via ingestion. Following entry into the intestinal epithelium via either M cells in Peyer’s patches or epithelia1 cells [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 [ 1541. 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 [ 1791. Direct entry via abrasions or lesions of the buccal mucosa, lips and nostrils, or conjunctiva may lead to encephalitis. Because entry into the dental terminals of the trigeminal nerve in sheep can cause an ascending neuritis and encephalitis [49,50], listerial en-

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cephalitis is most common in winter and early spring in sheep that are losing and cutting teeth [ 171. L. monocytogenes penetrates the buccal epithelium, accesses the 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 [174,154]. Abrasions of the eye by contaminated silage leads to ophthalmitis without any other clinical manifestations [283]. 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 not more than 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 muscles, and a stiff walk. 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. Excessive salivation often occurs because of the animal's inability to swallow. In advanced stages, muscular incoordination develops and is followed by inability of the animal to walk. Death usually occurs within 2-3 days after onset of clinical symptoms, with the illness seldom lingering beyond 10 days [239]. In the brain stem, Listeria antigens are characteristically variable but always sparse. Perivascular microabscesses with L. monocytogenes and microgranulomas in histopathological specimens of brain stems are characteristic. T lymphocytes (CD8' and CD4') and B lymphocytes contribute to the inflammatory process [ 1591. After ingestion and hematogenous spread to the gravid uterus, L. monocytogenes appears in the amniotic fluid and fetus within 48 h [ 1741. Listerial infections in pregnant sheep often result in premature birth and infectious abortions [ 109,I 8 1,2801. This illness seldom occurs concurrently with encephalitis [ 1351. Initially, pregnant ewes contract purulent metritis, from which most recover. Intrauterine transmission of L. monocytogenes via the placenta leads to a septic infection of the fetus, which in turn gives rise to abortion or premature delivery with most fatalities occurring 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 being high [154]. Injecting L. monocytogenes into pregnant ewes caused 10% of those animals to abort. Significant retardation of bone growth is seen in lambs born to experimentally infected ewes [ 1041. Septicemia is most frequent in neonates and lambs and appears 2-3 days after oral infection, although congenital and navel infection can also occur [ 1741. 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. Listeriosis is not a reportable disease in animals, thus precluding comparisons between countries. In addition, most infections in livestock are subclinical and therefore go undiagnosed [ 1541. In Hungary, 14% of sheep excreted Listeria [222], but the distinction between L. monocytogenes and L. ivanovii was not made. Although the incidence of L. monocytogenes in sheep and cattle has decreased in The Netherlands, which has been attributed in part to the change of silage production, in Great Britain modifications in silage production may have caused an increase in ovine listeriosis [ 1021. 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 [ 1021. In parallel with the rise in the incidence in the United

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Kingdom, there was also a change in the disease pattern with both encephalitis and abortion occurring in the same flock 102,179j. Listeriosis occurs most frequently in late autumn, winter, and early spring. Stress factors, such as abrupt changes in feed, concurrent disease, changes in dentition, and physical or viral damage to the epithelia] lining of the digestive tract may predispose to infection 11541. Introduction of new animals into the flock and overcrowding also are contributing fdctors [ 1531. Climatic changes, such as heavy rains [302], especially following a drought, which may spoil feed [228], or periods of extremely cold weather, which may cause animals to be housed indoors resulting in overcrowding [ 193,2851, are also determinants. A prospective study conducted on early lambing flocks in southwest England ( 1989- 1991) monitored 44 1 3 lambs from birth to slaughter for listerial meningoencephalitis. Two of three flocks developed clinical disease attributed to L. monocytogenes 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 [ I 101. 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 soil-based floors and thus easily contaminated. Before weaning, lambs were eating more silage, since ewes were producing less milk. Although all lambs were exposed to these risk factors, only an estimated 1.3% developed clinical infection with death occurring in 0.56% (21 of 4413) lambs from 4 to 32 days postweaning [109]. Quality of silage, as measured by digestibility ( D value), may influence the incidence of ovine listeriosis [88]. In an outbreak of ovine listeriosis in the United Kingdom, the D value of silage was below the optimum of 65-70% 12971. In Scotland, listeriosis caused by L. monocytogenes serotype 1/2 occurred in sheep which were reluctant to eat poorquality silage [ 1791. Silage analysis indicated pH > 4 and a high ash content, reflecting soil contamination. Despite antibiotic treatment, 19 ewes died, more than 60 developed vaginal discharges, and 94 were barren at lambing [ 179). The role of silage feeding in an epizootic of encephalitic listeriosis has been investigated. A British study found a significant association between silage feeding and development of ovine listerial encephalitis (relative risk of 3.8). In another report, excretion of L. monocytogenes by sheep was linked to diet, with animals being fed entirely on hay or manufactured diets not excreting detectable levels of L. monocytogenes. However, animals fed on silage commonly excreted the organism [297]. The feeding of poor-quality silage (pH 7.8) which was highly contaminated (10' L. monocytogeneslg) was the cause of an outbreak in Spain [272]. In this outbreak, the flock consisted of 450 animals (attack rate 11.8%) with a case fatality rate of 94.3%. Multiple strains of L. monocytogenes of the same or different serotype may be involved 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 [ 101, or pyrolysis mass spectrometry [ 1751. In the Spanish outbreak just mentioned, 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]. In addition, DNA fingerprinting by random amplified polymorphic DNA (RAPD) analysis and ribotyping have been used to affirm that L. monocytogenes strains from silage, farm equipment, and sheep brains were identical [295,296]. In contrast, examination of outbreaks of ovine listeriosis in Scotland indicated multiple strains of L. monocytogenes serovar I /2 could

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be recovered from the silage incriminated in the outbreak. By multilocus enzyme electrophoresis (MEE), the L. rnonocytogenes 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 the brains. This could reflect bias during sampling from the bales, thus missing a small virulent population of L. monocytogenes in the vegetation which, upon entry into the ovine host, preferentially replicated to become the dominant strain and thus cause disease [20]. Reports on the incidence of L. monocytogenes in ewes’ milk are limited. In Spain, L. monocytogenes was present in 2.2% of 1052 ewes’ milk samples representing 283 farms. Yet, L. monocytogenes was recovered from 18% of milk tanker samples in Spain. Tests of farm ewes’ milk samples indicated contamination by L. rnonocytogenes 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 resulting from either L. monocytogenes excretion in cows’ feces or common exposure to contaminated ensilage on premises shared by sheep and cattle [230]. Interestingly, no seasonal variation in milk contamination rates was evident. Although not widely practiced in the United States, vaccination with live attenuated strains of L. rnonocytogenes can effectively reduce ovine listeriosis [ 116,168,202,2821.In Norway, immunization of sheep with a commercially available live attenuated vaccine of serovars 1/2a and 4b reduced the incidence of listeriosis and abortions when compared with unvaccinated control farms [115,116]. In this study [115], half of the sheep in 70 flocks (total of 3 130 sheep) with a history of listeriosis received two attenuated strains of L. rnonocytogenes serotypes 12 and 4b, whereas the remaining sheep served as unvaccinated controls. 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. In 1984, a special license was issued to allow limited use of this vaccine in a 2-year field study [116]. After vaccinating approximately 8% of all Norwegian sheep (about 145,000 head), the incidence of listeriosis decreased from approximately 4% before introduction of the vaccine to 1.5% after vaccination began. The incidence of abortions was 0.7% in vaccinated compared with 1.1% in unvaccinated flocks. In another study, an experimental vaccine, consisting of L. rnonocytogenes serovars 1/2a and 4b which were attenuated via metabolic drift mutations, was tested in sheep, lambs, and ewes [168]. 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 kg vs 1.8 kg) than lambs from control unvaccinated ewes. In addition, L. rnonocytogenes was not isolated from milk samples of vaccinated ewes in contrast to controls in which 32% of milk samples yielded Listeria [ 1681. Although vaccination produced few adverse side effects, economic constraints suggest that vaccination of sheep should be confined to flocks that have exhibited recurrent listerial infections. The epidemiology and economics of clinical listeriosis were described for a flock of sheep in southern Illinois [202]. In that study, in which consumption of contaminated silage was a key factor, unvaccinated Rambouillet ewes were more at risk (odds ratio 4.6) than other ewes and yearling ewes were more at risk than older animals (odds ratio 4.1). Interestingly, use of a bacterin did not decrease the risk of L. rnonocytogenes in Rambouillets (odds ratio 0.8) but did among ewes of other breeds (odds radio 0.1). This indicates that the inherent susceptibility of Rambouillet ewes to L. rnonocytogenes cannot be modified by vaccination [202]. Serosurveys have been used to estimate the occurrence of listeriosis in sheep [250].

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However, antibodies to other gram-positive microbes cross react with Listeria antigens, thus giving rise to false-positive results. In 1990, Berche and coworkers [26j developed a more specific test for L. rnonocytogenes based on detection of antibodies to listeriolysin 0 (LLO) an antigenic protein of 58 kD. LLO is a virulence factor unique to L. monocytogenes that is required for the organism to escape from vacuoles and grow intracellularly. It is not found in other Listeria species, including L. ivanovii. Antibodies to LLO which are specific for L. monocytogenes and do not cross react with other Listeria species have been detected in lambs experimentally infected with L. monocytogenes [ 13,166,1771.Purified LLO was evaluated as a specific antigen to detect both humoral and cell-mediated immune responses of sheep infected with L. rnonocytogenes, L. innocua, and L. ivanovii [ 131. LLO antibodies were seen only in sheep infected with L. rnonocytogenes. Furthermore, in a blastogenesis assay and skin test, two indicators of cell-mediated immunity, only L. rnonocytogenes-infected sheep responded to LLO [ 131. Listeria ivanovii (formerly L. monocytogenes, serotype 5 ; [240]) was first described in association with ovine abortion and accounts for up to 8% of all animal listeriosis cases [ 101. Listeria ivanovii is a recognized cause of ovine abortions and stillbirths and unthrifty lambs [37,63,130,180,1811. Goats are not susceptible to L. ivanovii. To illustrate, L. ivanovii and L. rrzonocytogeneswere both reported in two migratory flocks of sheep and goats in Himachal Kadesh, India. Whereas L. monocytogenes was isolated from both sheep and goats, L. ivanovii was cultured only from sheep [244]. Experimental infections of sheep with L. ivanovii indicate that, unlike L. rnonocytogenes, abortions occur without encephalitis [ 1371. Factors which predispose to L. ivanovii abortions in livestock are similar to those described for L. monocytogenes: a lowering of ewes’ resktance to infection by nutritional stress, periods of cold and wet weather, feeding of poor-quality silage, and exposure to carrier animals [75,101,219]. Outbreaks of listeriosis caused by L. ivanovii have been reported to affect up to 45% of pregnant ewes [ 1371. One outbreak in New South Wales involved a total of 1 10 animals which aborted or died shortly after birth. Heavy grazing by sheep (1 20 sheep per hectare for 18 days) on pastures which had been cut for hay which had not been baled and which had spoiled as a result of heavy rains was presumed to be the source of initial contamination by L. ivanovii [242]. Multiple hepatic foci were seen in aborted lambs. L. ivanovii was cultured from liver, lung, and stomach contents [242]. A report of bovine abortions caused by L. ivanovii was associated with the grazing of cattle on pastures previously used by sheep [3]. Although rarely causing infections in humans [189], L. ivanovii has been reported as a cause of abortion [75] and septicemia in acquired immunodeficiency syndrome (AIDS) patients [56,164]. Even though L. monocytogenes is regarded as being more pathogenic, both L. ivanovii and L. monocytogenes invade mammalian cells in tissue culture, use actin filaments for intercellular spread, induce myometrial contractions in an in vitro uterine strip model, and elicit conjunctivitis after ocular inoculation into rabbits [ 1621.

The manifestations of clinical listeriosis in goats and sheep are essentially the same: encephalitis, septicemia, and abortion. As is true for other livestock species, asymptomatic infections also have been noted in goats [239]. After ingestion, L. rnonocytogenes penetrates the intestinal tract and sets up a transient bacteremia, which leads to dissemination to the central nervous system, viscera, or

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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 also may have diarrhea [ 1131. In the pregnant doe, L. rnonocytogenes may penetrate the placenta, enter the fetus where it replicates, and cause late-term abortion. In experimental studies with pregnant goats, localization of L. monocytogenes in the placenta led to an elevation in prostaglandin F2 and a decrease in progesterone levels. A slight decrease in secretion of estrone sulfate by the fetal-placental unit prompted myometrial contraction and abortion [80]. Meningoencephalitis is the most frequently reported form of listeriosis in goats with fatalities reaching 60% [ 1 131. The early signs of listerial encephalitis mirror the lesions to the respective cranial nerves indicated by roman numerals in parenthesis: drooped ears (VII), marked drooling (IX and X), protrusion of the tongue (XII), and cud retention from difficulty in swallowing (IX and X). Goats may be more susceptible than sheep to listerial encephalitis based on the severity of brain damage in goats [ 161. In an outbreak of encephalitis and abortion in a mixed herd of goats and sheep in Iraq, the morbidity (30 vs 17%) and mortality (21 vs 15%) was higher in goats than in sheep [302]. Heavy rains, cold weather, and susceptibility of pregnant animals may have contributed to the high numbers of encephalitis cases [302]. More frequent recovery of L. monocytogenes serotype 1/2b from goats than from sheep also has been documented in Sudan [246]. Listeriosis is linked to feeding silage [ 170,1741. 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 [ 1441. Heavy browse consumption may have led to oral lesions followed by penetration by L. monocytogenes into the dental pulp or buccal cavity that may have led to encephalitis [ 1441. Although not practiced in the United States, vaccination has limited the number of goat listeriosis cases [66,93,202]. Before vaccinating goats in Norway with live attenuated strains, the abortion rate in the test herd was 20-25%. However, after vaccination, the incidence rate of abortions decreased to 3% [157]. A live attenuated L. monocytogenes strain (strain Aer), obtained by three successive mutations in regard to streptomycin and erythromycin resistance, afforded some protection against abortion in vaccinated goats [93]. The optimal time for vaccinating goats and sheep may be shortly before the mating season [ 1571. 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 the does may be infected through the navel or through sucking on soiled teats [ 1141. Few studies describe the distribution of L. monocytogenes in raw goats’ milk. In one report [l 1 I], L. monocytogenes was detected in 3.6% of raw milk samples from cows with considerably lower recovery of L. monocytogenes from samples from goats’ (1%) and ewes’ (2%) milk. Serum antibody titers to L. monocytogenes may not indicate the immune status of the host [ 1951. Animals with septicemia develop high antibody titers, whereas animals with encephalitis have low titers, presumably because the brain is an immunologically privileged site. More recently, antibodies to LLO have been proposed as a specific gauge of antibody status. In experimentally infected goats, an increase in LLO antibodies is correlated with rapid clearance of L. rnonocytogenes from the gastrointestinal tract and thus predicts a favorable course of clinical infection [ 1961. DNA fingerprinting methods are clarifying the role of animals in human infection. To illustrate, 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

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made from that goat’s milk and an isolate from the refrigerator in which the cheese was kept. It is suggested that the cheese made from the infected goat’s milk may have contaminated the refrigerator shelves, thus serving as a reservoir for L. monocytogenes (741. 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 making zoonotic transmission less likely [%I.

The distribution of L. monocytogenes undoubtedly reflects the available pool of susceptible animals. In North America, most listeriosis cases occur in cattle (82%) with a smaller percentage in sheep (17%) and fewer still in pigs [21]. From 1993 to 1997, listeriosis cases (n = 253) submitted to the Iowa State University Veterinary Diagnostic Laboratory [161] showed a similar distribution and were from cattle (87%), sheep (9%), goats (2%), and a llama and a horse (0.4% each). In Iowa, 90% of listeriosis cases were diagnosed as encephalitis [161]. In marked contrast from 1975 to 1984, listeriosis cases in Great Britain were from sheep (63%), cattle (32%), and with pigs, goats, fowls and other species constituting less than 1 % each of the total submissions [297 I. The symptoms of bovine listeriosis include encephalitis, abortion, and septicemia with miliary abscesses [ 1471. In 1928, Matthews [ 1841 detailed an outbreak of encephalitis of unknown origin in cattle which, in retrospect, was probably bovine listeriosis. Listerial encephalitis has since been well documented [60,67,146,22 1,226,2391. However, even in acute outbreaks, generally no more than 8- 10% of a herd succumbs to infection. In Switzerland, after bovine spongiform encephalopathy, bovine listeriosis is the most frequently diagnosed neurological disease [ 1241, and thus may be the most common cause of bacterial infection of the central nervous system in adult cattle [226]. In Missouri [ 1431, from 1986 through 1994, encephalitic listeriosis cases were diagnosed in cattle (67%), goats (30%), and sheep (13%). Foodborne transmission is the main mode of infection in naturally occurring listeriosis in cattle with silage being most frequently implicated. As in sheep, following ingestion, Listeria is disseminated via hematogenous spread to the viscera, brain, and gravid uterus. In addition, since L. monocytogenes is present in soil, fecal material, and vegetation, it may enter via abrasions of the nostrils or the conjunctiva while grazing or via the teat of a lactating cow [ 1421. Direct injection of the conjunctiva, resulting in keratoconjunctivitis [215], has occurred as a result of contaminated silage particles falling into the faces of browsing cattle [ 1971. By travel along peripheral nerves (indicated by Roman numerals), 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 underlies the clinical presentation. For example, lesions of the fifth (V) cranial and mandibular nerves lead to inability to eat or drink or to retain food in the mouth. Excessive salivation from difficulty in swallowing (IX and X) and 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 nerves [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 1-2 days

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[239]. Histopathological lesions of the brain stem consist of foci of necrosis infiltrated with neutrophils, macrophages, and bacteria [ 1421. Perivascular cuffing with mononuclear cells is evident [267]. Unlike listerial encephalitis in sheep and goats, most cattle survive at least 4-14 days after the initial onset of symptoms, with a few reports of spontaneous recovery [22 11. Listeriosis in cattle is frequently associated with abortion [ 105,209,221,2391,which generally occurs during the last trimester of pregnancy. However, as demonstrated by Dora, an 1l-year-old cow with atypical mastitis, healthy calves can be born to chronic carriers that shed the pathogen in milk [72]. As was true for sheep, L. monocytogenes is transmitted to the placenta, and then into the fetus. Meningitis in neonates may follow intrauterine infection with the septicemic young animal dying shortly after birth [241]. In the US Midwest, Listeria spp. are the third most frequently encountered bacteria from bovine late-trimester abortion cases. L. rnonocytogenes accounted for 1.35% of bovine abortion and stillbirth submissions received by the South Dakota Animal Disease Research and Diagnostic Laboratory from 1980 to 1989 [ 1551. Ten years earlier, of 2544 bovine abortion cases examined in the Northern Plains region of the United States, 2.2% were attributed to listeriosis [ 1561. Similarly, in Germany, from 1984 to 1989, L. monocytogenes serotype 1/2 was recovered from 1.2% (122 of 993 1) and 1.8% (122 of 993 1) of bovine abortions [31] in the Erfurt and Cottbus regions, respectively. L. ivanovii is most often associated with sheep [20,37,63,130,137,180,181,2421 and is sometimes recovered from cattle [3,101]. In California, during a 3-year period, five cases of listerial bovine abortion were diagnosed among 243 fetuses submitted for evaluation. L. ivanovii was recovered from four cases, whereas L. monocytogenes was isolated from only a single bovine abortion. The pathological findings in these five listeriosis cases were similar [3]. L. rnonocytogenes may enter a herd through contaminated feeds, introduction of new stock, and rodents. Bovine abortions and stillbirths occur shortly after contaminated silage is fed [5]. Improvement in silage production and hay making resulted in a decrease (from 8.7 to 1.2%) in the number of listerial bovine abortion cases in The Netherlands [64]. A change in silage production also was linked to a reduction in the percentage of carrier animals (from 15 to 0.8%) based on fecal sampling [69]. An outbreak in Nigeria resulted in 35 cases of bovine encephalitis and four abortions in the same herd. Although the source of infection was unknown, the authors proposed that L. monocytogenes was introduced into the herd by introducing new animals [2]. As expected, the number of healthy carriers is lower on farms without overt disease than on farms with clinical listeriosis. To illustrate, L. monocytogenes was cultured from 2.0% of cows on farms without L. monocytogenes and from 6.7% of healthy animals on farms on which listeriosis had occurred [64]. 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 [5,107,153]. Although not particularly common, generalized listerial infections can give rise to mastitis [41,59,60,72,103,148,216,258,281].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 [300] isolated L. rnonocytogenes 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 penicillin-resistant strain of L. monocytogenes was cultured from one quarter of a 6-year-old dairy cow. Following acute onset, the condi-

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tion soon became chronic with shedding of L. monocytogenes in milk for 3 months. Prolonged excretion of L. monocytogenes in milk [65,66,135,209,2 10,2211, the apparently normal appearance of the milk, and consumption of raw milk on farms could be important factors in the transmission and epidemiology of milkborne listerial infections [ 1031. From a public health aspect, culling of L. monocytogenes-infected cows with clinical mastitis which do not respond to treatment is recommended [245]. After slaughter, cross contamination of the carcass with bacteria from the infected udder is possible through evisceration, meat inspection, or other manipulations [274]. L. monocytogenes strain Scott A (serotype 4b), a clinical isolate recovered from the New England outbreak, has produced experimental mastitis in dairy cows [38,291]. Following repeated intramammary inoculation of 34 Holstein cows with I 03- 107L. monocytogenes cells, 75% of the animals became chronically infected and shed Listeria in milk (about IO3-1Os L. monocytogenes cfu/mL of milk) intermittently for up to 8 months. The intramammary route of inoculation was used to simulate infection from contaminated bedding directly into the teat canal. Interestingly, one of these experimentally infected cows delivered a normal healthy bull calf. Bourry et al. [4] also induced mastitis via intramammary inoculation with a single dose of 300 cells of L. monocytogenes serotypes 4b and 112. L. monocytogenes was recovered from the supramammary lymph node but not from the spleen or liver, indicating clearance in the affected region. DNA profiles of isolates recovered throughout experimental infection confirmed the persistence of the inoculated strain [4]. As with sheep [7] and goats [ 1691, L. monocytogenes also is shed in milk by healthy dairy cattle with no indication of mastitis [64,73,86,103,12’7,134,135,234,278].Schultz [234] collected milk samples from 1004 cows and isolated L. monocytogenes from the milk of 10 animals (0.1%), 7 of which appeared perfectly healthy. Shedding of Listeria in milk by these animals was intermittent but continued for up to 12 months. Examination of dairy herds in Yugoslavia [247] also has demonstrated that clinically healthy cows can act as 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 on which listeriosis had been previously diagnosed [ 15 81. In addition, Kampelmacher [ 1481 reported that dairy cattle shed L. monocytogenes at levels of 10,000-20,000 cfu/mL of milk. Surveys of raw milk prompted by dairy-related outbreaks of human listeriosis in 1983, 1985, and 1987 confirmed that asymptomatic cattle are carriers of L. monocytogenes. Indirect contamination of bulk milk occurs from unhygienic rnilking practices, if L. monocytogenes is present in feeds, feces, udder surface, or bedding [87], or if an animal is recovering from a recent infection [ 104,1951. L. rnonocytogenes has been reported in raw cows’ milk with a distribution ranging from 0.1 to 45% [22,7 I ,86,87,94,111,122,173,234, 254,260,2651. Recoveries varied depending on whether individual cows, bulk tanks maintained on the farm, or milk tankers serving multiple premises were sampled. When available, data indicate that the incidence of L. monocytogenes from individual farms may be lower than that reported for processing centers or tanker trucks [120,230]. 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 from 0.2 to 4.270. However, 8.5% of bulk milk samples (n = 4451 were contaminated with L. monocytogenes [6]. Regional differences in the recovery of L. rnonocytogenes from milk have been documented. For example, Dominguez Rodriguez et al. [7 I ] found L. monocytogenes in 45.3% of 95 raw milk samples from a single bulk tank (80,000-L capacity). The

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dairy received raw milk from several small farms in western and central Spain over a 16month interval. In the northeastern United States, Hayes et al. [122] found L. monocytogenes in 12% of raw milk samples. L. monocytogenes was found in 4.4% of raw milk samples (n = 137) obtained from the Utrecht region of The Netherlands [22] and in 3.8% of raw milk samples in Scotland 1911. This parallels the recovery of L. monocytogenes in 4.1% of raw milk samples from Tennessee [231]. Interestingly, in that US study, consumption of raw bulk milk was reported by 35% of dairy producers [231]. A seasonal distribution of L. rnonocytogenes in raw milk, mirroring numerous determinants including a change of diet or weather-related stress, has been observed. Lovett et al. [ 1733 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, recovery of L. monocytogenes from Massachusetts samples was seasonal with the incidence being highest during cooler months and lowest in hot weather months. Interestingly, no seasonal trend was exhibited by samples collected from Ohio, Kentucky, and Indiana [173]. L. monocytogenes was cultured from 4.9% of raw milk samples obtained from 70 Irish farms with the incidence being higher in the winter when cows were housed indoors than in the summer [224]. A study of L. monocytogenes in raw milk in Nebraska indicated a seasonal distribution with 6% of raw milk samples harboring L. monocytogenes in February; 2% of samples were positive in July [ 1671. Seasonal variation may be related to silage feeding during the winter. In Scotland, a seasonal distribution was indicated for L. monocytogenes, which was present on 25 of the 160 farms surveyed (16%). Contamination was sporadic, with bacterial titers generally < 1 L. monocytogenes cfu per mL. Although more raw milk samples were positive for L. monocytogenes in January than at other sampling times throughout the year, the authors caution that no link to farm management practices was evident [90]. In Ontario, Canada, some geographical differences were observed, with the incidence of L. monocytogenes in raw milk being higher in the eastern region [86]. Despite the modest recovery of L. monocytogenes from raw milk in Ontario (1.3%, 6 of 455 samples), the incidence was lower during the winter and autumn than at other times [86]. In another report, L. monocytogenes was found in 5.14% of raw milk samples screened monthly in Ontario with no indication of seasonal differences [254]. Normal healthy cattle may intermittently shed Listeria in their feces, with prevalence rates ranging from a few percent to 52%, with some seasonality [87,131,271,2881. Fecal shedding may reflect levels of L. monocytogenes in feed. In one study, shedding of L. monocytogenes (52%) was related to feeding wet feed (e.g., silage of beet tops, oat, and pea straw); 67% of the samples were contaminated 12521. L. monocytogenes was isolated from the feces of 8.7% of nearly 4000 randomly selected dairy cows in Finland over a 2-year period [ 1331. Again, L. monocytogenes was recovered more frequently in bovine feces on farms where L. monocytogenes was in feed than on premises where feed (silage or pasture grass) was negative for L. monocytogenes [ 13 1,1331. Switching cattle from grazing to a diet of silage increased fecal shedding of L. monocytogenes. The distribution was seasonal with L. monocytogenes recovered from feces more frequently during the indoor season (9.2%) than when animals were on pasture (3.I %). Expectedly, the seasonal occurrence of L. monocytogenes in milk reflected the frequency of this pathogen in feces but not in grass silage or pasture grass, thus inferring fecal contamination during milking [131]. Serosurveys have been used to monitor distribution of L. monocytogenes infections in dairy cows. Infection rates in cattle can be estimated by measuring antibody levels to whole cells as well as antibodies specifically targeting LLO [ 12,331. Agglutination titers

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of serum and whey were evaluated in experimentally infected dairy cattle ( n = 34). By the eighth week postinfection, 80% of the cows exhibited serum titers of > I :20,480. Whey titers, which reflected the local mucosal immunity to L. monocytogenes in the mammary gland, rarely exceeded I :256, because of the lower immunoglobulin concentration in milk versus blood [72,293]. Since up to 33% of dairy cattle continued to shed L. monocytogenes despite high serum titers, antibody levels did not accurately predict the presence of L. monocytogenes in milk [72,257,276,293]. As with humans [26], sheep 1 13,166,1771, and goats [ 1061, antibodies to LLO have been used to evaluate the immune response of experimentally infected cattle. [ 12,331. A positive response to ILL0 specifically confirmed previous or current infection with L. rnonocytogenes in dairy cows [ 12,331. Stress-related immunosuppression associated with change of diet, weather, transportation [89], 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 administrated to cows experimentally infected with L. monocytogenes, dexamethasone increased shedding of the pathogen in milk by up to 100-fold [291]. Increased levels of L. monocytogenes in 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 distance!; significantly increased the level of fecal excretion of L. monocytogenes. However, contamination of the resultant cattle and sheep carcasses was minimal [92]. 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 [ 1401. Furthermore, a comparison of 33 isolates from bovine mastitis and 27 human clinical isolates recovered in Denmark 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 had cows infected with L. monocytogenes strains which were similar to human clinical strains 11411. L. monocytogenes ribotypes common to both dairy processing and the farm environment (dairy cattle, raw milk, silage) were also reported in the United States, thus suggesting that the farm may serve as a reservoir for L. monncytogenes strains capable of entering the dairy processing facility [9]. The findings also verify the ubiquitous distribution of the pathogen. Although foodborne listeriosis in humans is more frequently linked to consumption of contaminated dairy products than to beef, 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 postinfection; none was recovered at 6 or 54 days after inoculation [ 1451. Thus culled dairy cattle may be an insignificant source of L. monocytogenes contamination in meat. Epizootics have been observed in both feedlot and beef cattle herds (5,299). Transport of cattle over long distances increased the level of fecal excretion of L. monocytogenes, but contamination of carcasses was not high [89]. Yet in this study, L. monocytogenes was detected in 9 1 % (2 1 of 23) of minced beef samples, demonstrating that processing significantly increases the level of Contamination compared with that of the whole carcass 1891. This hypothesis is further strengthened by tracking L. monocytogenes strains by multilocus enzyme dectrophoresis. L. monocyto-

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genes strains of electrophoretic type (ET) 1 have been implicated in major human foodborne epidemics and are found coincidentally in livestock. L. monocytogenes strains of ET 1 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 [32]. In contrast, environmental strains such as ET 19 contaminate the carcass during processing. 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. Overall these findings indicate that contamination of meat occurs during processing by L. monocytogenes strains which are resident in the packing plant rather than by strains indigenous to animals [32]

Porcine listeriosis manifests itself 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 [ 118,1941. Unlike its frequent occurrence in ruminants (cattle, sheep, and goats), listeriosis is rare in monogastric swine. Slabospitskii [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 [ 1521 recovered Listeria from the liver of a pig with no clinical signs of encephalitis. In Norway, Hessen [125] reported listerial septicemia in piglets raised on a farm where sheep had died of listeriosis several weeks earlier. Whether transmission was from sheep to pigs or the result of common exposure is unknown. In natural and experimental infections, listeriosis is more severe in young animals [30,42,150]. 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 [ 1721 and spring. Listerial encephalitis seldom occurs in pigs. Symptoms of central nervous system disturbance, including incoordination and progressive weakness followed by death, are characteristic of listeriosis in 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 include severe monocytic infiltration. Numerous blood vessels, particularly those in the pons, reveal perivascular cuffing [29,108,219,2391. Although listerial meningoencephalitis in swine is infrequent, several such outbreaks have been reported, including one in India in which 27 of 75 pigs died [220]. In England, the Veterinary Investigation Center reported only 14 listeriosis cases in swine between 1975 and 1982 as compared with 666 cases in sheep and 472 cases in cattle [ 1021. Listeriosis in pigs is also reported to be uncommon in The Netherlands [201]. Porcine listeriosis comprised 1% of listeriosis cases in western Canada [21]. In Iowa, a major hog-producing state, of a total of 253 listeriosis submissions to the state veterinary diagnostic laboratory from 1993 to 1997 none were from pigs. In that same interval, 87% of listeriosis cases in Iowa were from cattle [161]. Earlier, Blenden reported that cattle and sheep accounted for 274 of 281 (98%) of listeriosis cases submitted to the Missouri Diagnostic Laboratory; only 1 case was from pigs [29]. Although few surveys are available describing the prevalence of L. monocytogenes

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in healthy pigs, its distribution can be estimated from surveys of fecal excretors and recoveries from tonsils and carcass swabs collected at slaughter. L. monocytogenes was cultured from 5% of rectal swabs and from 1.9% of hog carcasses in Trinidad [ 11, indicating minimal carcass contamination during processing. L. monocytogenes was present in 13% of hog tonsils and in 2% of lymph tissues in pigs in Togo, West Africa [ 1291. In Belgium, 16% of fresh pig feces (n = 25 samples) harbored L. monocytogenes [271]; 5.9% of swine fecal samples were positive in Germany [288]. In Yugoslavia, L. monocytogenes was cultured more frequently from hog tonsils (25%) than from fecal samples ( 5 % ) taken from the same animals [277]. In Scotland, L. monocytogenes was not found in either pig feces before slaughter or on swine carcasses [92]. Likewise, L. monocytogenes was not recovered from hog carcasses in Norway or Sweden [204]. Asymptomatic carriers of Listeria may be more prevalent in eastern Europe. To illustrate, L. monocytogenes was recovered from 25.6% of swine feces in Hungary [222]. Ralovich [221] reviewed studies in which a fecal recovery rate of 47% in individual animals and in 11 of 12 among farms was described. Yet in Yugoslavia, 45% of all pigs examined harbored L. monocytogenes in the tonsils, whereas only 3% were fecal excretors [40]. A high infection rate in pigs has led to speculation that swine may be important reservoirs of L. monocytogenes [ 1081. Husbandry practices such as feeding pigs dry feed or silage, rearing in closed houses, and maintaining specific pathogen free (SPF) herds as well as differences in sampling sites (tonsils versus feces) may account for the variation in the incidence of healthy porcine carriers reported. For example, in Yugoslavia, L. monocytogenes was recovered more frequently from tonsils of pigs raised on silage (61%) than from animals raised on dry feed (29%). Interestingly, in that study, L. monocytogenes was also recovered from more than 19% of pork meat products tested [40]. Although not found in fecal samples in SPF herds, L. rnonocytogenes was cultured from 2.2% of fecal samples from non-SPF herds in Denmark [252]. Norrung et al. [206] detected Listeria spp. in 29% of tonsils removed from market weight hogs and in 75% of pig feed samples tested in Denmark. Unfortunately, data on the specific distribution of L. monocytogenes in this study were not provided. L. monocytogenes was recovered from the lymph nodes of 5% of slaughtered pigs and from 8% of pork samples in Bosnia and Hercegovina [ 1711. Of L. monocytogenes recovered in that survey, 76% were from hog carcasses sampled during the autumn and winter months [ 1711. Skovgaard et al. [252] reported that only 1.7% of pig fecal samples yielded L. monocytogenes; however, the pathogen was detected in 12% of ground pork samples tested in Denmark indicating dissemination of Listeria during processing. In Yugoslavia, where L. monocytogenes was isolated more frequently from ground pork (69%) than from deep muscle (O%), contamination occurs during pork processing [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 [ 1381. 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.

Avian listeriosis was first described in 1935 [238], 3 years after TenBroeck isolated L. monocytogenes (then Bacterium monocytogenes) from diseased chickens. Both wild [227]

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and domestic avians, including turkeys [24,12 1,2051, ducks [ 106,2391, geese [ 106,2391, and pheasants [ 1061, are the largest group of asymptomatic carriers [ 106,1081. A survey of healthy urban rooks indicated carriage with L. monocytogenes (33%), L. innocua (24%), and L. seeligeri (8%) [35]. Likewise, L. monocytogenes was detected in 9.5% of fecal samples from apparently healthy, ring-billed gulls (Larus delawarensis) in Montreal. In contrast, samples from pigeons in Barcelona were negative [46], whereas L. monocytogenes was found in 1% of pigeons examined in Germany [2881. Up to 33% of all healthy chickens may asymptomatically shed L. monocytogenes in fecal material [67,68,252]. 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 which were housed in stables where chicken litter was used as bedding. L. monocytogenes serogroup 4b was recovered from the bovine brains, litter, and the intestinal contents of 4.1% of the donor birds 1671. In another study, the increased incidence of L. monocytogenes in rooks coincided with the nesting season and the peak of ovine listeriosis, which in turn was linked to consumption of contaminated silage [88]. Thus, although the true incidence of listeriosis in birds and other forms of domestic livestock is undoubtedly much higher than published reports, clinical listeriosis is uncommon in domestic fowl. 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 [ 106,107,108]. For example, listerial infections were discovered in only 13 of more than 38,000 chickens submitted for examination in Pennsylvania between 1960 and 1965 [236]. Furthermore, large-scale outbreaks of listeriosis in chickens appear to be uncommon [200,212]. In an outbreak in India involving young chicks, death (mortality rate of 60%) was sudden and usually with no prior symptoms. Some birds exhibited symptoms of weakness and lassitude and a tendency to stand in an isolated dark place [200]. 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 [108]. In 1988, an encephalitic form of listeriosis was reported in broiler chickens in California [54]. Predisposing conditions which may have precipitated the outbreak included recent debeaking and vaccination with a modified live viral arthritic vaccine which was 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 and affected 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 precipitated this outbreak [54]. Septicemia, the most frequent manifestation of listeriosis in chickens and other domestic fowl, is characterized by focal necrosis within the viscera, particularly the liver and spleen [ 1081. Although not present in all cases [200], cardiac lesions frequently develop, which in turn lead to engorgement of cardiac vessels, pericarditis, and increased amounts of pericardial fluid [ 108,2231. 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 [ 1081.

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Unfortunately, chickens and other domestic fowl that suffer from listerial septicemia normally exhibit few overt signs of disease other than progressive emaciation and usually die within 5-9 days of infection. Although far less common than the septicemic form of listeriosis, L. monocytogenes also can produce meningoencephalitis in domestic fowl. Domestic birds suffering from listerial meningoencephalitis exhibit several striking behavioral changes, including incoordination, tremors, torticollis, unilateraUbilatera1 toe paralysis, and dropped wings, all of which directly relate to disturbances of the central nervous system [ 181. Such infections are virtually always fatal. Postmortem examination often reveals congestion and necrotic foci in the brain [ 181 along with many of the aforementioned conditions that are characteristic of listerial 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 can colonize both chick embryos and young birds with older birds appearing more resistant [ 108,117]. Following oral challenge of chickens with 102or 106 L. monocytogenes cells, Bailey et al. [ 151 detected the pathogen more frequently in ceca, spleen, liver, and cloacal swab samples from 1-day-old chicks rather than 14- or 35-dayold 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 2-5 days postinoculation. Milder symptoms such as anorexia and drowsiness were also observed in several animals. At 5 days postinfection, 100% of the cecal samples yielded L. monocytogenes. However, the percentage of L. monocytogenes-positive birds decreased, and by day 28, L. monocytogenes was recovered from the ceca of only 10% of experimentally infected birds [132]. In another report, with tests on a smaller number of birds, L. monocytogenes was only found on the first day following infection in 15% of fecal samples [186]. These data suggest that L. rnonocytogenes is cleared rapidly from infected birds, indicating that chicks are transiently infected and unlikely reservoirs of L. monocytogenes. Interestingly, following artificial infection, Pustovaia [2 I 81 found that Columbiformes (pigeons, doves), Passeriformes (perching birds), and Galliformes (turkeys, pheasants) were susceptible, whereas Falconiformes (falcons, hawks) and Strigiformes (owls) were resistant [ 1281. L. monocytogenes has been used to investigate macrophage function in retroviral infection and the cell-mediated immune response in susceptible and resistant chickens exposed to Marek’s disease virus 145,571. Viral infection depressed the resistance of 10day-old chickens to experimental infection with an avian osteopetrosis virus [57]. When compared with virus-free chickens, the dual infected birds were less efficient in clearing L. monocytogenes from their spleens [57). L. monocytogenes can be recovered from infected chicks by inoculation of 1O-dayold embryos [62]. Thus, egg inoculation has been suggested as an assay to replace the mouse test to gauge the virulence of L. monocytogenes [ 1 11. For example, L. monocytogenes and L. ivanovii are fatal to experimentally inoculated 10-day-old chick embryos. In contrast, embryos infected with the nonpathogenic species, L. innocua, L. seeligeri, and L. welshimeri, generally survived [266]. L. monocytogenes is present in 0-33% of healthy birds [67,68,76,99,127,136,288], with contamination in retail poultry ranging from 17 to 70% [ 14,32,76,92,99,186,208].A link between transport stress and fecal shedding of L. rnonocytogenes has been suggested.

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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. However, no data on the status of L. monocytogenes in these birds before shipment are provided [252]. The presence of Listeria on retail poultry probably results from contamination during processing rather than from the bird. In an effort to trace the source of L. monocytogenes in retail poultry, low levels of natural carriage ( 5 % ) were reported in cecal samples from parent flocks providing broilers. L. monocytogenes was not cultured from cecal samples from over 2000 broilers (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 [208]. Once processing is initiated, the numbers (and percentage) of Listeria-positive samples increase [92]. Studies in poultry slaughterhouses failed to detect L. monocytogenes in several organs, including the intestinal tract and ceca. Yet it was found in processing water, in mechanically deboned meats, and on the hands and gloves of 34% of the meat cutters, indicating cross contamination during processing [99]. Later studies used DNA profiles of L. monocytogenes collected during processing to indicate significant environmental contamination during processing [32] Sporadic human cases of listeriosis have been epidemiologically linked to consumption of undercooked poultry products [235]. Analysis of risk factors associated with sporadic human listeriosis in the United States indicated that cancer and immunocompromised patients, in whom 69% of listeriosis cases occur, were more likely than controls to have eaten undercooked poultry (odds ratio = 3.3) [233].

Minor Species Listeriosis has been diagnosed in several minor livestock species, such as horses, llamas, animals raised commercially for pelts, companion animals, deer, and primates. The routes of transmission and symptoms parallel those of cattle, sheep, and goats. As in other livestock species, Listeria infection in horses can cause abortion [289], septicemia [25,51,79,112], and encephalitis [ 1821. In contrast to cattle and sheep, few cases of equine listeriosis are reported [ 160,182,185,239,263,264,2681.A survey of fecal samples from 400 German horses indicated a carrier rate of 4.8% for L. monocytogenes, 6% for L. innocua, and 1.5% for L. seeligeri with less than 1% harboring L. welshimeri [288]. The few surveys describing L. monocytogenes-seropositive horses should be interpreted cautiously in the light of possible cross reactivity between antibodies of other bacterial species and Listeria. Prior contact with cattle and feeding on silage may explain sporadic cases of equine listeriosis [ 1871. L. monocytogenes was reported from four Welsh and two Shetland ponies housed together with cattle, one of which was diagnosed with listeriosis, and given poorquality silage [79]. At necropsy, L. monocytogenes was cultured from the equine liver, spleen, heart, kidneys, and lungs [79]. In Tasmania, abortions occurred in two mares which were allowed to graze on a pasture which 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 that L. monocytogenes infection does not lead to permanent infertility [ 1831. Equine abortion, preceded by mild respiratory tract infection

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caused by L. rnonocytogenes serotype 4, was reported for a mare which had wintered with cattle and had consumed ensilage [289]. L. monocytogenes was cultured from the fetal liver, lung, spleen, and stomach. Neonatal septicemia was documented in a 3-day-old foal whose mare was housed indoors and fed poor-quality contaminated hay [126]. As with other species, the origin of infection in equine listeriosis cases may be unknown. To illustrate, L. monocytogenes was recovered from the brain stem of a 16year-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 [182]. A 21-day-old Appaloosa filly was examined because of diarrhea of 2 weeks’ duration [284]. As the animal’s condition deteriorated, gentamicin sulfate and procaine penicillin G were administered. Septicemia was diagnosed based on the presence of L. monocyrogenes cultured from the blood. No sources of infection were evident, thus leading to speculation that L. monocytogenes was transmitted to the foal via contaminated mare’s milk. As is true for humans, listeriosis also occurs more frequently in immunocompromised livestock with defects in both the humoral and cell-mediated immune systems [269]. Listeriosis was described in an Arabian foal with combined irnmunodeficiency [ 5 I]. The I-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 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 asymmetrical vestibular disease in adults [ 191. Most affected animals are weaned and grazing or consuming roughage but not silage. Multifocal suppurative encephalitic listeriosis was diagnosed in two adult llamas, both of which were pregnant. L. monocytogenes was cultured from one of two animals and was observed in brain stem lesions of both llamas by fluorescein-conjugatedantibody to L. monocytogenes [43]. In another report, L. monocytogenes caused fatal meningoencephalomyelitis in a 3- to 5-monthold llama. The animal displayed unilateral peripheral disease progressing to encephalitis 12701. The source of infection for cases detailed in these two reports is unknown [ 19,431. Listeriosis can have an economic impact on commerciall pelt farms. An outbreak of disseminated visceral listeriosis in chinchillas in Nova Scotia [95] was associated with consumption of contaminated sugar beet pulp, although L. monocytogenes was not isolated from the feed. This outbreak occurred in a colony with a 239‘0 mortality rate of breeding chinchillas [298]. 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 (2981. Hay contaminated with rodent, bird, or ruminant feces has been implicated in previous outbreaks of listeriosis among chinchillas with removal of contaminated feed often interrupting the cycle of transmission [47,95]. L. monocytogenes could have been transmitted by coprophagia, since animals defecated in dust bath pans and the pans were transferred from cage to cage [298]. In an enzootic outbreak of listeriosis in a rabbitry, L. monocytogenes 1/2a was cultured from feed samples and from a doe which had died of septic metritis 12141. The early literature describes L. monocytogenes in nondomesticated ruminants, including reindeer, roe deer, and a Grant’s gazelle that had previous contact with Listeriainfected sheep [23,81,83,151,205,2861. L. monocytogenes has been recovered at necropsy from ruminants housed in zoological parks [8 133,2861. Meningoencephalitis occurred during the winter and early spring in 42 of 1800 deer in a Danish park. This was preceded, in the previous spring, by death of six deer which exhibited circling and appeared to be

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blind. The following year, the first sign of illness was a drooping ear, caused by paralysis of the facial nerve, and a slight inability to follow the herd. No external source of L. monocytogenes was evident and stress resulting from a poor beech-mast crop, an increased stocking rate of animals resulting in overcrowding with possible introduction of asymptomatic carriers, and a sudden change in the weather were all potential contributing factors [811. Listeriosis is rarely reported in dogs and can cause encephalitis, including circling [232], and abortion [261]. In a survey of domestic animals, L. monocytogenes was detected in 1.3% of dog fecal (n = 300) samples and 0.4% of cat fecal (n = 275) samples [288]. The low recovery may indicate that companion animals are not important in the epidemiology of listeriosis in humans [287] or that shedding is sporadic. In contrast, serosurveys which indicate that up to 90% of dogs may be seropositive [48,25 11 should be interpreted cautiously because of the cross reactivity inherent in agglutination tests. A single report on possible transmission of L. monocytogenes from humans to dogs [262] warrants reevaluation, since the isolates were later identified as L. innocua. Nevertheless, recovery of a single species of Listeria from both humans and dogs in close proximity could reflect substantial environmental contamination rather than human-to-dog passage. Listeriosis also can occur in non-human primates where it manifests itself as septicemia [303], meningoencephalitis [61], and stillbirths [ 188,2731, as documented in a large outdoor breeding colony in California [2 131. Transmission may occur by consumption of contaminated foods [273]. Attempts have been made to experimentally infect Cynomolgus monkeys (Macacafascicularis) through feeding [85] and exposure to aerosols [ 1491. Although these monkeys shed L. monocytogenes in their feces for up to 21 days after ingesting I O9 Listeria, neither septicemia nor encephalitis were reported indicating that normal healthy primates are resistant to L. monocytogenes.

Fish and Crustaceans Demand for seafood is growing because of its popularity, and the aquaculture industry is responding by providing fish raised on controlled fish farms rather than depending on the availability of fresh-caught products. In 1980, in New Zealand, Lennon et al. [163] reported a cluster of 22 perinatal human listeriosis cases. A weak association between these cases and consumption of contaminated raw fish and shellfish was established. Facinelli et al. [84] described a case of sporadic listeriosis, in which clinical isolates and those from the undercooked fish in the patient’s refrigerator were identical by DNA fingerprinting. The first report of Listeria in fish came from Romania in 1957 [259]. In that study, L. monocytogenes was isolated from viscera of pond-reared rainbow trout that presumably became infected after consuming contaminated donkey meat. The results of current surveys indicate that Listeria is absent from live saltwater fish but is present in live freshwater species. The bacteria could not be detected in the intestinal tract, skin, and gills of 10 live salmon [77]. Likewise, L. monocytogenes was not found in either salmon (n = 199) or in environmental samples taken from a fish farm in Bergen, Norway [78]. In contrast, L. monocytogenes was recovered from two of the five intestinal tracts of market-purchased fresh water fish in India [ 1231. Channel catfish (Zctalurus punctatus) is the most widely cultured species in the United States with most commercial ponds being located in the southeastern region. A study of catfish, water, and feed collected from university ponds in Alabama indicated the presence of L, monocytogenes on skin and viscera (mean presumptive count = 1.99

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log cfu/g wet weight). L. monocytogenes was not detected in the water or feed. Unfortunately, the authors provided no data on the percentage of L. monocytogenes-positive fish, but they concluded that bacterial concentrations in the viscera suggest cross contamination is possible during evisceration [ 1651. 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 of 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 3-7 days before harvest [ 1391. Similarly, L. monocytogenes was not recovered from the skin, gills, intestines, tank water, diet of striped bass grown in recirculating water tanks [203]. Experimental infection of zebrafish (Bruchydunio rerio) indicated the LDSowas higher in fish than in mice and that L. monocytogenes did not multiply in fish [ 1921. Experimental infection increased production of granulocytes and monocytes. In contrast to L. monocytogenes, strains of L. welshimeri, L. innocuu, and L. seeligeri killed more than 50% of the fish 7 days postinfection [ 1921 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 runoff all may potentially contribute Listeriu spp. to the aquatic environment. In addition, the presence of L. monocytogenes in sea gulls may be another source of shellfish contamination [88]. 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 [198]. The pathogen was detected in 11% of unprocessed shrimp (n = 74) but not in oysters (n = 7 9 , although some of the oysters were harvested from prohibited shellfishgrowing sites. In a parallel study conducted in freshwater tributaries off the HumboldtArcata Bay in Northern California, L. monocytogenes was detected in freshwater samples (61%), some of which received runoff from nearby farms. However, L. monocytogenes was not found in oysters in that study [52] nor in oysters kept in live holding tanks in seafood markets in Seattle [53]. Albeit a modest number of shellfish were examined, L. monocytogenes was not detected in fresh shellfish (shrimp, cuttlefish, clams) tested in Cochin, India [98]. Overall, these data indicate that live fish and shellfish are not likely carriers of L. monocytogenes. Polymerase chain reaction (PCR) tests have been used to detect L. monocytogenes in experimentally contaminated marinated rainbow trout [82]. With the increased interest in L. monocytogenes in fish and seafoods 1771, this sensitive technique may be useful for 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 Listeriu spp. detected in the retail product most likely resulted from postharvest contamination.

TREATMENT Poor animal husbandry, consumption of contaminated feed, and stress are important factors in precipating listeriosis. Thus identifying and eliminating these problems are critical to preventing reoccurrences. In general, since antemortem diagnosis is rarely made, treatment is seldom attempted. Since listerial 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

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of a cure. 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 1-2 weeks [96]. If signs of encephalitis are severe, death usually occurs in spite of treatment. Supportive therapy, which is usually reserved for valuable animals, including fluid and electrolyte replacement, 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 listerial encephalitis in sheep, it may be prudent to consider vaccinating animals against listeriosis, particularly if they are being raised in areas prone to listerial infection [202]. In birds, tetracyclines (5-10 mg/kg body weight daily for 1 week) are efficacious in both acute and subacute cases. Treatment of chronic listeriosis is unsuccessful. As with other livestock species, rigid sanitation and disinfection procedures with culling and isolation of affected birds may be helpful [97]. Prompt treatment of animals with listeriosis is clearly beneficial, with early diagnosis dependent on observation of clinical symptoms. In cattle and sheep, 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 humane euthanasia as an alternative.

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195. 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. Microhiol. 28:340-343. 196. Miettinen, A., and J. Husu. 1991. Antibodies to listeriolysin 0 reflect the acquired resistance of Listcria rnonocytogenes in experimentally infected goats. FEMS Microbiol. Lett. 77: 18I 186. 197. Morgan, J.H. 1977. Infectious keratoconjunctivitis in cattle associated with Listeria monocytogenes. Vet. Rec. 100:1 13- 114. 198. Motes, M.L. 1991. Incidence of Listeria spp. in shrimp, oysters and estuarine waters. J. Food Prot. 54:17-173. 199. Mouton, R.P., and E.H. Kampelmacher. 1966. Proceedings of the 3rd International Symposium on Listeriosis, Bilthoven, Netherlands, pp. 425. 200. Nagi, M.S., and J.D. Verma. 1967. An outbreak of listeriosis in chickens. Indian J. Vet. 44: 539-543. 201. Narucka, V., and J.F. Westendorp. 1973. Het voorkomen van Listeria monocytogenes by slachtvarkens. Tijdschr. Diergeneesk. 98: 1208. 202. 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. 203. 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. 204. 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. 205. Nilsson, A., and K.A. Karlsson. 1959. Listeria monocytogenes isolations from animals in Sweden during 1948 to 1957. Nord. Vet. Med. 11:305-315. 206. Norrung, B., M. Solve, M. Ovesen, and N. Skogaard. 199 1. Evaluation of an ELISA test for the detection of Listeria spp. J. Food Prot. 54:752-755. 207. Odegaard, B., R. Grelland, and S.D. Henricksen. 1952. A case of Listeria-infection in man, transmitted from sheep. Acta Med. Scand. 67:23 1-238. 208. 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. 209. 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. 210. 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. 21 1. Owen, C.R., A. Meis, J.W. Jackson, and H.G. Stoenner. 1960. A case of primary cutaneous listeriosis. N. Engl. J. Med. 262: 1026-1028. 212. Paterson, J.S. 1937. Listerella infection in fowls-preliminary note on its occurrence in East Anglia. Vet. Rec. 49: 1533- 1534. 213. Paul-Murphy, J., J.E. Markovitz, I.V. Wesley, and J.A. Roberts. 1990. Listeriosis causing stillbirths and neonatal septicemia in outdoor housed macaques. 41 st Ann. Mtg. Am. Assoc. Lab. Anim. Sci. 40547. 214. Peters, M., and G . Scheele. 1996. Listeriosis in a rabbitry. Dtsch. tierarztl. Wochenschr. 103: 460--462. 215. Pohjanvirta, R., and T. Huttunen. 1985. Some aspects of murine experimental listeriosis. Acta Vet. Scand. 26563-580. 216. Potel, J. 1953/ 1954. Atiologie der Granulomatosis Infantiseptica. Wiss. Z. Martin Luther Univ.-Halle, Wittenberg 3:341. 217. Price, H.H. 198 1. Outbreak of septicemic listeriosis in a dairy herd. Vet. Med. Small Anim. Clin. 76:73-74. 21 8. Pustovaia, L.F. 1970. Susceptibility of wild fowl to listeriosis. Vet. Bull. 41 533-534.

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219. Radostits, O.M., D.C. Blood, and E.E. Gay, eds. 1994. Diseases caused by Listeria spp. In: Veterinary Medicine: A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses. Bailliire Tindall, London, pp. 660-666. 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. Domjiin-Kovacs. 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. 73:33 1-336. 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. Rocourt, J., and P. Cossart. 1997. Listeria monocytogenes. In: M.P. Doyle, L.R. Beuchat, and T.J. Montville, eds. Food Microbiology Fundamentals and Frontiers. Washington, DC, ASM Press: pp. 337-352. 230. Rodriguez, 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. Schroeder, H., and I.B. van Rensburg. 1993. Generalized Listeria monocytogenes infection in a dog. J. S. Afr. Vet. Assoc. 64:133-136. 233. Schuchat, A., K.A. Deaver, J.D. Wenger, B.D. Plikaytis, L. Mascola, R.W. Pinner, A.L. Reingold, and C.V. Broome. 1992. Role of foods in sporadic listeriosis. I. Case-control study of dietary risk factors. J.A.M.A. 267:2041-2045. 234. Schultz, G. 1967. Untersuchungen uber das Vorkommen von Listerien in Rohmilch. Monatsh. Veterinaermed. 22:766-768. 235. Schwartz, B., C.A. Ciesielski, C.V. Broome, S. Gaventa, G.R. Brown, B.G. Gellin, A.W. Hightower, and L. Mascola. 1988. Association of sporadic listeriosis with consumption of uncooked hot dogs and undercooked chicken. Lancet 2:779-782. 236. Schwartz, J.C. 1967. Incidence of listeriosis in Pennsylvania livestock. J. Am. Vet. Med. ASSOC.15111435-1437. 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. 62:203212. 239. Seeliger, H.P.R. 1961. Listeriosis. New York: Hafner. 240. Seeliger, H.P. 1984. Modern taxonomy of the Listeria group relationship to its pathogenicity. Clin. Invest. Med. 7:217-22 1. 241. Seimiya, Y., K. Ohshima, H. Itoh, and R. Murakami. 1992. Listeria septicemia with meningitis in a neontal 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.

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Listeriosis in Humans LAURENCE SLUTSKER AND ANNESCHUCHAT Centers for Disease Control and Prevention,

Atlanta, Georgia

INTRODUCTION Listeria monocytogenes has been recognized as a human pathogen since 1929 [74]. This organism is found in multiple ecological sites throughout the environment, including soil [ 1051, water, and decaying vegetation [ 104,1061. Recent studies of epidemic and sporadic cases of listeriosis have increased our knowledge of important sources of L. monocytogenes in human illness. Epidemiological investigations during,the last 15 years have shown that epidemic listeriosis is a foodborne disease. [ 10,17,23,27,38,44,56,66,80,85]. Similarly, recent studies have suggested that a substantial proportion of sporadic cases of listeriosis are also caused by consumption of the organism in foods [72,89,92]. Development of improved laboratory techniques to detect and subtype L. monocytogenes has also contributed to an improved understanding of human listeriosis [7,9,11,82,83]. Human disease caused by L. monocytogenes usually occurs in certain well-defined high-risk groups, including pregnant women, neonates, and immunocompromised adults but may occasionally occur in persons who have no predisposing underlying condition (Table 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. 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% [34]. This high case-fatality rate, along with the heightened awareness of listeriosis as a foodborne disease and increasing clinical concern 75

76

Slutsker and Schuchat

TABLE 1 Clinical S y n d r o m e s Associated with Infection with L isteria rnonocytogenes

Population Pregnant women

Newborn s <7 days old 2 7 days old

Clinical presentation

Diagnosis

Fever, _t myalgias, & diarrhea Preterm delivery Abortion Stillbirth

Blood culture 5 Amniotic fluid culture

Sepsis, pneumonia Meningitis, sepsis

Blood culture Cerebrospinal fluid culture Culture of blood, cerebrospinal fluid, or other normally sterile site Stool culture in selective enrichment broth

Nonpregnant adults

Sepsis, meningitis, focal infections

Healthy adults

Diarrhea and fever

Predisposing conditions or circumstances

Prematurity Immunosuppression, advanced age

Possibly large inoculum

about the importance of illness caused by this organism in the expanding population of highly susceptible persons, has 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 as examples of epidemic disease among humans, a more exhaustive treatment of foodborne listeriosis appears in Chapter 10.

HUMAN INFECTION AND CLINICAL MANIFESTATIONS OF LlSTERlOSlS Asymptomatic Carriage L. monocytogenes is distributed throughout the environment and can be frequently recovered from a broad spectrum of foods [26,78] and from the gastrointestinal tract of healthy people. Numerous fecal carriage studies of L. monocytogenes among different populations have been done with widely varying rates reported (Table 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. Using cold enrichment culture techniques, investigators in Denmark determined that

Listeriosis in Humans

77

TABLE 2 Reported Fecal Carriage Rates of Listeria monocytogenes Year

Population

1972

Slaughterhouse workers Hospitalized adults Hospitalized children Hospitalized adults with diarrhea Household contacts of persons with listeriosis

1972

Laboratory workers handling L. mo-

No. studied

% with L. monocytogenes

1147

4.8

1034

1.2

195

0

595

1.0

34 1

26.0

26

77.0

26

62.0

54

2.0

60

3.4

1000

0.6

2000

0.8

177

5.6

80

2.5

171

1.8

18

5.6

60

8.3

nocytogenes

1986

1990

199 1

1992

Office workers with no L. monocytogenes contact Healthy pregnant women Healthy nonpregnant women Persons with diarrhea Healthy food handlers Renal transplant patients

Home hemodialysis patients Outpatients with gastroenteritis Pregnant women with listeriosis Household contacts of pregnant women with listeriosis

Method

Reference

Point prevalence Point prevalence Point prevalence Point prevalence Cumulative prevalence over 6 months; up to 8 cultures per person Cumulative prevalence over 8 weeks; up to 8 cultures per person Same

13

Point prevalence Point prevalence Point prevalence Point prevalence Cumulative prevalence (mean 2.5 specimens per patient) Point prevalence Point prevalence Point prevalence Point prevalence

52

54

68

59

62

78

Slutsker and Schuchat

TABLE 2 Continued Year

1993

1993

Population Age-, race-, and hospitalmatched controls Cheese plant employees Household contacts of cheese plant employees Household contacts of patients with listeriosis (persons) Households of patients with listeriosis with at least one carrier Healthy pregnant women

No. studied

% with L. monocytogenes

Method

7

0

Point prevalence

31

9.7

94

10.6

Point prevalence Point prevalence

82

21 .o

Point prevalence

28

21.0

Point prevalence

147

2.7

Cumulative prevalence (mean 2.4 specimens per patient)

Reference

88

40

4.8% of 1147 healthy slaughterhouse workers had stool cultures yielding the organism [ 131. Similar surveys by the same researchers documented L. monocytogenes fecal carriage rates of 1.2% among 1034 hospitalized adults, none of 195 hospitalized children, and 1% of 595 hospitalized adults with diarrhea. Kampelmacher et al. reported high rates of fecal carriage among laboratory workers having daily contact with L. monocytogenes (77% of 26) as well as 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 [52]. Subjects had L. monocytogenes isolated an average of 1.3 times out of the eight serial specimens collected. Among other populations, a large stool survey conducted in Germany found that 6 of 1000 (0.6%) fecal specimens collected from persons with diarrhea yielded L. monocytogenes, giving a point prevalence similar to the 0.8% found in 2000 healthy food handlers from the same area during the same time period [68]. In 1987, MacGowan et al. surveyed 177 renal transplant recipients with multiple fecal samples obtained over a 1-year period. Overall, 10 (5.7%) individuals had positive stool cultures, and a positive culture was associated with ranitidine use or consumption of three or more types of cheese since the beginning of the year [59]. 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. Among pregnant women, Lamont and Postlethwaite reported a fecal carriage rate of L. monocytogenes of 2% among 51 women early in pregnancy (10-16 weeks), similar to the 3.4% rate observed among 59 nonpregnant women attending the same clinic [54].

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Timing of carriage was examined among 147 women attending an antenatal clinic in the United Kingdom [40]. 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. 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 vs 0 of 7, respectively, P = NS) [62]. Household contacts of patients with listeriosis also have been surveyed. In Denmark, fecal specimens from 26% of 34 household contacts of persons with listeriosis yielded L. monocytogenes [13]. In this study, among 14 households sampled, 5 (36%) had at least 1 household member with a positive stool culture; however, only two family members had the same serotype of L. monocytogenes as the patient. IJp to eight specimens were collected from each household contact, suggesting that the carriage rate in this study may not be directly comparable to others. In the United States, 82 household contacts of 28 patients with invasive listeriosis were identified through active surveillance and investigated [88]. 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. 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 [62].

Invasive Disease in Nonpregnant Adults Various clinical conditions are reportedly associated with listeriosis in nonpregnant persons; these include malignancy, organ transplants, immunosuppressive therapy, infection with the human immunodeficiency virus (HIV), and advanced age [4,35,77,91]. The first reported correlation between listeriosis and cancer appeared in 1967, and it emphasized the severity of manifestations, high fatality rate, and association with lymphoreticular malignancies [581. Subsequent reports quickly confirmed this observation [ 16,951. In a review of 148 adult listeriosis cases reported from 1968 to 1978, malignancy was the underlying condition in 25% (25 of 102) of cases of meningitis and 33% (15 of 46) of cases of primary bacteremia caused by L. monocytogenes [73]. Similarly, in 261 human listeriosis cases among nonpregnant adults and juveniles reported in Britain from 1967 to 1985, 105 (40%) had a malignant condition. Of these 105 patients, 32% had leukemia, 29% had lymphoma, and 39% had other types of malignancies [65]. Elderly patients and those who are receiving immunosuppressive therapy may also account for a substantial proportion of listeriosis cases [65,71,73,96]. For example, in a series of 84 cases reported in 1994 from Australia, 39% of the 59 cases among nonpregnant adults were over the age of 60, 46% were on at least 10 mg of prednisone daily, 29% were on azathioprine or cyclosporin, and 20% had undergone chemotherapy for malignancy [76]. Other disorders accounting for a lower proportion of cases in nonpregnant adults include alcoholism, diabetes, cirrhosis, chronic renal disease, collagen vascular diseases, sarcoidosis, ulcerative colitis, aplastic anemia, and conditions associated with iron overload [34,4 1,53,67,72,73,913. 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 pregnancy-associated

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occurred in persons with at least one underlying illness, although some were conditions such as heart disease that are not traditionally considered immunosuppressive [89]. The most frequently identified diseases or conditions were heart disease (33%), corticosteroid therapy (3 1%), 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. In an earlier report, 30% of patients with meningitis and 11 % of those with bacteremia caused by L. rnonocytogenes had no recognized predisposing condition [72]. In this latter study, however, cases were largely identified through a literature review, and thus may not be comparable to those identified through population-based surveillance. There have been many reports of patients with HIV infection or AIDS who countracted listerial meningitis or bacteremia [ 12,22,24,42,611. 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 persons 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 [89]. Using similar methodology, a prospective, population-based 2-year study in metropolitan Atlanta estimated that the annual incidence of listeriosis was 52 and 115 cases per 100,000 patients for those with HIV infection and AIDS, respectively; these rates were 62 and 145 times the rate among nonpregnant adults not known to be infected with HIV [51]. A 1995 prospective study in Los Angeles estimated the annual incidence to be 9 and 96 cases of listeriosis per 100,000 patients in persons with HIV infection and AIDS, respectively, compared with a rate of 1 per 100,000 in the total population [25]. Differences in risk estimates between these last two studies likely resulted from use of different methods to estimate the total numbers of HIV-infected persons in the study areas. Nonpregnant adults with listeriosis most frequently present with sepsis, meningitis, or meningoencephalitis. Although meningitis is usually reported to be the most common form of listeriosis in adults, recent reports have documented bacteremia to be even more common. 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 [35]. Presenting symptoms in nonpregnant adults with central nervous system listeriosis may include fever, malaise, ataxia, seizures, and altered mental status. Listerial brain stem encephalitis (rhombencephalitis) occurs infrequently and is characterized by asymmetrical cranial nerve deficits, cerebellar signs, and hemiparesis or hemisensory deficits [5,97,101]. Fever is generally present in patients with bacteremia; other nonspecific symptoms such as malaise, fatigue, and abdominal pain may also occur. In meningitis caused by L. monocytogenes, 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. In addition to sepsis, meningitis, and meningoencephalitis, a variety of other clinical manifestations of infection with L. rnonocytogenes have been described. 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 endo-

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carditis [8,33]. Focal infections are rare and usually result from seeding during a preceding bacteremic phase. Several different sites of involvement have been reported, including endophthalmitis [6], septic arthritis [70], osteomyelitis [45], pleural infection [63], and peritonitis [72]. Cutaneous infections without bacteremia have been reported in persons handling infected animals [7S] and in accidentally exposed laboratory workers [2].

Listeriosis During Pregnancy Listeriosis may occur at anytime during pregnancy but is most frequently documented during the third trimester [ 141. Because bacterial cultures are not routinely obtained from spontaneously aborted fetuses or stillborn neonates, it is diflicult 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 [36]; other estimates have varied [3]. 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 (601. Symptoms associated with listeriosis during pregnancy may be nonspecific, and they often manifest as only a mild flu-like 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, However, these women were no more likely than controls to report backache or sore throat [87]. Approximately two thirds of pregnant women with listeriosis during pregnancy have this flu-like prodromal syndrome [ 14,651. These symptoms are associated with the bacteremic phase of infection and represent the optimal time to obtain diagnostic blood cultures. 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. 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 [30,46,56]. Neonatal infection can be prevented by antibiotic treatment during pregnancy. 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 should be given on avoiding high-risk foods. Given the potential adverse consequences of maternal listeriosis and the availability of effective treatment, it is prudent to evaluate all febrile episodes during pregnancy with blood cultures.

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 listeriosis. These clinical forms parallel the pattern of the disease seen in 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

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life. Between 45 and 70% of neonatal listeriosis is of early onset [31,64]; this disease often presents with sepsis rather than meningitis [35]. Less frequently, infants with earlyonset 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 [39]. 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 [35,102]. Infants are usually born healthy and full term to mothers who have had uncomplicated pregnancies. Similar 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 [64]; similarly, active surveillance for listeriosis in the United States in 1986 documented meningitis as the presenting syndrome in 88% of late-onset cases [35]. Mortality rates for both earlyand late-onset disease are usually 20-30% [ 15,57,64]. 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 late-onset disease have been reported following cesarean delivery. Clusters of late-onset disease have been identified in newborn nurseries, suggesting that some nosocomial transmission also occurs [48,55,69,94]. In one outbreak of late-onset disease in Costa Rica, infection was linked to contaminated mineral oil used to bathe newborns [90].

Noninvasive Disease: Gastrointestinal Illness It has been postulated that a noninvasive gastrointestinal illness may occur in normal hosts that consume foods contaminated with an infectious dose of L. monocytogenes, but this has been difficult to establish. Because the organism is commonly found in many foods, in an outbreak setting, recovery of L. monocytogenes from implicated foods is seldom sufficient to verify the source of infection. Moreover, stool specimens from persons with diarrhea are rarely cultured for L. monocytogenes. When such cultures are obtained, the frequency of positive cultures among ill persons must be compared with the proportion of positive stool cultures from well controls, because a substantial minority of persons are asymptomatic fecal carriers of L. monocytogenes. Information from several outbreak investigations suggests that L. monocytogenes may cause a febrile gastroenteritis in normal hosts [86]. The most persuasive evidence comes from a recent investigation of an outbreak of gastroenteritis and fever linked to consumption of chocolate milk served at a picnic [23]. Picnic attendees drank chocolate milk that was later found to be heavily contaminated with L. monocytogenes (109cfu/ mL); 79% of 58 persons who consumed implicated milk from the picnic reported diarrhea, and 72% reported fever. The median incubation period was 20 h (range 9-32 h). The

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same subtype of L. monocytogenes was isolated from the stools of ill persons and the chocolate milk. I11 persons were more likely than well persons to have elevated antilisteriolysin 0 levels and to have stool cultures that yielded L. monocytogenes. None of the individuals involved in this outbreak had a chronic illness or immunodeficiency. Of particular note, three cases of sporadic invasive listeriosis in two other states were also linked to consumption of the implicated chocolate milk; the same subtype of L. monocytogenes as the outbreak strain was isolated from these patients. Other outbreak investigations also support the concept of febrile gastroenteritis being caused by L. rnonocytogenes. In an outbreak of invasive listeriosis in Philadelphia in 1986 and 1987, case-patients were significantly more likely than controls to have reported fever, vomiting, or diarrhea in the week before the case’s positive culture [93]. In another outbreak investigation, two pregnant women who attended a catered party each delivered infants infected with the same strain of L. monocytogenes [80]. Diarrhea or fever was reported by 22% of the 36 party attendees. However, stool cultures were not obtained until several weeks after the party, and the outbreak strain of L. monocytogenes was isolated from only one other party attendee, so a correlation between L. monocytogenes stool carriage and gastrointestinal symptoms could not be definitively established. Finally, in an outbreak of gastroenteritis among immunocompetent adults attending a supper party in Italy, diarrhea and fever occurred in over 70% of the ill party-goers, and two developed bacteremia caused by L. rnonocytogenes [84]. The median incubation period from time of the supper to onset of gastrointestinal symptoms was 18 hours. Although the same strain of L. rnonocytogenes that was isolated from the patients was also cultured from several foods leftover from the supper, no stool specimens collected from ill persons yielded L. rnonocytogenes. The frequency of febrile gastroenteritis caused by L. monocytogenes remains undetermined, as does the infectious dose and characteristics of’the host that are associated with this syndrome. Clinicians and public health officials should consider examining stool cultures for L. rnonocytogenes 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.

EPIDEMIOLOGICAL PATTERNS OF LlSTERlOSlS 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 15 years through epidemiological studies of both epidemic (Table 3) and sporadic illness.

Epidemic Listeriosis The first convincing evidence that listeriosis can be a foodborne disease comes from a 1981 outbreak in Nova Scotia [85]. Thirty-four pregnancy-associated cases and seven cases in nonpregnant adults occurred over a 6-month period in the Maritime Provinces. Twenty-seven percent of the infants who were born alive 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

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TABLE 3 Foodborne Outbreaks of Invasive Listeriosis

Reference

Year

Place

No. of cases (deaths)

20 (5) Massachusetts, US 41 (18) Nova Scotia, Canada 49( 14) Massachusetts, US California, US 142 (48)

44

I979

85

1981

27

I983

56

1985

10 66 80

1983- I987 Switzerland 1988- 1989 United Kingdom 1989 Connecticut,

38

1992

France

84 23

1993 1994

Italy Illinois, US

us

Implicated (or likely) vehicle

0

4b

Coleslaw

83

4b

Pasteurized milk

14

4b

66

4b

-

53

4b 4b

33

4b

0

4b

0 0

1l2b 1 l2b

(Raw vegetables)

Mexican-style cheese 122 (34) Soft cheese Piit6 10 (0)

% of perinatal L. monoctogenes cases se rotype

(Shrimp)

279 (85) Pork tongue in jelly 18 (0) Rice salad 48" (0) Pasteurized chocolate milk

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

patient, and later from two unopened packages of the product. On review of the production process, it was 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 (441. 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 one 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 [27]. Forty-nine cases occurred over a 2-month period, 42 in immunosuppressed adults and 7 in pregnant women; the overall

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case-fatality rate was 29%. Case-patients 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 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 [ 181. In this outbreak, contamination likely occurred during post-pasteurization handling. The largest North American outbreak of listeriosis occurred in Los Angeles County, California, in 1985; 142 cases were detected over an 8-month period (561. Pregnant women accounted for 93 cases, and nonpregnant adults 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 both likely contributed to the contamination. This outbreak provided valuable data on the incubation period of invasive listeriosis, since food histories were available for four patients who had a single known exposure to the implicated cheese. The median incubation period in these patients of 31 days (range 11-70 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 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 [10]. Over one-half of the nonpregnant adult cases had no underlying predisposirig condition; the case-fatality rate for nonpregnant patients was 32% [ 171. 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. Two recent outbreaks in Europe were associated with ready-to-eat meats. In En-

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gland, 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 1987-1989,30-54% were of two subtypes of L. monocytogenes serovar 4b (4bX and 4b phage type 6,7) that had occurred less commonly before and after this period [66]. A microbiological survey showed that L. monocytogenes contamination in meat pit6 from one manufacturer was more frequent (48% of 107 samples of pit6 from the implicated manufacturer vs 4% of 781 samples of pit6 from other manufacturers) and heavy (1 1% of samples of pit6 from manufacturer A with 21000 organismdg vs 0.6% of samples of pit6 from other manufacturers with 2 1000 organisms/ g). Ninety-six percent of pgt6 isolates from manufacturer A 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 pit6 than patients infected with other strains. Warning about pit6 consumption and removal of manufacturer A’s pit6 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 [38]. 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 in the outbreak; 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 [47]. 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 pit&)[37]. 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 [37].

Sporadic Disease-Incidence Although much has been learned about epidemic listeriosis, most cases of human listeriosis occur sporadically. In the United States, voluntary disease reporting and hospital discharge data have been used to estimate the number of sporadic listeriosis cases [21]. However, such methods are generally insensitive and result in underestimates of the true incidence of listeriosis in the population. Beginning in 1986, active surveillance for listeriosis in the United States has been done by the Centers for Disease Control and Prevention (CDC) in several well-defined populations representing different geographical areas. 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 [35,89]. The study population ranged from 19 to 34 million depending on the number of study sites participating each year [99]. In 1986, in an aggregate population of 34 million persons, the annual incidence of

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listeriosis was 7 cases per million; of the 246 cases that occurred, 67 (27%) were perinatal and 179 (73%) were nonperinatal [35]. 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. The estimated sensitivity of the surveillance system was 93%. From 1988 to 1990, in an aggregate population of 19 million, the annualized incidence of listeriosis was 7.4 per million. Incidence rates varied by geographical site, with the highest rate being observed in San Francisco (9.3 per million) and the lowest rate in Oklahoma (4.8 per million) [89]. No clear seasonal trends were noted. In the most recent analysis of combined data from 1989 to 1993 for a study population of 19 million, the annualized incidence of listeriosis decreased from 7.9 per million in 1989 to 4.4 per million in 1993; this decrease was distributed uniformly throughout the different geographical areas [99]. Based on these data, projected estimates of the number of cases and deaths from listeriosis in the entire United States population were 1965 cases and 489 deaths in 1989, decreasing to 1092 cases and 248 deaths in 1993. Case-fatality rates (23-25%) did not vary significantly by year. Pregnancy-associated cases accounted for about one third of all cases each year. Both perinatal and nonperinatal case rates showed similar decreases over the 4-year period. Serotypes 4b (43%), 1/2b (35%), and 1/2a (20%) accounted for almost all infections. The observed decrease in incidence may have resulted from enhanced listeriosis prevention efforts by the US food industry, including enforcement by regulatory agencies of a zero-tolerance policy for processed meat, and intensified clean-up programs in meat-processing facilities. Published dietary recommendations for consumers may also have contributed to the decreased disease incidence [20,28,29]. Numerous reports of listeriosis incidence rates in other cities and countries have been published. For example, based on a search of hospital records, the estimated incidence of listeriosis in the English city of Bristol from 1983 through 1992 was 3.5 per million [50]. In England, Wales, and Northern Ireland in 1991, the estimated annual incidence based on passive case reporting was 1.8 per million [71]. In Denmark, monitoring of laboratory-diagnosed cases during the 1980s resulted in an estimate of six to seven cases per million per year [49]. Listeriosis incidence estimates from a number of countries have been recently summarized [81]. These rates should be interpreted in the context of the methods used for case ascertainment and reporting in each country.

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- 1987 study, 82 patients and 239 controls were enrolled. 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 [92]. 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 isoenzyme 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,

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and unopened packages of turkey frankfurters at a retail store [ 191. Subsequent investigation of the turkey frankfurter production facility found that cultures from a conveyor belt transporting finished frankfurters yielded the case strain of L. monocytogenes [ 1071. Systematic culturing at various points in the production process identified likely points where L. monocytogenes was being introduced into the product and suggested appropriate control points for reducing contamination in such food processing 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 [89]. 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 [78]. L. monocytogenes was isolated from at least one food in the refrigerators of 64% of patients. Highest contamination rates among the 20 13 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 enzyme 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 pgt6 were the only risk factors identified [49]. 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 [79]. Since 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 [ 1001.

Diagnosis of listeriosis depends on isolation of L. monocytogenes from a normally sterile site such as blood or cerebrospinal fluid. Since 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 usually not helpful, since asymptomatic carriage occurs. 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

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5% sheep, horse, or rabbit blood. The organism is usually identified within 36 h. Isolation of the organism from other sources such as stool specimens that contain large numbers of competing microorganisms is 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 0 have been useful in some epidemiological investigations [23] and have been used to support the diagnosis in culture-negative listeriosis of the central nervous system [32].

TREATMENT Controlled trials to determine the optimal antibiotic therapy for listeriosis have not been done. Bacteriostatic drugs such as chloramphenicol or tetracycline have been associated with high treatment failure rates, and they are not recommended [98]. Generally, ampicillin or penicillin has been recommended as the drug of choice. However, relapses have been reported in immunosuppressed patients after 2 weeks of penicillin therapy [ 1031. 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 [98]. Intracellular concentrations of penicillin may be insufficient for complete eradication. Since many immunosuppressed patients have a decreased ability to clear infected cells, antibiotic treatment for 3 to 6 weeks may be prudent [4]. 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; 2-3 weeks for neonatal listeriosis; 2-4 weeks for nonimmunosuppressed adults with meningitis and bacteremia; and longer for complicated infections such as endocarditis. Although experimental evidence suggests that aminoglycosides are synergistic with ampicillin or penicillin in vitro, they penetrate cells poorly and may be ineffective in the living host. L. monocytogenes continues to grow in cells despite high extracelluar concentrations of aminoglycosides [43]. Trimethoprim-sulfamethoxazolereadily enters cells and kills L. monocytogenes, and it may be the most effective treatment. This drug combination has proved effective in patients with listeriosis who have hypersensitivity to penicillin [ 5 ] .

Since L. monocytogenes is commonly found in the environment, avoiding exposure presents a difficult challenge. 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 [20]. For persons who are at increased risk for listeriosis, including those who are pregnant or immunocompromised, there are specific dietary measures that can be taken to decrease risk. Such persons should avoid foods epidemiologically linked with listeriosis, including p2t6 and soft cheeses such as Brie, Camembert, blue-veined, or Mexican-style cheese. In

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addition, ready-to-eat foods such as frankfurters and leftover foods should be cooked until steaming hot before being eaten. These persons may also choose to avoid delicatessen foods or thoroughly reheat cold cuts before eating. 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 that are consumed without further cooking. The food industry should continue to develop and implement hazard analysis critical control point programs (HACCP) to minimize the presence of L. monocytogenes at important points in the processing, distribution, and marketing of processed foods [ 11.

REFERENCES 1. Anonymous. 1991. Listeria monocytogenes: recommendations by the National Advisory Committee on microbiological criteria for foods. Int. J. Food. Microbiol. 14:185-246. 2. 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. 3. Anton, W. 1934. Kritisch-experimentaller Beitrag zur Biologie des Bakterium monocytogenes. Zentralb. Bakteriol. Mikrobiol. Hyg. A 131:89-103. 4. Armstrong, D. 1995. Listeria monocytogenes, In G.L. Mandell, J.E. Bennett, R. D o h , eds. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. New York, NY: Churchill Livingstone, pp. 1880- 1885. 5. Armstrong, R.W., and P.C. Fung. 1993. Brainstem encephalitis (rhombenephalitis) due to Listeria monocytogenes: case report and review. Clin. Infect. Dis. 16:689-702. 6. Ballen, P.H., F.R. Loffredo, and B. Painter. 1979. Listeria endophthalmitis. Arch. Ophthalmol. 97:lOl-102. 7. 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. 8. Bassan, R. 1986. Bacterial endocarditis produced by Listeria monocytogenes: case presentation and review of the literature. Am. J. Clin. Pathol. 63522-527. 9. 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. 10. Bille, J. 1990. Epidemiology of human listeriosis in Europe, with special reference to the Swiss outbreak. In: A.J. Miller, J.L. Smith., and G.A. Somkuti, eds. Foodborne Listeriosis. Amsterdam: Elsevier, pp. 7 1-74. 11. Bille, J., and J. Rocourt. 1996. WHO international multicenter Listeria monocytogenes subtyping study-rationale and set-up of the study. Int. J. Food Microbiol. 32:25 1-262. 12. Bizet, C., D. Mechali, J. Rocourt, and F. Fraisse. 1989. Listeria monocytogenes bacteremia in AIDS. Lancet 1:501. 13. Bojsen-MQller, J. 1972. Human listeriosis: diagnostic, epidemiological, and clinical studies. Acta Pathol. Microbiol. Scand. 229 (Sect B. suppl):72-92. 14. Bortolussi, R. 1990. Neonatal listeriosis. Semin. Perinatol. 14(suppl.):44-48. 15. Boucher, M., and M.L. Yonekura. 1984. Listeria meningitis during pregnancy. Am. J. Perinatol. 1:312-318. 16. Buchner, L.H., and S.S. Schneier. 1968. Clinical and laboratory aspects of Listeria monocytogenes infection, with a report of ten cases. Am. J. Med. 45:904-921.

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Pathogenesis of Listeria monocytogen es MICHAEL KUHNAND WERNER GOEBEL University of Wurzburg, Wurzburg, Germany

INTRODUCTION Studies which aimed to unravel the pathogenicity of Listeria monocytogenrs and its interaction with host cells on the cellular, molecular, and genetic levels were initiated only 10 years ago. The early studies used transposon mutagenesis and infection of primary and established cell lines (epithelia] cell, fibroblast, and macrophage) to obtain insights into the interaction of L. monocytogenes with eukaryotic host cells (reviewed in refs. 1 1 1 and 174). Development of new genetic tools now allows manipulation of L. monocytogenes, which has, together with the cell culture models, greatly broadened our understanding of the molecular and cell biology of L. monocytogenes infections. Most studies on the cell biology of L. monocytogenes infections used epithelia-like and macrophage-like cell lines [55,143,194]. Macrophages actively ingest L. monocytogenes, but internalization of the bacterium by normally nonphagocytic cells is triggered by L. moncicytogenes-specific products. Besides the internalization step, the intracellular life cycle of listeriae in phagocytes or normally nonphagocytic mammalian cells is, however, very similar. The pathogen first appears in a vacuole, which is subsequently lysed by most of the ingested bacteria allowing L. monocytogenes to escape into the cytoplasm. Whereas listeriae begin to replicate in the cytoplasm, cells remaining in the phagosome are killed and digested. Concomitant with the onset of intracellular replication, L. monocytogenes induces nucleation of host actin filaments which form a cloud around the bacterial cell. The actin filaments are then rearranged to a polar tail which consists of short actin 97

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FIGURE1 The intracellular life cycle o f Listeria monocytogenes. See text for details. (Adapted from Refs. 14 and 194 and kindly provided by J. Kreft.)

filaments and other host actin binding proteins which stabilize this structure. Formation of the actin tail at one pole of the bacterial cell produces the propulsive force which moves the listeriae through the cytoplasm of the host cell. This bacterial movement requires continuous de novo actin polymerization. Listeriae which reach the surface of the infected host cell induce formation of pseudopod-like structures with the bacterium at the tip and the actin tail behind. These pseudopods are taken up by neighboring cells. The bacteria thus entering the neighboring cells are within a vacuole that is surrounded by a double membrane which is subsequently lysed to release the listeriae into the cytoplasm of the new host cell (Fig. 1). Most 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 PrfAdependent virulence gene cluster. The cluster comprises six well-characterized genes, p$A, plcA, hly, mpl, actA, and plcB (Fig. 2 and Table 1) and three small open reading frames (ORFs) of unknown functions downstream of plcB, called X, Y, and Z. 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, is located the prs gene encoding a phosphoribosyl-pyrophosphate synthetase [72,115]. The Zdh gene coding for lactate dehy-

FIGURE2 The virulence gene cluster from L. monocytogenes. Black boxes represent PrfA-boxes and arrows represent transcripts. (Kindly provided b y F. Engelbrecht.)

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Pathogenesis of Listeria monocytogenes

TABLE 1 Features of the L. rnonocytogenes Virulence Determinantsa Gene P@Ah plcA hly' mplh actA plcB' inlA' inlB' inlCh iap lmaAh lmsodh cat' clpc'

Protein

ORF bp

MA PI-PLC LLO MPl ActA PC-PLC Internalin InlB InlC P60 LmaA SOD Catalase ClpC

705 95 1 1617 1533 1917 867 2400 1890 89 1 1452 5 10 606 1464 2478

mRNA (kb) 0.912.1 1.112.1 1.8 1.615.7 2.915.7 2.915.7 2.915 5 1 1.5

2.5 14.5

AAh

235 317 529 5 10 639 289 800

630 297 484 170 202 488 826

MWcal'

MWd

PI'

27.1 36.3 58.6 57.4 67.0

27 34 58 67/35 92 32129 95 65 30 60 21 24 67

7.3 10.1

86.0 71.1 29.6 50.7 18.0 22.6 55.9 91.0

See text for references. Amino acids (including signal sequence). Molecular weight (MW) in kD (including signal sequence) and isoelectric point (PI) as calculated from sequence data. Molecular weight in kDa as calculated from SDS-PAGE Presence of signal sequence. M A regulated expression. g Temperature-regulated expression. From strain L. monocytogenes EGD. From strain L. monocyfogenes L028. From L. seeligeri.

a

I

J

6.6 4.4 10.1 6.7 10.0 4.2 7.8

Sig. Seq.'

+ + + -

+ + + + +

PrfA Reg'

+ + + + + + + + +

Temp. Reg .g

-

-

+

100

Kuhn and Goebel

drogenase together with the orfs A and B [72,196] mark the “right” border of the gene cluster downstream from plcB and the small orfs X, Y, and Z. The products of these virulence genes are: listeriolysin (encoded by hly), a phosphatidylinositol-specific phospholipase C (pZcA),a phosphatidylcholine-specificphospholipase C (pZcB),a metalloprotease (mpl), ActA, a protein involved in actin polymerization (actA), and the positive regulatory factor PrfA (prfA). The internalin genes inlA, inlB, and inlC coding for internalin, InlB, and InlC, respectively, the iap gene coding for p60, and other genes suggested to play a role in virulence are located outside the virulence gene cluster. Most of these are, however, connected to the virulence cluster genes, as they are also regulated by the transcriptional activater PrfA (see below).

MOLECULAR ASPECTS OF ADHESION AND INVASION Uptake of L. monocytogenes by macrophages of different origin is well documented [ 113,127,1521.Invasion by L. monocytogenes of different, normally nonphagocytic mammalian cell types including murine and human fibroblasts [47,83,113,152], murine and human epithelial cells [4,47,55,152], murine hepatocytes [40,20 I], and human endothelial cells [45,108,175,176] also has been described along with invasion and survival of L. monocytogenes in protozoa of the genera Acanthamoeba and Tetrahymena [ 1261.

Invasion of Nonprofessional Phagocytic Cells Proteins Internalin (InlA), InlB, and lnlC Transposon mutagenesis and an appropriate in vitro invasion assay using Caco-2 epithelial cells resulted in identification of internalin, a surface protein of L. monocytogenes mediating bacterial invasion into epithelial cells (541. The mutants identified exhibited a lower invasive capacity than the wild-type strain when tested on different cells. In addition to reduced invasiveness, mutants also lost the ability to adhere to eukaryotic cells. Transposon insertions were mapped and 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. This experiment shows that the inlA gene product is necessary and sufficient to mediate invasion. Internalin is an acidic protein of 800 amino acids [4 1,541 which possesses two extended repeat domains. Domain A consists of 15 repeats of 22 amino acids each, whereas domain B consists of 2.5 repeats of about 70 amino acids each. The InlA protein has a typical N-terminal transport signal sequence, and in the C-terminal part, it has a cell wallspanning region followed by a hydrophobic sequence which represents a putative membrane anchor (Fig. 3) [41]. Internalin was originally shown to be a L. monocytogenes surface protein [54], but substantial amounts of the protein are also found in the supernatant liquid [42]. Recently it was shown that surface location is necessary for internalin to mediate entry of L. monocytogenes into epithelial cells by facilitating direct contact between the bacterium and the host cell [ 1 181. The eukaryotic receptor for internalin was identified as E-cadherin by a biochemical approach using matrix-bound purified internalin to isolate the internalin ligand from epithelial membrane proteins [ 1361. E-cadherin, a member of the cadherin family, is mainly expressed at the basolateral site of enterocytes [ 191. It binds internalin directly, and its location on the basolateral membrane of epithelial cells is in line with previous observa-

Pathogenesis of Listeria monocytogenes

29

N

650

711

LR

8 SR

C*

I

I

I

238

30 63

InlC

462

357

SS

InlB

101

I

1

I

399

466

559

8 SR

SS

N

C

I I

34 62

I 234

FIGURE 3 Schematic structure of the members of the internalin family: InlA, InlB, and

InlC. SS, signal peptide; SR, short leucine-rich repeat; LR, long repeat; MA, membrane anchor.

tions suggesting the basolateral membrane as an entry site for L. monocytogenes [ 1901. Antibodies directed against the leucine-rich repeat region of internalin block entry of L. monocytogenes into cells expressing E-cadherin, thereby uriderlining the importance of the repeat regions of internalin for its function as an invasin [ 1361. InlB, a 630-amino acid protein, also carries an N-terminal transport signal sequence and repeat domains, but, in contrast to InlA, has no obvious membrane anchor and no cell wall-spanning region (Fig. 3) [41]. Nevertheless, InlB is a listerial surface protein, but the mechanism(s) which target it to the bacterial surface are unknown [40]. Recently it was shown that inlB mutants still expressing inlA are invasive for human enterocytelike Caco-2 cells. InlB is required but is obviously not sufficient to promote entry of L. monocytogmes into hepatocytes 1401, but results concerning its role in the entry of epithelial cells are controversial [40,125]. Invasion of fibroblasts by L. monocytogenes seems to be independent of either inlA or inlB and even double mutants are still invasive for fibroblasts, suggesting that different cell type-specific adhesion and invasion systems are present in I,. monocytogenes [41,125]. The high sequence sirnilarity of inlA and inlB indicates that the two genes originate from a common ancestral gene and represent members of a gene family in L. monocytogenes. One additional member of this gene family, called inlC, was cloned and characterized and encodes a small (297 amino acids), secreted protein (Fig. 3) which is mainly expressed at later stages of the intracellular life cycle and obviously not involved in the entry process into epithelia1 cells [48]. InlC was identified independently and called Irp (the gene irpA), and it also was found present in the supernatant liquid of the closely related species L. ivanovii [39,124].

Protein p60 Rough mutants of L. monocytogenes expressing reduced amounts of a 60-kD extracellular protein, termed p60, show appreciably reduced uptake by 3T6 fibroblast cells [ 1091. These “p60 mutants” (also referred to as R-mutants because of their rough colony appearance) from long cell chains which possess double septa between the individual cells. Treatment of L. monocytogenes R-mutants with partially purified p60 protein disassociates these cell chains into normal-sized single bacteria which are again invasive for fibroblasts. Ultrasonication, which leads to physical disruption of the cell chains, produces similar single cells

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102

which are, however, noninvasive. On treatment with wild-type p60, these ultrasonicated mutant cells are again able to invade fibroblasts [16,109]. Reduced invasiveness of 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 on disruption of the bacterial cell chains by ultrasonication without addition of p60 [ 161. Protein p60 is a major secreted protein of all L. monocytogenes isolates [16,109], but it is also found on the cell surface of L. monocytogenes [163]. In contrast to other virulence factors, p60 is also an essential metabolic enzyme of L. rnonocytogenes, since it possesses murein hydrolase activity which appears to be involved in a late step of cell division [202]. The gene coding for this obviously bifunctional protein, called iap (invasion associated protein), was cloned from a L. monocytogenes gene bank using an anti-p60 antiserum and sequenced [103]. Its expression is controlled on the posttranscriptional level by a yet unknown mechanism [102]. The amino acid sequence of p60 predicts 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 which are 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 [103,202]. A stretch of 50 amino acids in the N-terminal part of the protein which is also present in p60 proteins of the other Listeria species shows homology to sequences found in an autolysin of Streptococcusfaecalis (Enterococcusfaecalis). In this species, the sequence is thought to represent a possible murein binding site. Interestingly, the sequence motive occurs twice in the p60 protein of L. monocytogenes [202].

Protein ActA The listerial surface protein ActA (Fig. 4), a major virulence factor primarily involved in actin-based motility [38,99] (see below for details), was recently suggested also to play a role in internalin-independent uptake of L. monocytogenes by epithelial cells [3,108]. Analysis of the invasive capacity of an inZA deletion mutant and mutant PKP-1 without the virulence gene cluster genes [48] complemented with multiple copies of PrfA strongly suggest that PrfA-dependent proteins from the virulence gene cluster may cause invasion of Caco-2 cells in the absence of InlA [ 1081. Such an ActA-promoted attachment and invasion of Chinese hamster ovary (CHO) epithelia-like cells as well as IC-21 murine macrophages was mediated by interaction of the listerial surface protein ActA with a heparan-sulfate proteoglycan receptor [3]. Electrostatic interactions between heparan sulfate and positively charged residues in the N-terminal part of ActA could presumably result in low-stringency binding to cell surface proteoglycan receptors which are widely distributed in mammalian cells [3]. Whether the proposed low-stringency binding of L. monocytogenes to heparan sulfate proteoglycan receptors triggers uptake directly or results in adequate presentation of other bacterial factors to the host cell membrane which ultimately lead to phagocytosis remains to be clarified.

29

128

151

263

390

613 630

FIGURE4 Schematic structure of the bifunctional protein ActA. SS, signal peptide; AP, region critical for actin polymerization; PRR, proline-rich repeat; TA, transmembrane domain.

Pathogenesis of Listeria monocytogenes

103

Uptake by Professional Phagocytic Cells Macrophages of different origin were used in in vitro studies to analyze mechanisms of L. rnonocytogenes uptake by macrophages, which are generally assumed to take up L. rnonocytogenes by conventional phagocytosis involving actin-polymerization. Complement factors C I q and C3 are deposited on the bacterial surface and stimulate L. monocytogenes uptake by binding the bacteria to the respective receptors [2,27,43]. The kinetics of uptake and intracellular killing of L. monocytogenes by macrophages were determined [30,3I , 1551. Macrophages ingest L. rnonocytogenes very rapidly, and intracellular killing starts shortly after phagocytosis and leads to destruction of most of the ingested bacteria. In a single macrophage, both 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 listeriae and their escape from the acidified phagosome before phagosome-lysosome fusion occurs. The result of this competition is a population of cytoplasmic listeriae able to grow inside the macrophage. The route of uptake by macrophages may also be important for the fate of invading bacteria. As demonstrated by Drevets et al. [44], the mode of uptake is critical for subsequent survival, since L. rnonocytogenes taken up in the presence of complement C3 leads to enhanced killing of the bacterium. Whether the surface protein InlA contributes substantially to triggering of phagocytosis of L. monocytogenes by macrophages is still under debate. Using bone marrowderived macrophages, InlA had only a slight effect on invasion, since an inlAB mutant still showed more than 60% invasion when compared with the wild-type strain [85]. Uptake of L. rnonocytogenes by the mouse macrophage-like cell line J774A.1 was inhibited by at least 50% by the pretreatment of L. rnonocytogenes with anti-InlA antibodies, and recombinant InlA specifically was bound to the macrophages [ 1661. Recently it was suggested that the listerial protein p60 might enhance phagocytosis by macrophages. Salmonella typhimuriurn, expressing and secreting p60, seems to be more invasive for phagocytic cells but not for enterocytes [85]. In line with this assumption is that pretreatment of L. rnonocytogenes with a polyclonal anti-p60 antiserum inhibits uptake of listeriae by a macrophage-like cell line [85]. Another factor that could be involved in attachment and invasion of 1,. rnonocytogenes in macrophages is the listerial cell wall polymer, lipoteichoic acid. L. rnonocytogenes binds strongly to the macrophage scavenger receptor most likely via lipoteichoic acid [46,73]. This interaction may also trigger conventional receptormediated phagocytosis of L. rnonocytogenes.

Tissue Tropism and Mechanism of Invasion The cellular mechanisms of L. rnonocytogenes invasion are still largely unknown. Uptake of L. rnonocytogenes by macrophages and other mammalian cells is dependent on functional actin microfilaments, since invasion is blocked by treatment with actin-depolymerizing drugs such as cytochalasins [55,113]. Additionally, entry can be blocked by tyrosine kinase inhibitors such as genistein [ 160,189,I971 and the tyrosin phosphatase inhibitor vanadate [ 1071. For epithelia] cell invasion, the signaling events are probably triggered by binding of internalin to its receptor, E-cadherin. Links between cadherins and signaling pathways have recently been reported [ 1301. Identification of E-cadherin as the receptor for internalin and electron microscopic observations strongly support the basolateral membrane of the epithelia] cell as the site for listerial attachment and entry [ 136,1901.However,

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Kuhn and Goebel

entry by the apical surface of polarized Caco-2 cells was also observed [91]. Attachment of wild-type L. monocytogenes to these host cells induces structural modification(s) in microvilli which are not observed with less invasive p$A mutants of L. monocytogenes (see below) [66,91]. Whether the apical route of infection of polarized cells is also internalin-dependent is not known, and the overall significance of each of these two proposed entry sites of epithelial cells is still under debate. The picture of listerial invasion is becoming more and more complex, since aforementioned data point to different invasion sites on the same cell as well as to different mechanisms involved in invasion of different cell types. These observations are in line with the idea of tissue tropism, with different known and unknown bacterial factors like internalins and p60 being responsible for invasion of different tissues during infection. Presently the data can be summarized as follows: Protein p60 is clearly not involved in epithelial cell invasion, but it may play a role in fibroblast invasion [16,109]. SalrnoneZZa strains expressing p60 are taken up significantly better as the control strains by macrophages and hepatocytes, also indicating a role of p60 for invasion of these cell types [85]. Internalin is an important factor in epithelial cell and hepatocyte infection, but the capacity of InlA alone to promote epithelial cell entry is still under debate, since conflicting results have been published [40,54,125]. InlB, originally proposed as a specific factor for hepatocyte invasion [40], is now suggested also to be critical for epithelial cell invasion, but both internalins play no role in fibroblast invasion [ 1251. The eukaryotic receptor for InlB is not known, but the recent report of InlB-dependent stimulation of phosphoinositide-3kinase being required for efficient L. monocytogenes invasion of nonprofessional phagocytic cells [87] underlines the importance of InlB in the invasion process. High-efficiency binding to and invasion of human endothelial cells by L. monocytogenes is dependent on one or both inZAB gene products. However, low-level invasion in the absence of both internalins was also observed [45]. Internalin-independent invasion was also reported for the dentritic cell line CB1 [76] and at low levels also for Caco-2 epithelial cells [57]. The role of the small internalin family member InlC (Irp), if any, in invasion is unclear [48]. L. monocytogenes can spread from macrophages to endothelial cells by direct transfer of the bacteria from one cell to the other [45]. Because of its expression in the late stages of the infectious cycle, InlC was thought to be involved in this type of heterologous cellto-cell spreading event [48]. The in vivo significance of these listerial proteins is even less clear. InZAB as well as iap mutants are clearly impaired in virulence in the mouse model [40,85]. However, an inlAB mutant was only transiently impaired in persistence in the liver and behaved like the wild-type in spleens and lymph nodes of infected mice [40]. In a different study, however, inZAB mutants were only rarely found inside hepatocytes, compared with the wild-type strain, indicating a role for the inlAB locus in hepatocyte invasion in vivo [58]. In contrast, Gregory et al. [74] found that the inlAB operon of L. monocytogenes is not required for entry into hepatic cells in vivo.

ESCAPE FROM THE PHAGOCYTIC VACUOLE AND INTRACELLULAR GROWTH Hemolytic activity detected around colonies of L. monocytogenes growing on blood-agar plates was long assumed to represent a major virulence determinant, since all clinical isolates of L. monocytogenes show this hemolytic phenotype. The hemolytic activity re-

Pathogenesis of Listeria monocytogenes

105

sults from the action of a cytolysin, called listeriolysin 0 (LLO). In experimental infections, all virulent strains were hemolytic, whereas nonhemolytic strains were avirulent. Nonhemolytic mutants which were obtained after transposon rnutagenesis using the conjugative transposons Tn1545 or Tn916 [56,92,152] always proved to be avirulent in the mouse model. Virulence is restored in hemolytic revertants, which have lost the transposon insertion or by introduction of the cloned hly gene into a nonhemolytic L. monocytogenes transposon mutant [25]. Despite the clear correlation between hemolysis and virulence, the level of hemolysin production in vitro is not directly proportional to virulence of producing strains in the mouse [94], suggesting that synthesis of LLO under intracellular conditions is different from that observed under extracellular growth conditions. Listeriolysin 0, a secreted protein of 58-60 kD, belongs to a family of pore-forming, sulfhydryl-activated cytolysins for which streptolysin 0 is the prototype [ 1801. All members of this family are inhibited by low concentrations of cholesterol and oxygen and activated by reducing agents like DTT. Cholesterol is considered as a receptor for these cytolysins, since this component inhibits pore formation and toxicity. On addition to erythrocytes, toxin monomers oligomerise in the target cell membrane to form stable pores which can be visualized by electron microscopy [ 1481. Listeriolysin 0 has been purified to homogeneity and its toxicity, as determined by intraperitoneal injection in the mouse, shows a LDSoof 1.7 pg per mouse. Optimal hemolytic activity is found at pH 5.5, a pH value which is much lower than that determined for the other SH-activated cytolysins [60], a property which is in accord with the function of LLO in the acidified phagosome (see below). The gene encoding LLO, hly, was cloned from strains of different serovars of L. monocytogvnes and sequenced [35,132,137]. The deduced amino acid sequence for LLO yielded 529 amino acids, including a N-terminal signal sequence of 25 amino acids. As expected, the sequence shows extended homologies with the protein sequences of other SH-activated cytolysins. The highest homology is observed in the C-terminal part and includes a highly conserved undecapeptide containing the unique cysteine which was thought to be essential for cytolytic activity. Site-directed mutagenesis revealed, however, that cysteine is not essential for hemolytic activity. In contrast, a tryptophan residue, in close vicinity to cysteine, appears to be required for both hemolytic activity and virulence [139]. 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 following the fate of the listeriae in liver and spleen. In contrast to the wild-type strain, nonhemolytic mutants are eliminated from these organs within a few hours without eliciting protective immunity [25,56,92,152]. The role of LLO in intracellular survival was determined using different mouse and human cell lines. In the human enterocyte-like cell line Caco-2 [55], mouse 3T6 fibroblasts [ I 131, and mouse CL.7 fibroblasts [152], nonhemolytic L. monocytogenes mutants were as invasive as isogenic wild-type strains. The nonhemolytic mutants are, however, incapable of intracellular growth and survival within these host cells and also in mouse peritonea1 macrophages [ 1 131, mouse bone marrow-derived macrophages [ 1521, and the mouse macrophage-like cell line 5774 [ 1521. Electron microscopy of infected macrophages and epithelia1 cells reveals that nonhemolytic L, monocytogenes mutants which are found inside cells are unable to open the phagosome to escape into the cytoplasm of the host cells [55,194]. Bafilomycin treatment inhibits vacuolar acidification and prevents L. monocytogenes from escaping phagosomes of infected Caco-2 cells. These findings further support the importance of the low pH activity

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optimum of LLO for its role as a vacuole opener [24]. Taken together, these results suggest that hemolytic activity is indispensable for lysis of the phagosomal membrane. Additional evidence for LLO being essential for lysis of the phagosomal membrane and for intracellular growth was obtained by infection of macrophages with a Bacillus subtilis strain expressing LLO [7]. 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. Recently, Portnoy and coworkers [ 88,891 analyzed the role of LLO by constructing L. monocytogenes strains which secrete the closely related extracellular cytolysin perfringolysin instead of LLO. Such a strain escaped from the vacuole but damaged the host cell. Using an elegant selection procedure, mutants were isolated which did not damage the host cell on perfringolysin expression in the cytoplasm. The mutated perfringolysins were either less hemolytic at neutral pH, generally less active, or had a shorter half-life in the cytoplasm. Thus the low activity of LLO at neutral pH values and its short half-life in the cytoplasm are critical parameters of its suitability as a phagosome opener without concomitant cytotoxicity. Strains expressing mutated perfringolysins which allow intracelMar growth without cell damage are, however, totally avirulent. This unexpected finding points to additional functions for LLO in converting the host cell cytoplasm into a suitable growth compartment [ 64,891. Fusion of L. monocytogenes-containing phagosomes with endosomes has been observed in electron microscopy studies [ 1941.However, it is not known whether such an event is necessary for L. monocytogenes to progress through its intracellular life cycle. The recent description of rab5-regulated fusion of L. manocytogenes-containing phagosomes with endosomes and the observation that live L. monocytogenes upregulates this process by recruiting rab5 to the membrane strongly argues for phagogosome-lysosome fusion as being an important step in the life cycle of L. monocytogenes [l]. Listeriolysin 0-independent escape of L. monocytogenes from primary vacuoles in human epithelia1 cells [ 1521 is mediated by the phosphatidylcholine-specific phospholipase C (PC-PLC) and a metalloprotease [ 1 291. The phosphatidylinositol-specific phospholipase C (PI-PLC) contributes to vacuole escape in other cells like bone marrow-derived macrophages [ 181. Phospholipase activity of L. monocytogenes cultures was first observed as a zone of opacity surrounding colonies on egg yolk agar [53]. Transposon mutants of L. monocytogenes lacking phospholipase activity were identified by formation of small plaques on fibroblast cell monolayers [ 1871 and by reduced hemolysis on blood-agar plates [93], indicating a participation of phospholipase activity in hemolysis. One transposon insertion was mapped in an ORF located adjacent to the hly gene on the chromosome of L. monocytogenes [ 138,1871. The gene, plcA, was cloned 17,119,13I ] and it encoded a protein of 34 kD which exhibits high homology to several gram-positive phospholipases and contains a typical transport signal sequence. The enzyme called phosphatidylinositolspecific phospholipase C (PI-PLC) was purified from culture supernatant liquids of an overexpressing L. monocytogenes strain [ 681 and was highly specific for phosphatidylinositol with no detectable activity on phosphatidylethanolamine, phosphatidylcholine, or phosphatidylserine. It also does not cleave phosphatidy linositol-4-phosphate or phosphatidylinositol-4,5-bisphosphate,but it is active, albeit with low specific activity, on glycosylated phosphatidylinositol-anchored proteins [59]. Besides the highly specific PI-PLC, L. monocytogenes produces a second phopholipase C which hydrolyzes phosphatidylcholine (lecithin), and it is thus a phosphatidylcho-

Pafhogenesis of Listeria monocytogenes

107

line-specific phospholipase C (PC-PLC) or lecithinase [611, also called broad-spectrum phospholipase C. A 32-kD protein was detected in the supernatant liquid of a L. monocytogenes culture which showed phospholipase activity on egg yolk overlays [93]. The protein was purified to homogeneity [61,67] and was a zinc-dependent phospholipase C of 29 kD. The pH optimum of this enzyme is between pH 6 and 7, and its activity is stimulated by 0.5 M NaCl and 0.05 mM ZnSO,. Besides 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 [61]. The gene plcB, encoding PC-PLC, is part of the lecithinase operon which consists of mpl, actA, plcB, and the three small ORFs, X, Y, and Z. The gene was cloned and sequenced [196], and the deduced amino acid sequence yielded 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-kD precursor of PC-PLC occurs after secretion, since both forms of the protein can be found in the supernatant liquid and is obviously accomplished by the metalloprotease of L. rnonocytogenes [ 153,1541. 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 [ 181. 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. However, cooperative membrane permeabilization by PI-PLC and LLO in in vitro assays is independent of phospholipid hydrolysis, since composition of artificial membranes used as targets does not influence the permeabilization activity of PI-PLC when acting together with LLO [69]. To further assess the role of PI-PLC, the plcA gene was expressed in L. innocua, which lacks the prJA-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 5774 macrophages, but it exhibits limited intracellular growth inside vacuoles which appear to be structurally intact [ 1691. These data suggest that PI-PLC alone is unable to open the phagosomal membrane but affects the vacuole in a way which alters its function but not its structure. The role of PC-PLC in escape from vacuoles is not clear and differs between cell types. In bone marrow-derived macrophages, PC-PLC has no role in lysis of the vacuole [ 1771. However, in the human Henle 407 epithelia-like cell line, where escape of L. monocytogenes occurs at low efficiency independent from LLO [ 129,1521, 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 [129]. The way in which the metalloprotease contributes to pathogenicity and intracellular replication of L. rnonocytogenes is still unclear. Transposon mutants in the rnpl gene are less virulent but grow normally inside mammalian cell lines [ 1541. The reduced virulence was attributed to the lack of proteolytic processing of the 32-kD PC-PLC proform [153,183]. However, mutants within frame deletions in rnpl, also impaired in PC-PLC maturation and ActA degradation, are as virulent as the isogenic wild-type strain when injected intraperitoneally in the mouse (D. A. Portnoy, personal communication) [9]. The

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way in which Mpl contributes to lysis of the vacuole in Henle 407 cells is not known, but the most favorable hypothesis suggests that Mpl is necessary to activate PC-PLC as shown in broth culture [ 1541. Located immediately downstream of the hly gene, the mpl gene [36,134] encoding a zinc-dependent metalloprotease, is the first gene of the lecithinase operon [36,134,196]. The deduced amino acid sequence of this protease shows high homology to several metalloproteases from Bacillus species and yields 5 10 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 35-kD mature protein [36,134]. A 60-kD protein is detected with an antiserum raised against Bacillus stearothermophilus thermolysin which probably represents the proform of the metalloprotease. Only small amounts of the postulated 35-kD mature form of the protein were detected in the supernatant liquid of a L. monocytogenes culture [36]. The role of the virulence gene cluster products LLO, PI-PLC, PC-PLC, and Mpl in escape from the phagocytic vacuole and in intracellular growth has been analyzed in some detail during the last decade, as just described. A ClpC ATPase of L. monocytogenes was recently identified as a new type of virulence factor being involved in intracellular multiplication. The gene encoding the ClpC ATPase, called clpC, was identified by Tn917 mutagenesis with selection of mutants dependent on iron [161]. The clpC mutants are highly susceptible to stress from 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 [ 1621. Molecular mechanisms by which the ClpC ATPase of L. rnonocytogenes protects against stress and promotes intracellular multiplication are unknown, but obviously the PrfA-dependent virulence machinery (e.g., the virulence gene cluster products) is not significantly affected in clpC mutants [162].

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. [ 1431 and Tilney and Portnoy [ 1941. Their work was followed by a series of studies describing the cell biology of the process. It was shown that L. monocytogenes moves rapidly through the cytoplasm at a speed of up to 1.5 pm/s with help of formed actin tails. The rate of actin assembly, which occurs at the barbed ends of actin filaments near the bacterial surface, equals the rate of actin-based motility with actin polymerization providing the propulsive force for intracellular movement. Such motility also takes place in cytoplasmic extracts from Xenopus oocytes [28,164,19 1,192,1931. Mutants defective in intracellular motility were obtained by transposon mutagenesis. These mutants have either lost the ability to initiate actin polymerization [ 1871 because of an insertion into a gene, called actA, or are unable to rearrange the polymerized actin filaments into actin tails [ 1141. The gene actA located downstream from mpl in the lecithinase operon codes for a proline-rich protein (ActA) of 639 amino acids (Fig. 4). Its apparent molecular weight determined by SDS-PAGE is 92 kD [38,196]. ActA is a surface protein consisting of three domains: the N-terminal domain with the transport signal sequence, the central prolinerich repeat region, and the C-terminal part which includes a membrane anchor [38,99,196]. Mutations in the actA gene resulted in avirulence in mice [38], cessation of intracellular

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actin polymerization around bacteria, and lost intracellular motility [38,99]. Inside host cells, the actA mutant forms microcolonies which are located near the nucleus [38]. To elucidate the role of ActA in actin filament assembly, actA was transfected into mammalian cells [52,150,1511. Expression of the complete ActA protein (including the membrane anchor) results in targeting of the protein to mitochondria, which subsequently recruits actin to these organelles, suggesting that ActA alone is sufficient to polymerize actin. However, the mitochondria did not move intracellularly [ 150,1511. Expression of ActA lacking its signal sequence and its membrane anchor resulted in increased amounts of F-actin in the transfected cells. ActA fused to a plasma membrane anchor which targeted the fusion protein to the plasma membrane resulted in actin polymerization and formation of aberrant protuberances on the cell surface [52]. From these assays, it appears that ActA is sufficient to induce actin assembly. To prove that ActA is also sufficient to promote intracellular movement, the nonmotile species L. innocua was engineered to express ActA. The recombinant bacterium produced actin tails and moved in cytoplasmic extracts as did the wild-type L. monocytogenes strain. In all parameters tested, the recombinant L. innocua strain expressing ActA was indistinguishable from L. monocytogenes [ 10I]. The ActA protein is distributed asymmetrically on the surface of L. monocytogenes but is not found within the actin tail [145]. After cell division, it is not present at the new bacterial pole but is concentrated at the old pole [100,190). Using streptococci coated asymmetrically with genetically engineered ActA protein, this asymmetrical distribution of the ActA protein was shown to be required and sufficient to direct actin-based motility [178]. In a cell-free system, these streptococci, but not uniformly coated ones, moved efficiently in cytoplasmic extracts [ 1781. The precise mechanisms by which ActA allows actin recruitment and intracellular movement are still unknown. However, expression of mutated forms of ActA either in mammalian cells [ 1513 or in L. rnonocytogenes [ 1 16,1791made it possible to define regions of the ActA protein with specific functions in actin polymerization and movement. Deletion of the N-terminal domain of ActA was followed in both systems by total abolishment of actin polymerization and intracellular movement, thus showing the absolute necessity of this domain in ActA function. In contrast, deletion of neither the proline-rich repeat domain nor the C-terminal domain prevented actin assembly. However, the actin tails produced by L. rnonocytogenes strains expressing ActA without proline-rich repeats were appreciably shorter, and the number of repeats deleted corresponded with reduction in speed, pointing to a stimulatory function for this region. Earlier work suggested a more prominent function of the proline-rich repeats, since polyproline peptides, peptides representing one internal ActA repeat, or a naturally occuring polyproline peptide, blocked actin assembly and motility after microinjection into L. nzonocytogenes-infected cells [ 185,1861. It was speculated that the polyproline peptides would bind profilin [ 1861, which in turn was suspected to be directly involved in actin-mediated motility of L. rnonocytogenes [ 1921, thereby inhibiting movement by inhibiting the association of profilin with the repeat region. Among actin binding proteins localized on actin tails--a-actinin, tropomyosin, vinculin, talin, fimbrin, villin, ezrinkadixin, profilin, the vasodilator-stimulated phosphoprotein (VASP), Mena, and Arp3 [20,28,34,63,98,190,192,200]-only profilin, Mena, and VASP are associated with the surface of moving bacteria and colocalize with ActA. VASP, a natural ligand of profilin [156], recently was shown to bind directly to the proline-rich repeats of ActA [20] and can stimulate actin assembly by binding to ActA and enhancing

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the profilin concentration near 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 [63]. However, this model is questioned by results of studies in which profilin was depleted from cytoplasmic extracts. In such experiments, profilin-depleted extracts still supported actin assembly and bacterial movement [ 1281. The recently described Arp2/3 complex consisting of eight host cell proteins found in actin tails of moving bacteria may represent the host-cell actin polymerization machinery [200]. The pure complex is sufficient to initiate ActA-dependent actin polymerization on the surface of L. monocytogenes in a cell-free system and is thought to interact at least transiently with ActA. Identification and purification of the Arp2/3 complex as a constituent of actin tails represents a great step forward toward the full in vitro reconstitution of L. monocytogenes motility with purified components. As recently shown, the 92-kD ActA surface protein is cleaved by the listerial metalloprotease (see above) resulting in a major 72-kD degradation product and, dependent on the strains, additional smaller degradation products [66,145]. These products are either found on the bacterial surface or in the supernatant fluid as 65- and 30-kD fragments [ 1451. Whether degradation also occurs inside the cytoplasm of the infected host cell is unknown. Additionally, the ActA protein is phosphorylated inside host cells, which yields three distinct forms of this protein with slightly different sizes in SDS-PAGE [15]. However, a genetically engineered ActA variant which was fully functional but lacking the C-terminal region is no longer phosphorylated inside host cells, suggesting that phosphorylation may not be necessary for movement [ 1 161. As just mentioned, L. monocytogenes can spread from cell to cell without leaving the cytoplasm by forming microvilli-like protrusions on the host cell surface which are phagocytized by neighboring cells. The mechanism of microvilli formation and of induction of phagocytosis by neighboring cells are totally unknown. In cells infected with Shigella flexneri, which uses a similar mechanism of cell-to-cell spread, proteins of the cadherin family are critical for the spreading mechanism [ 1651. Whether this is also true for L. monocytogenes remains to be clarified. Once inside the double membrane-bound vacuole, bacteria again have to escape into the cytoplasm. On monolayers of 3T3 fibroblasts, pZcB mutants form only small plaques, suggesting that the cell-to-cell spread is impaired in these mutants. Electron micrographs of pZcB mutants inside mammalian cells [ 1961 show numerous bacteria possessing actin tails which are trapped in vacoules surrounded by a double membrane. This indicates that these pZcB mutants cannot lyse the double membrane of the vacuole which is formed when listeriae spread from cell to cell. Plaque formation capacity (which is thought to be a strong indicator of intercellular spread) of different mutants revealed that in addition to the broad-spectrum phospholipase PC-PLC, PI-PLC and the metalloprotease also contributed to plaque formation, most likely by supporting lysis of the double-membrane vacuole [ 1771. The importance of LLO in this step has not yet been revealed. L. ivanovii, a species pathogenic only to animals, is also invasive to most mammalian cells tested, and the intracellular life cycle of this bacterium is similar to that of L. monocytogenes. Inside host cells, L. ivanovii polymerizes F-actin-like L. monocytogenes albeit at a reduced rate, with actin tail formation and cell-to-cell spread also being observed [90]. Recent cloning and sequencing of the actA-related gene from L. ivanovii [72,105] showed surprisingly little sequence homology with the actA gene of L. monocytogenes. On the protein level, some homology exists at the N- and C-termini and in the prolinerich repeat sequences between the two proteins which are both active in actin polymeriza-

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tion [105]. The ActA-related protein of L. ivanovii is larger in size (1044 amino acids) than ActA of L. monocytogenes (639 amino acids) because of two insertions which are missing in ActA of L. monocytogenes and an increased number of proline-rich repeats [72,105]. Despite the overall low-sequence similarity of the two ActA proteins, the mechanism of actin polymerization seems to be similar, since host microfilament proteins that bind to L. monocytogenes ActA also bind to L. ivanovii ActA [20,62]. Additionally, L. ivanovii actA can replace L. monocytogenes actA in an L. rnonocytogenes actA mutant [711.

PRFA AND REGULATION OF VIRULENCE GENE EXPRESSION IN L. MONOCYTOGENES Positive Regulatory Factor A 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 carried deletions in a region upstream from the hly gene [70,121]. Cloning and 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 k D [122,133] regulates all virulence genes of the virulence gene cluster. The p$A deletion mutants can be complemented in trans by introduction of the cloned p$A gene again to yield a wild-type phenotype [ 1221. Site-specific mutations or transposon insertions in the prfA promoter or in the prfA coding region block transcription of the entire gene cluster (i.e., plcA, hly, mpl, actA, and plcB [21,133]), indicating that the p$A gene encodes a transcriptional activator required for expressing 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 ~511. A plrfA-like gene with high sequence similarity to pr3';4 from L. monocytogenes is also present in the closely related species L. ivanovii, where it also controls a set of virulence genes similar to those of L. monocytogenes [ 1151. The amino acid sequence of PrfA suggests that it is a member of the Crp/Fnr family of transcriptional activators which have been primarily identified in gram-negative 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 containing a leucine zipper and a second helix-turnhelix motif at its N-terminus [14,115,172] (Fig. 5). A 14-bp palindromic sequence which was first identified in the promoter region of the hly gene [ 1381 was present in promoters of all PrfA-dependent genes and located about 40 bp upstream from the transcriptional start site. The 14-bp palindrome is, however, not perfectly conserved in all promoters, and these differences could contribute

PrfA

Nm I I HTH

7 30

HTH LZ

C

I

I

169 194

FIGURE5 Schematic structure of PrfA from L. rnonocytogenes Sv 1/2a EGD. HTH, he I ix-tu r n- he I ix m ot iv; LZ, I e u c i ne-zi p pe r.

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to differential regulation of the adjacent genes by PrfA [ 14,104,1731. Meanwhile, it was shown that PrfA binds directly to these palindromic sequences [8,172], thereby activating virulence genes. Purified PrfA alone is, however, not able to bind specifically to the target sequence, but it does so after addition of PrfA-free extracts from various Listeria species, indicating that it requires additional factor(s) for binding [8]. The putative PrfA-activating factor (Paf) is most likely a protein which is negatively influenced in its activity by iron [8]. This iron regulation of Paf might also be the key to explain observations of either iron-repressed LLO expression or iron-induced internalin expression [23,26].

PrfA-Dependent Promoters and TranscriptsThe PrfA Regulon Transcriptional organization of the virulence gene cluster is complex. The listeriolysin gene, hly, is the only gene transcribed in a monocistronic mRNA from two PrfA-dependent promoters, P1 and P2, located in the intragenic region between hly and plcA [138]. A third hly promoter, P3, downstream from P1 and P2, recently identified and shown to be PrfA-independent, results in low-level transcription of the hly gene [37]. Three transcripts of the p$A gene were identified: a long (2.1 kb) transcript, which is cotranscribed with the plcA gene and autoregulated by PrfA, and two shorter transcripts (0.8 and 0.9 kb) transcribed from three distinct promoters located in front of the pr$A gene [50]. Transcription of pr$A from one of the promoters seems to be negatively regulated by PrfA. Synthesis of the bicistronic plcA-pr$A transcript depends on the activity of the plcA promoter [49], and is necessary for full expression of PrfA-dependent genes [18]. Translation of the monocistronic pr$A transcript(s) appears to be very inefficient, since PrfA-dependent virulence genes are poorly expressed even in the presence of large amounts of this transcript when the synthesis of the PrfA-regulated bicistronic transcript is blocked [65]. The three genes of the lecithinase operon are transcribed from at least two PrfA-regulated promoters: one, located in front of the mpl gene, yields a 5.4- to 5.7-kb transcript comprising mpl, actA, and plcB and an additional mRNA of 1.8 kb comprising mpl alone [ 111. A second promoter, located directly in front of the actA gene, leads to a 3.6-kb bicistronic transcript comprising actA and plcB [ 1I]. Early in the infectious process, it is believed transcription of p$A via the p$A promoters results in synthesis of a limited amount of PrfA sufficient to activate the high-affinity PrfA-dependent hly and plcA promoters. This, in turn, would allow synthesis of plcA-p$A transcripts which leads to enhanced PrfA synthesis. Higher cellular levels of PrfA activate the mpl and actA promoters, which seem to have a lower affinity to PrfA because of base mismatches in their palindromic “PrfA-boxes” [50]. A high cellular level of PrfA also leads to downregulation of the monocistronic pr$A transcripts [18,49,50,65]. Recent results [lO], however, suggest that both the amount of the PrfA protein and the quality of the palindromic binding sites are critical parameters for PrfA-mediated gene expression. The quality of the PrfA protein itself, which differs in its C-terminal part between different L. monocytogenes strains, seems to influence PrfA function. The only known genes which do not belong to the virulence gene cluster but are also regulated by PrfA are inlC [39,48] and inlAB [42]. Of the multiple promoters upstream from inlA, which result in 2.9- and 5 .O-kb monocistronic and bicistronic transcripts, only

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one is PrfA dependent and harbors a rather incomplete palindromic PrfA-box [ 10,421. The inZC gene is, however, transcribed from a single PrfA-dependent promoter which contains a conserved PrfA-box at position -40 from the transcriptional start point. A second possible PrfA binding site is located downstream from the transcriptional start site in a position different from those of all other known PrfA-boxes in the promoter regions of the PrfA-dependent genes [48]. The significance of this second PrfA-box for regulation of inlC expression, however, is unknown.

Environmental Signals Affecting Virulence Gene Expression Pathogenic bacteria living in the free environment must sense and adapt to their surroundings by regulating expression of genes needed for living both inside or outside of their host. Facultative intracellular pathogens also must be able to recognize 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 ref. 14). These signals can be classified into either 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 are either unknown or only poorly understood in some instances, but 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 prfA transcription. A shift in temperature to 37°C results in the onset of pr$A expression followed by transcription of virulence cluster genes [42,120]. Treating a culture medium with activated charcoal probably depletes the medium of a signal molecule which in turn would result in increased transcription of prfA and the PrfA-dependent genes [ 1571. Carbohydrates modulate virulence gene expression in a complex and poorly understood manner. Glucose directly influences prfA gene expression and thereby interferes with PrfA regulation. Increasing the glucose concentration in the medium leads to acidification which reduces LLO expression by unknown mechanisms [29,50]. The disaccharide cellobiose inhibits hZy and pZcA expression without a reduction in prfA mRNA levels, probably by reducing the amount of active PrfA by posttranscriptional mechanisms [97,146]. The mechanisms of stress-mediated altered gene expression are even less understood. Heatshock conditions increase hZy, pZcA and actA expression. p60 expression is inhibited by heat shock and also by oxidative stress (H202)[ 181,1821. Shift of L. munocytugenes from a rich medium to a nutritionally stressful minimal essential medium (MEM) induces expression of virulence cluster genes as well as other surface-associated proteins [ 1571. The pattern of induction of known PrfA-regulated transcripts in MEM indicates that the PrfAcontrolled genes are differentially regulated in the presence of apparently constant levels of PrfA. Phagocytosis and intracellular localization are two natural stress factors, and numerous proteins are selectively induced during phagocytosis of L. monocytogenes by macrophages [79]. A genetic assay to isolate genes preferentially expressed inside mammalian cells resulted in identification of genes involved in nucleotide biosynthesis, an arginine transporter, and pZcA [97]. Experiments directly measuring bacterial mRNA levels inside host cells revealed that the genes hZy, actA, and inZC are heavily expressed inside the

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mammalian cell, most likely by PrfA-dependent mechanisms [ 1 1,481. The complexity of regulation of gene expression by environmental stimuli is, despite all progress, far from being understood.

ROLE OF SUPEROXIDE DISMUTASE AND CATALASE IN VIRULENCE Possible roles of catalase and superoxide dismutase in virulence of L. monocytogenes have been reviewed recently [78,11 I]. Both enzymes act in concert to detoxify potentially harmful superoxide radicals. Superoxide radicals generated by the oxidative burst in a phagocytic cell are converted into hydrogen peroxide by action of superoxide dismutase (SOD), which is then cleaved by catalase into water and molecular oxygen. Bacterial catalases and superoxide dismutases were long suspected of being important virulence factors of intracellular bacteria, but no correlation of superoxide dismutase expression with virulence was found in L. monocytogenes [ 1991. The gene for SOD from L. monocytogenes [ 121 was recently cloned in Escherichia coli. The nucleotide sequence of the gene, called lmsod, revealed an ORF coding for a protein of 202 amino acids with high homology to the manganese-containing SODS from other organisms. The gene, which is conserved in all other Listeriu species, was mutagenized and virulence of the mutant was tested in mice, but no difference in survival of the bacteria in the spleen and liver of infected animals was observed between the lmsod mutant and the wild-type strain [ 131. Catalase mutants obtained by transposon mutagenesis show wild-type virulence in infected mice [ 1 171. Whereas catalase-negative L. monocytogenes mutants are killed by mouse resident macrophages already at low serum concentrations, killing of the wild-type bacteria requires high serum concentrations, suggesting that resistance to fully activated macrophages is partially mediated by catalase activity [ 1951. Meanwhile, the catalase gene (cut) of L. monocytogenes was cloned and sequenced [13]. Recent construction of frame catalase and SOD mutants as well as a catalase/SOD double mutant has shown that catalase and SOD alone obviously are dispensible for the bacterium. The double mutant, however, is drastically reduced in its ability to grow inside liver and spleen of infected mice and is also unable to grow inside mouse bone marrow-derived macrophages, suggesting a role for both enzymes in virulence and intracellular survival [13].

HOST CELL RESPONSES TO INFECTIONS BY L. MONOCYTOGENES Differential Regulation of Cytokine and Cytokine Receptor Expression In this chapter, we will not discuss the immunological aspects of cell-mediated immunity against L. monocytogenes which were reviewed recently [95], but we will concentrate on what is known about the response of mammalian host cells to L. monocytogenes infection on the molecular and genetic levels. We will focus on host genes involved in signal transduction and altered gene expression during infection by L. monocytogenes. The initial studies on host response done with primary mammalian cells and established cell lines infected with L. monocytogenes primarily determined cytokine activities whose importance in nonspecific and T-cell-mediated immunity during experimental listeriosis is well documented [ 1401. However, these studies could not discriminate between

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release of preformed proteins and de novo synthesized proteins. More recent studies determined expression of the affected host genes more specifically by semiquantitatively measuring expression on the transcriptional level using the highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) method. The early reports showed that primary mouse embryonic fibroblasts infected with L. monocytogenes released interferon-a@ (IFN-a@) into the culture medium [83]. Interleukin-1 (IL- 1) production by mouse peritoneal macrophages was observed with viable, virulent L. rnonocytogenes strains but not with killed or avirulent Listeria. Northern blot analysis further showed that the increase in IL-1 secretion correlates with an increase in IL- 1a-specific mRNA after infection of macrophages with L. monocytogenes [ 1411. Appreciable amounts of tumor necrosis factor (TNF) and IL-6 are secreted in alveolar macrophages after infection with viable L. monocytogenes [84]. IL-6 is induced in embryonic fibroblasts even by heat-killed L. monocytogenes albeit to a lesser extent than by infection with viable bacteria. TNF is secreted only after treatment with killed but not with viable L. monocytogenes [841. Differences between killed and viable Listeria in induction of TNF-a also were observed after infection of mouse peritoneal cell preparations consisting mostly of macrophages [203). Release of the proinflammatory cytokines, IL-lp, IL-6, and TNF-a, occurs in human polymorphonuclear granulocytes and in the human epithelial cell line HEp-2 after L. monocytogenes infection [4]. The granulocytes secrete all three cytokines in response to infection, whereas the HEp-2 epithelial cells secrete IL-6 and small amounts of TNF-a but no IL-lp. Transcription of the respective genes also is induced in these host cells. Using the human epithelial cell line Caco-2, we could only detect IL-6-specific mRNA expression on L. monocytogenes infection which was, however, already induced by adherent L. monocytogenes, since cytochalasin D treatment did not inhibit IL-6 expression [ 1081. Mouse peritoneal macrophages also secrete IL-6 after L. monocytogenes infection [ 1061. Hemolytic L. monocytogenes strains are less efficient in IL-6 induction than are nonhemolytic mutants, probably because of cell damage caused by the high level of secreted listeriolysin. Inhibited maturation of IL-lp by L. monocytogc’nes in mouse peritoneal macrophages was recently proposed as a novel mechanism of how L. monocytogenes may escape the host cell response, since infection of the macrophages by L. monocytogenes results in intracellular accumulation of unprocessed IL- 1 p precursor [61. In a recent study using the mouse macrophage-like cell line P388DI and different well-defined mutants of L. monocytogenes to analyze cytokine induction after infection, we showed [ I 101 that viable L. monocytogenes rapidly induced IL- 1 a, IL- 1p, IL-6, and TNF-a mRNAs in these host cells, whereas killed L. monocytogenes only induced IL- 1 p mRNA. Nonhemolytic mutants which cannot escape into the cytoplasm and which do not multiply are unable to induce I L - l a , IL-6, and TNF-a but still induce IL-lp mRNA. In most instances, the amount of cytokines in the culture supernatant liquid of infected macrophages correlates well with levels of induced mRNAs. The exception is IL- 1a, of which only low levels are found in the supernatant liquid despite an appreciable induction of IL- l a l p mRNA. Mouse bone marrow-derived macrophages infected with L. monocytogenes also induce proinflammatory cytokines [ 1 101. However, in these cells, a nonhemolytic L. monocytogenes mutant induces the same types and amount of cytokines as the wild-type strain, indicating that intracellular growth is not necessary for transcriptional induction of these cytokines in bone marrow-derived macrophages [33].IL-6 is produced in the bone marrow-derived macrophages independently of IL-1 and TNF [33]. The immunomodulating cytokines, IL- 10, IL- 12, and the IL- 1 receptor antagonist, also are in-

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duced in bone marrow-derived macrophages after L. monocytogenes infection [33,112]. Large amounts of the chemokine, IL-8, are secreted by the polarized human colon epithelial cell line TE4in response to L. monocytogenes infection [47]. Induction of IL-8 secretion is caused by an increase of IL-8-specific mRNA synthesis and is not mediated by secreted TNF-a and occurs preferentially at the basolateral side. IL-8 may be an initial signal for the acute inflammatory response after bacterial invasion of mucosal surfaces. The transcription of cytokine receptor mRNAs in mouse bone marrow-derived macrophages infected with L. monocytogenes seems to be regulated inversely to the respective cytokines. TNF receptor type I and IFN-y receptor are transcriptionally downregulated shortly after infection of mouse bone marrow-derived macrophages by L. monocytogenes, whereas IL-1 receptor type I1 mRNA is unaffected [33]. However, infection with the closely related but nonpathogenic species L. innocua does not alter cytokine receptor expression in bone marrow-derived macrophages. Consequently, these events might diminish the ability to activate L. monocytogenesinfected macrophages by cytokines, such as TNF-a and IFN-y, at least in vitro. This mechanism probably allows L. monocytogenes to evade the killing mechanisms of infected macrophages in vivo, since L. monocytogenes surmounts this barrier of defense and escapes to other cell types in the course of infection.

Expression of Stress Genes, MHC Genes, and Other Genes Identified by Differential PCR Invasion of a mammalian cell by bacteria growing rapidly inside the cytoplasm is a likely stress factor for the host cell. Immediately after infection, the macrophage-like cell line 5774 responds with enhanced synthesis of heat-shock protein 70 (HSP70) mRNA and HSP70 protein [168]. Phagocytosis of the bacteria is necessary for this induction, since cytochalasin D treatment, which blocks invasion, also prevents induction of HSP70 mRNA. The amount of HSP60 and HSP90 mRNAs is only slightly enhanced during infection. Another stress response protein, MAP kinase phosphatase (MKP-1 or PTP), which seems to participate in signal transduction pathways, is significantly induced 1 h postinfection and stays at an induced level for several hours in infected macrophages [107]. Transcription of both HSP70 and MKP-1 mRNAs is much less induced when macrophages are infected with nonhemolytic mutants of L. monocytogenes, suggesting that escape of bacteria into the cytoplasm is required for induced transcription of the stress genes. MKP-1 mRNA is transiently induced in L. monocytogenes-infected HeLa cells where it might contribute to dephosphorylation of the MAP kinase [ 1981 (see below). Since immunity to L. monocytogenes, which replicates in the cytoplasm of the infected macrophage, is mainly dependent on major histocompatibility complex (MHC) class I-restricted CD8 T lymphocytes [95], expression of the MHC class I molecules is critical for macrophage antigen presentation. We analyzed the expression of H-2K and I-AP mRNA in 5774 and P388Di macrophage-like cell lines infected with L. monocytogenes compared with noninfected cells [ 1671. Expression of both genes was repressed on infection of P388D1 but not 5774 macrophages. Class I1 MHC gene transcription was already repressed at early stages of infection when most bacteria were inside a phagosome. In contrast, class I MHC transcription decreased 2 to 4 h postinfection when the bacteria replicated inside the cytoplasm [ 1671. L. monocytogenes infection not only repressed MHC class I and I1 expression in resting P388Di macrophages but also lowered responsiveness of macrophages to IFN-y treatment [ 1671, which is known to induce MHC I and I1 expres-

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sion [ 1471. MHC I and I1 expression also was repressed by L. rnonocytogenes infection in P388D1 macrophages previously activated by IFN-y treatment. This suppression of MHC expression in activated macrophages was, however, only detectable on infection with wildtype L. rnonocytogenes but not with a p$A mutant unable to escape efficiently from the vacuole into the cytoplasm [ 1671. Suppression of MHC gene transcription may represent an important mechanism allowing L. rnonocytogenes to reduce macrophage-mediated antigen presentation followed by T-lymphocyte activation. Using a modification of the previously described procedure of differential PCR [ 123,1701, induction or repression of host genes after infection by L. rnonocytogenes was determined in a more general way. This method allows isolation of cDNAs representing fragments of genes which are expressed differently in infected and uninfected macrophages. By this procedure we obtained several cDNA clones derived from macrophage genes that were either transcriptionally activated or downregulated after infection by L. monocytogenes [ 123,1701. Some of the cloned cDNAs were sequenced and subsequent homology searches revealed that some of the sequences did not show any significant homologies to known genes [ 1081. One of the cloned cDNA fragments showed more than 99% homology to murine mitogen-activated protein kinase phosphatase (MKP-1) [ 1701 which was earlier described as being upregulated in macrophages on L. rnonocytogenes infection [ 1681.

Activation of Signal Transduction Pathways Enhanced expression of MKP-1 in macrophages and HeLa cells is also an indication that signal transduction pathways are modulated by a L. rnonocytogenes infection. Phosphorylation of MAP kinase is observed after infection of Caco-2 and HeLa cells with L. rnonocytogenes and is mediated by the pore-forming activity of listeriolysin 0 [ 188,189,1981. MAP-kinase is a part of signal transduction pathways which link extracellular signals to gene expression [158]. We have recently shown that the entire Raf-MEK-MAP kinase cascade is transiently activated on L. rnonocytogenes infection of HeLa cells [ 1981. Further studies are needed to elucidate the role of this activation and to clarify if enhanced cell proliferation occurs, probably for the benefit of invading bacteria. In this respect, expression of listeriolysin 0 in mammalian cells also results in a dramatic change of cell morphology and formation of foci consisting of tightly connected cells. The hly-transfected mammalian cells also exhibit an appreciably enhanced proliferation rate [ 3 2 ] .Because of the presence of its transport signal sequence, we assume that listeriolysin is transported via the Golgi system to the cytoplasmic membrane of the host cell where it may trigger the Raf-MEK-MAP kinase cascade which may ultimately lead to enhanced cell proliferation. Besides LLO-mediated activation of the Raf-MEK-MAP kinase pathway, LLO in synergism with PI-PLC also triggered synthesis of phosphatidylinositol phosphates (IP3 and IP4] and diacylglycerol in endothelial cells [ 175,1761. The molecular mechanisms as well as their significance during a L. monocytogenes infection remain to be unraveled. Differential gene expression is mediated by transcription factors like nuclear factor KB (NF-~€3)[5]which are particularly involved in transcription of many immunologically relevant genes, including cytokine genes. We studied NF-KB DNA binding activity in response to L. rnonocytogenes infection in the macrophage-like cell line P388DI [82]. A rapid invasion-independent enhancement of the NF-KB DNA binding activity was observed in these host cells within 10-20 min after adding the Listeria. NF-KB is induced in biphasic kinetics on L. rnonocytogenes infection: The first transient induction of NF-

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KB requires only adhesion of L. monocytogenes to P38SDI cells. This induction also occurs

with avirulent mutants of L. monocytogenes and the avirulent L. innocua with similar efficiency. This event likely involves lipoteichoic acid, the cell wall component of L. monocytogenes, since purified lipoteichoic acid shows the same transient triggering of NF-KB. A second, but permanent, induction of NF-KB occurs after release of listeriae into the cytoplasm of the host cell. This event occurs exclusively with virulent L. monocytogenes strains and requires the bacterial phospholipases PI-PLC and PC-PLC that are produced in the infected host cell’s cytoplasm [Sl]. Activation of NF-KB also occurs in Caco-2 cells after infection with L. monocytogenes but with slower kinetics than seen in the macrophages [SO]. The DNA binding activity of two other transcription factors, AP1 (activator protein-]) and NF-IL6, is not changed after infecting the P388D1 cell line with L. monocytogenes, indicating that the observed activation of NF-KB by L. monocytogenes is a specific event [82].

Apoptosis Programmed cell death, or apoptosis, induced by pathogenic bacteria was first documented for Shigella jexneri using the mouse macrophage-like cell line 5774 [204]. Induction of apoptosis was later shown for several facultative intracellular bacteria, including Salmonella typhimurium [ 1421, Bordetella pertussis [96], Legionella pneumophila [ 1441, and L. monocytogenes [75,159]. L. monocytogenes, originally described as being unable to induce apoptosis in 5774 macrophages [204], was recently shown to induce apoptosis in hepatocytes [159] and in dendritic cells with listeriolysin 0 being thought to trigger apoptosis [75]. L. monocytogenes-infected hepatocytes undergo apoptosis in vitro as well as in infected mice, and it was suggested that events of hepatocyte apoptosis which are linked to neutrophil recruitment eliminate infected cells rapidly and thereby inhibit L. monocytogenes spread [ 1591. In most instances, the mechanisms of apoptosis induction by bacteria are totally unknown. However, for S. Jexneri, IpaB invasin was reported to bind directly to an interleukin-converting-enzyme (ICE) protease and thereby interfere with the apoptosis-controlling network of the host cell [22].

CONCLUSIONS The last years have seen an enormous increase in our understanding of the molecular basis of infectious diseases. Our knowledge of the genes determining virulence of L. monocytogenes and the role which the virulence gene products play in the infectious process is rapidly expanding. However, many problems concerning virulence of L. monocytogenes still remain unsolved. For instance, L. ivanovii, a species largely nonpathogenic for humans [ 1711, resembles L. monocytogenes in its intracellular life cycle [90]. Genes homologous to most of the known virulence genes of L. monocytogenes are also detectable in this species [72,77,105], and the complete PrfA-regulated gene cluster identified in L. monocytogenes apparently also is present in L. ivanovii 1721. However, L. ivanovii is only virulent for animals and avirulent for humans with an experimental L. ivanovii infection in mice yielding a different outcome than that by L. rnonocytogenes [86]. 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 infected cells or differences in specific activity of known virulence gene products? Are

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there as yet unknown virulence factors in L. monocytogenes which are absent in L. ivanovii or vice versa? 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 already complex under in vitro growth conditions and regulated by PrfA-dependent and PrfA-independent mechanisms. Very little is known about how PrfA and other putative regulatory factors control these genes while the bacteria reside inside host cells and tissues. Preferential synthesis of listeriolysin inside the phagosome and of ActA and InlC inside the cytoplasm has been described [ 1 1,481. However, the precise timing in expression of virulence genes as well as cellular signals and bacterial sensors which may control their intracellular expression are largely unknown. Most analyses of listerial virulence factors were done with commonly used laboratory strains such as serotype 1/2a strain EGD or serotype I /2c strain L028. However, many human infections and most foodborne outbreaks have been associated with serotype 4b strains [ 1491. In the future, differences in structure, function, and especially regulation of virulence factors of different L. monocytogenes serotypes [ 1841 and clinical isolates will likely gain much more interest. Analysis of host cell responses to a L. monocytogenes infection is now becoming a topic of major interest, as it represents a suitable model system for studying the molecular basis of pathogen-host cell interactions. Research now concentrates on identification of new host genes which are differentially expressed during various steps of a L. monocytogenes infection. Characterization of such host genes may help us to understand better the strategies which these two partners are using in their intimate and sometimes very severe cross talks. The molecular mechanisms of this intimate cross talk which require signal transduction from pathogens to their host cells and vice versa are now being analyzed. Elucidation of the interaction of bacterial and host cell proteins also will shed new light on the coevolution of bacteria and their hosts. These central questions of pathogenesis of facultative intracellular bacteria pertain not only to L. monocytogenes but also to several gram-negative bacteria, such as Shigella, Yersinia, and Salmonella, and may lead to exciting answers in the near future.

ACKNOWLEDGMENTS We thank A. Demuth for carefully and critically reading this manuscript, J. Kreft and F. Engelbrecht for providing figures, and all members of our laboratory for allowing us to quote their unpublished results. We apologize to all who contributed to our current knowledge on the infection biology of L. monocytogenes but were not mentioned in this chapter. Work from the group at the University of Wiirzburg was supported by the Deutsche Forschungsgemeinschaft through the grant SFB 165-B4.

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Characteristics of Listeria monocytogenes Important to Food Processors YUQIANLou Bil Mar Foods, Inc., Zeeland, Michigan

AHMEDE. YOUSEF The Ohio State University, Columbus, Ohio

INTRODUCTION Today’s food manufacturer relies on a variety of processing and preservation techniques to produce a safe and wholesome product with a suitable shelf life. Preservation methods ensure the safety and stability of food by inactivating or inhibiting growth of foodborne spoilage and pathogenic microorganisms. Methods currently used in food preservation involve physical, chemical, and biological factors. Physical preservation includes heating, cooling, freezing, and irradiation. Chemical treatments include addition of antimicrobial agents such as benzoates, propionates, and sorbates, acidifying agents such as acetic, and lactic acids or curing agents such as sodium chloride and sodium nitrite. Preservation by biological means (biopreservation) includes fermentations which control spoilage and pathogenic microorganisms through gradual lowering of pH. Combinations of these preservation factors also are applied simultaneously or sequentially in food processing. In addition to these conventional preservation methods, novel nonthermal processing technologies are being investigated to meet increasing consumer demands for minimally processed food with fresh-like taste and texture. Ultra-high pressure, pulsed light or electric fields, and oscillating magnetic fields are examples of these novel techniques. 131

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New nonthermal technologies when combined with mild conventional preservation methods can produce microbially safe minimally processed foods. Treatment combinations are commonly used in food processing and such use 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. Throughout this chapter, some basic terms and concepts are used which must first be defined for clarity. The expression log refers to the microbial count in Log&FU/ml or g. When a measurable increase in count occurs, it is described as such unless multiplication of the microorganism is reported; in this instance, the increase will be described as growth. Microbial growth is enhanced when the lag period decreases, the generation time decreases, and/or the gain in count attained after a given growth period increases. In contrast, growth inhibition, or simply inhibition, results from a reversal in the aforementioned growth parameters. Growth inhibitors may also be described as bacteriostatic, and for Listeria as listeriostatic agents, with such agents usually not causing measurable inactivation. A decrease in count will be described as inactivation and defined in decimal reduction time (D-value) or a decrease in log count, when such information is available. An agent that causes microbial inactivation will be called bactericidal, but it may also be reported as listericidal when it is active against Listeria. The D-value is the time of exposure to a deleterious factor (e.g., heat) required to inactivate 90% (i.e., 1 log) of the population of a given microorganism. Hence, the D-value is a measure of resistance of the microorganism to the deleterious factor; the larger the D-value, the greater 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 the ability of the microorganism to maintain its viability during treatment.

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

Growth Temperatures Microorganisms grown at optimum incubation conditions exhibit short initial lag periods, short generation times during exponential growth, and high cell counts or densities at the stationary phase. Incubation at temperatures different than the optimum extends the lag period and/or the generation time and may decrease the maximum attainable population. In this chapter, growth parameters of Listeria monocytogenes as a function of incubation temperature will be described. The temperature range within which L. monocytogenes grows is of particular interest to food processors, since this pathogen has common features of both psychrotrophic and mesophilic bacteria. Under laboratory conditions, L. monocytogenes was originally reported to grow at temperatures between 3 and 45°C [160], with optimal growth occuring between 30 and 37°C [300,340]. In 1972, Wilkins et al. [394] examined the temperature range for growth of L. monocytogenes in a medium containing 1% tryptone, 1% yeast

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extract, 0.3% K2HP04, and 0.1% glucose. Extrapolation of data from an Arrhenius plot of exponential growth rates collected at various temperatures indicated that the bacterium had maximum, optimum, and minimum growth temperatures of 45-5OoC, 38"C, and 3"C, respectively, which generally agree with the growth temperatures most frequently mentioned in earlier textbooks. However, Bergey 's Manual of Systematic Bacteriology [341] gives the minimum growth temperature as 1°C. In support of this change from 3 to l°C, Junttila et al. [ 1911 found that growth of 78 L. monocytogenes strains on tryptose soy agar occurred at a mean minimum temperature of l.l(t0.3)"C, with 10 and 2 strains of L. monocytogenes serotype 1/2 growing at 0.8 and OSOC, respectively. In contrast, L. innocua (19 strains), L. murrayi (1 strain), and L. grayi (1 strain) failed to grow at temperatures below 1.7 (tr0.4), 2.8, and 3.OoC, respectively. Although researchers in Florida also observed slight growth of some L. monocytogenes strains in laboratory media at 1"C, Walker et al. [382] confirmed the ability of this pathogen to multiply at even lower temperatures, with three L. monocytogenes strains exhibiting generation times of 62- 131 h in chicken broth and pasteurized milk, respectively, during extended incubation at -0.1 to -0.4"C. Lowest growth temperature was reported by Hudson et al. [ 1781. L. rnonocytogenes and two other ps ychrotrophic pathogens, Aeromonas hydrophila and Yersinia enterocolitica, grew at - 1.5"C in vacuum-packaged sliced roast beef with calculated lag times of 174, 110, and 49 h and generation times of 100, 33, and 32 h, respectively. Growth of L. monocytogenes in laboratory media at 1°C is very slow. However, when incubated at 3-6"C, the growth rate of the pathogen increases with final populations of approximately 1OS CFU/mL attained after several weeks of incubation [ 1 151. In a study by Bojsen-Moller [39] in 1972, flasks containing Tryptose Phosphate Broth (TPB) were inoculated with one of several L. monocytogenes strains and incubated at different temperatures. L. monocytogenes exhibited average generation or doubling times of 12.0, 5.0, and 2.6 h during incubation at 4, 10, and 15"C, respectively. In a more recent study [27], the average generation times for 39 L. monocytogenes strains growing in Tryptic Soy Broth (TSB) supplemented with 0.6% yeast extract were 43, 6.6, and 1.1 h at 4, 10, and 37"C, respectively, whereas the respective average lag times were 151, 48, and 7.3 h. In the latter study, L. monocytogenes strains reached maximum OD600-valuesof 0.74, 0.92, and 0.97 after incubation at 4, 10, and 37°C for 336, 113, and 16 h, respectively. Since many other bacterial species fail to grow at refrigeration temperatures, extended cold storage of clinical, environmental, and food samples previously diluted in a nonselective medium such as Tryptose Broth (TB) often was successful for isolating L. monocytogenes. This Listeria-detection procedure, which forms the basis for cold-enrichment, was widely used until the mid 1980s. Significant growth variation among 39 strains of L. monocytogenes, especially at refrigeration temperature, also was observed by Barbosa et al. [27]. The lag phase for 39 strains varied from 69.8 to 270 h at 4°C and from 36.5 to 68.9 h at 10°C. Scott A, the strain most extensively used in Listeria-related research, had the longest (209 h) and the second longest (62.8 h) average lag periods at 4 and 10°C, respectively. However, when strains of I;. rnonocytogenes were grouped according to serotypes, few differences in growth parameters among serotypes were noticed. Therefore, the choice of L. munocytugenes strains for use in challenge studies, particularly at refrigeration temperatures, may affect the results and conclusions regarding food safety. Greater safety margins will be obtained if the hardiest L. monocytogenes strains are used in such studies. Because of the safety concerns, researchers attempted to find nonpathogenic indicator microorganisms which can replace L. monocytogenes, particularly for studies done in pilot plants or food processing facilities. L. innocua, which is nonpathogenic and has

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similar or higher growth rates and resistance to common food preservation methods than L. monocytogenes, was considered a suitable substitute for L. monocytogenes [97,143,299]. L. innocua PFEI, a strain with antibiotic resistance which aids its enumeration in foods, is reported to be a good thermal-resistance indicator of L. monocytogenes [143]. When incubated in Brain Heart Infusion (BHI) broth at 2-46"C, this L. innocua strain grew faster below 42"C, slower above 42"C, and had a shorter lag phase below 8°C than L. monocytogenes, and thus was generally considered a good indicator of the growth behavior of L. monocytogenes [97]. Similar or faster growth of L. innocua in a laboratory broth and a cheese sauce, compared with that of L. monocytogenes, was also noted by Petran and Swanson [299]. Prior treatment of a microorganism affects its behavior during subsequent growth. Gay et al. [149] found that the lag phase of L. monocytogenes (Scott A and V7) and L. innocua increased by a low inoculum ( 10' vs 1O3 CFU/mL) cold storage and preincubation at 30" rather than 14°C. Listeria strains tested by these investigators exhibited a slower first logarithmic growth phase and a faster second phase under most of the conditions tested. The food medium can also influence growth and calculated growth parameters of L. monocytogenes. Rosenow and Marth [322] measured growth parameters of four L. monocytogenes strains in autoclaved samples of skim, whole, and chocolate milk and whipping cream that were stored at 4-35°C. Listeria growth rates were generally similar in all four products at a given temperature and increased with an increase in incubation temperature. Generation times in hours for listeriae in all four products were 29.7-45.6 at 4OC, 8.7- 14.6 at 8OC, 4.5-6.9 at 13OC, 1.7-1.9 at 2 1 OC, and a uniform 0.68 at 35°C. L. monocytogenes reached maximum populations of 1 07- 1O9 CFU/mL in all products that were incubated 30-45 days at 4"C, 11-14 days at 8"C, 5.0-5.8 days at 13"C, 2.1 days at 21 "C, and 1 day at 35°C. In addition, numbers of listeriae failed to decrease substantially in the four products during extended storage. Donnelly and Briggs [92] also reported rapid growth of five L. monocytogenes strains in inoculated samples of whole, skim, and reconstituted nonfat dry milk (1 1% total solids) during incubation at 4, 10, 22, and 37°C. Growth of L. monocytogenes at low temperatures is also stimulated by presence of certain solutes in growth media. Such compounds include glycine betaine or carnitine [209,352], which are known as osmoprotectants, osmolytes, or compatible solutes. Similar osmolytes may exist in foods at measurable levels. Such osmolytes usually accumulate in microbial cells during periods of osmotic stress. Compatible osmolytes may stabilize the otherwise unstable physiological functions of cytoplasmic proteins or other structures under osmotic stress [398]. KO et al. [209] found that osmolytes accumulated in cells of L. monocytogenes at low temperatures and during chill stress. When L. monocytogenes was surface-plated on a defined medium containing 130 pM glycine betaine, colonies were observed after 32 days at 7"C, whereas no colonies were visible without this osmoprotectant. When L. monocytogenes was grown in the defined liquid medium at 4"C, addition of 130 pM glycine betaine nearly doubled the specific growth rate [209]. Virulence of Listeria is increased when the bacterium is grown at low rather than high temperatures. Durst [99] reported that 7 of 36 weakly virulent L. monocytogenes strains became markedly virulent to mice by intraperitoneal injection after the cultures were maintained on agar slants for 6 months at 4°C. Similarly, Wood and Woodbine [397] found that one strain of L. monocytogenes was more virulent to chick embryos when grown at 4 rather than 37°C. Thus the possibility exists that cold storage may enhance virulence of some L. monocytogenes strains isolated from refrigerated foods.

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Freezing Numerous reports of Listeria-contaminated frozen foods in the United States and elsewhere have prompted investigators to examine viability of L. monocytogenes during frozen storage. Survival of L. monocytogenes in laboratory media, buffers, and milk during freezing and frozen storage was assessed by Hof et al. [ 17 I], El-Kest and Marth [ 107,108,109] and El-Kest et al. [ I 121.

Viability in Frozen Culture Media Although L. monocytogenes does not grow below - 1.5"C, this pathogen can readily survive at much lower temperatures. Nontheless, freezing and frozen storage will cause a limited reduction in the viable population of L. monocytogenes. Using TPB, Hof et al. [ 17 1] found that viable populations of L. rnonocytogenes, L. ivunovii, L. innocua, L. seeligeri, and L. welshimeri decreased by 50, 90, 90, 40, and 50%, respectively, following 3 weeks of frozen storage at -20°C. These findings suggest that L. monocytogenes can remain viable as long as or longer than most other Listeria slip. during extended storage at subzero temperatures. Survival and injury during frozen storage depends on L. rnonocytogenes strains used, freezing menstruum, and freezing rate [ 106- 108,I 121. Milk or TB provided better protection of L. monocytogenes against death and injury than did phosphate buffer. After 4 weeks of frozen (- 18°C) storage, death and injury rates for three strains of L. monocytogenes were 9 1-99% and 52-78%, respectively, when the cells were suspended in phosphate buffer. 42-92% and 33-56% in TB, and 38-61 % and I I-67% in milk [ 1081. Addition of 2-4% glycerol or 2% milk components to phosphate buffer markedly decreased the extent of cell death and injury. Simulated milk ultrafiltrate, when compared with phosphate buffer, caused almost no change in death rate but decreased cell injury during the first 24 h of frozen storage at - 18°C 11071. Slow freezing at -- 18°C was more lethal and injurious to this organism than rapid freezing at - 198°C; however, freezing at - 198°C followed by storage at - 18°C resulted in cell death and injury rates that were similar to those caused by combined freezing and storage at - I 8°C [ I 121. No evidence of cell injury or death was observed when L. monocytogenes cells were suspended in phosphate buffer and stored at - 198°C for I month. When TB was used in place of phosphate buffer, the extent of cell injury increased for suspensions stored at - 198°C but decreased for similar suspensions stored at - 18°C [ I 121. El-Kest et al. [ 1 121 found that after 1 month of frozen storage in TB, at - 18"C, 45% of the population was sublethally injured compared with 72430% of L. monocytogenes cells in a virtually identical study [ l58]. As expected, multiple freezekhaw cycles were more detrimental to survival of listeriae than was a single cycle. Such treatment was far more damaging to the pathogen when done at - 18°C (>99% lethality and no detectable injury) rather than - 198°C (16-34% lethality and 1 1-27% injury) with generally similar behavior observed using TB and phosphate buffer [ I 121. Although freezing and frozen storage cause a limited decrease in viability of L. munocytogmes, such treatments can produce injury and thus sensitize L. monocytogenes cells to antimicrobial agents. Damage to several sites on L. monocytogenes cells caused by freezing and frozen storage at - 18°C was observed by El-Kest and Marth [ I 101. Freezing increased sensitivity of L. monocytogenes to lysozyme and lipase, which are two enzymes occurring naturally in some foods. Treatment with both enzymes resulted in a greater effect than when each enzyme was used alone [ 1 10,I 1 I]. However, adaptation of L. mono-

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cytogenes to sublethal levels of certain environmental stresses, such as low pH, ethanol, NaCl, heat shock, or starvation can increase survival of this pathogen during freezing, frozen storage, and freezelthaw cycles [237].

Viability in Frozen Foods Oscroft [282] found that frozen storage of three L. rnonocytogenes strains in carrot or chicken homogenate at - 18°C for 29-84 days did not appreciably reduce the viable cell counts. Results from other studies [ 164,284,2881demonstrated that L. rnonocytogenes populations decreased only <1-3 logs in inoculated samples of packaged fish and shrimp, canned milk, 10%Karo corn syrup, ground beef, ground turkey, frankfurters, carined corn, and ice cream mix during 2-3 months of frozen storage at - 18 to -20°C. A somewhat greater decrease in viability was observed in samples of frozen tomato soup, possibly because of the lower pH of the product. Gianfranceschi and Aureli [155] investigated survival of two L. rnonocytogenes strains in five foods (spinach, mozzarella cheese, cod fish, chicken breast, and beef hamburger) during initial quick freezing at -50°C for 57 min and subsequent frozen storage at - 18°C for 240-300 days. Quick freezing and subsequent storage reduced viable Listeria populations by only 0.1- 1.6 and 0- 1.O log, respectively. Injury among survivors ranged from a nondetectable level to 90%. In contrast, Palumbo and Williams [284] did not recover injured listeriae from a variety of frozen foods tested except for tomato soup.

Thermal Inactivation Thermal processing is the most widely used method to preserve food and to destroy harmful microorganisms, thus rendering food safe for human consumption. The established association of L. monocytogenes with raw milk in the 1950s gave rise to several early studies dealing with the possible resistance of this organism to pasteurization. In 1983, interest in this topic was revived as a result of a listeriosis outbreak in Massachusetts that was epidemiologically linked to consumption of pasteurized milk. The literature now contains a wealth of information on thermal resistance of L. rnonocytogenes in a wide variety of foods. Although data concerning heat resistance of L. monocytogenes in fluid milk will be presented now, the discussion regarding thermal inactivation of listeriae in other foods has been reserved for other chapters which deal with the incidence and behavior of Listeria spp. in meat, poultry, seafood, and products of plant origin. Early studies on the thermotolerance of L. rnonocytogenes gave controversial results because of the methods used in measuring heat resistance. The 1983 outbreak suggested that L. rnonocytogenes, at levels that may exist in milk, can survive minimal high-temperature short-time (HTST) pasteurization. Subsequent studies on freely suspended cells showed that minimal HTST pasteurization is adequate; however, results from investigations on resistance of intracellular L. rnonocytogenes were in conflict. Further efforts by the US Food and Drug Administration (FDA) [44,63,240], Centers for Disease Control and Prevention (CDC) [ 13,14,18], and the World Health Organization (WHO) [393] support HTST pasteurization as a safe process. The following is a discussion of these aspects of thermal inactivation of L. monocytogenes given in the sequence just outlined.

Conflicting Results in Early Literature Numerous conflicting reports concerning the unusual heat resistance of L. rnonocytogenes in milk can be found in the early literature. In 1951, Potel [307] demonstrated that L.

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monocytogenes died rapidly in milk held at 80°C. However, the following year, Ozgen [283] reported that L. monocytogenes survived 15 s at 100°C. In 1955, Stenberg and Hammainen [364] published results of a study which examined the heat resistance of five L. monocytogenes strains in milk at different pasteurization temperatures. Using smalldiameter capillary tubes filled with inoculated milk, these researchers demonstrated that L. monocytogenes was not completely inactivated until the milk was held at 65°C for 5 min, 75°C for 2 min, or 80°C for 3-5 min. Thermal resistance of L. monocytogenes also was studied by Stajner et al. [362]. When milk contained approximately 5 X 105L. monocytogenes CFU/mL, the organism survived heat treatments of 71 and 74°C for 42 s but did not survive heating at 85 and 95°C for 15 and 5 s, respectively. In 1957, Dedie and Schulze [82] examined thermal resistance of 54 strains of L. nzonocytogenes in milk using 0.2- to 0.3-mm diameter capillary tubes. According to their results, L. rnonocytogenes survived 30--40 s at 65"C, 10 s at 75OC, and -1 s at 85°C. Ikonomov and Todorov [182] used a pilot plant-sized tubular glass pasteurizer to examine heat resistance of Listeria in milk obtained from ewes and cows. The milk was pasteurized (63-65"C/30 min), inoculated to contain 107- 108L. monocytogenes CFU/ml, and then repasteurized at temperatures between 63 and 74°C. They found that the pathogen survived 20 min at 63"C, 10 rnin at 65OC1, 3 rnin at 68"C, 1 rnin at 7OoC, 20 s at 72"C, and <20 s at 74°C. Thus results of virtually all these early studies indicate that L. monocytogenes can survive HTST pasteurization at 71.6"C for 15 s.

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Variability in Results Caused by Thermal Inactivation Methods Several different approaches have been used to determine thermal resistance and have given rise to conflicting results. Findings from the early pasteurization study of Bearns and Girard [28], which included an "open-tube" heating procedure became highly suspect during the 1980s. Their experimental approach involved inoculating 20 X 150-mm screwcapped test tubes of sterile skim milk with approximately 5 X: 10' to 5 X 107L. monocytogenes CFU/ml. All tubes were placed in a water bath at 61.7"C so that the milk surface was 3-4 cm below the water level in the water bath. Tubes were held in a wire test tube rack attached to a mechanical shaker and were allowed to bounce in the rack to aid in mixing. Results obtained from direct plating of milk on Tryptose Agar (TA) indicate that L. monocytogenes survived 35 min at 61.7"C provided that the organism was present at an initial level 2 5 X 104CFU/mL. From these data, the authors calculated a D61.70C value (i.e., the time necessary to decrease the population 90% at 61.7"C) of 10.9 min, which indicates that L. monocytogenes, if present at populations > 103CFU/mL, can survive vat pasteurization at 61.7"C for 30 min. Using the method of Beams and Girard [28], Donnelly et al. [94] demonstrated that complete inactivation of L. monocytogenes in milk with an initial population of 106-1O7 Listeria CFU/mL cannot be accomplished within 30 rnin at 62, 72, 82, or even 92°C. Extensive tailing of survivor curves was observed after an initial 3- to 4-log decrease during the first 5 rnin of heating. These investigators concluded that the open-tube method of Bearns and Girard [28] is unreliable to determine thermal inactivation rates of microorganisms, and they offered several explanations for their conclusion. One explanation is that condensate and splashed cells accumulated on the test tube cap, which was above the level of water in the water bath and, therefore, not exposed to thermal-inactivation temperatures. Condensate containing listeriae would be expected to drip back into the heating menstruum, thus eventually establishing a constant low population of survivors.

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A more likely explanation is that the test tube walls were coated with cells of Listeria during initial mixing. The test tubes were not completely submerged in the water bath; therefore, cells on the test tube wall would not be exposed to thermal-inactivation temperatures. Since a constant surface area is presumably coated with listeriae, low levels of survivors likely would be detected throughout the inactivation process. Concurrent studies by Donnelly et al. 1941 using a “sealed-tube” method demonstrated that L. monocytogenes was rapidly inactivated in milk at 62°C. The sealed-tube method involved adding I .5 ml of sterile whole milk inoculated to contain 107L. monocytogenes CFU/mL to a 2-ml vial, sealing it, and then submerging the vial in a water bath at the desired temperature for various times. In contrast to results of Bearns and Girard [28], thermal-inactivation profiles obtained by the sealed-tube method were linear for three strains of L. monocytogenes during the entire inactivation period and gave rise to D620C values between 0.1 and 0.4 min depending on the strain of bacterium. From the aforementioned results, it is apparent that the inactivation rate for L. monocytogenes at pasteurization temperature depends on the method used to study heat resistance of the bacterium. In 1987, Beckers et al. [29] compared thermal resistance of L. manmytogenes in TB using an open-tube and “sealedbag” method and obtained results similar to those of Donnelly et al. [94] just described. Heating in sealed tubes may produce anaerobic conditions in the heating menstruum. It is well known that anaerobic recovery of heat-injured cells of many pathogens, including L. monocytogenes, results in higher D-values [208,234]. Knabel et al. [208] found that after heating a Listeria-broth mixture in thermal death time (TDT) tubes, the broth at the bottom of the tubes became anaerobic, as indicated by the color change of the redox potential indicator, resazurin. Although no color change was found in milk in TDT tubes, some degree of anaerobiosis may also exist. The shortcomings of the open-tube procedure can be eliminated by using the capillary tube method [ 122,238,356,357,3661. In this method, a small sample of culture or inoculated liquid food (e.g., 40 1 L ) is introduced into a sterile capillary tube (e.g., 0.81.10 X 100 mm) and both ends are carefully heat sealed. The sample is then heat treated for a specified time after which the capillary tube is rapidly cooled, sanitized, and crushed inside a large test tube. The released sample is then diluted appropriately and plated to enumerate survivors. Compared with oiher methods, the capillary tube procedure allows uniform heating of the sample and minimizes come-up and cooling-down times. When compared with the capillary tube, other methods require a larger sample size and thus a significant part of the heat treatment occurs during come-up and cooling-down times. The amount of heat the sample receives during the coming-up and cooling-down times must be calculated to avoid inaccurate D-values. The capillary tube method was used by several investigators to determine the thermal resistance of L. monocytogenes in liquid media and foods [ 122,2381. Overall, thermal inactivation rates for L. rnonocytogenes were linear throughout the entire course of heating in the range of 50-75°C.

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Thermal Inactivation of Freely Suspended L. monocytogenes As a result of the 1983 listeriosis outbreak in Massachusetts that was epidemiologically linked to consumption of pasteurized milk, Bradshaw et al. [42] investigated thermal resistance of freely suspended L. monocytogenes in raw milk. A culture of L. rnonocytogenes strain Scott A (serotype 4b, clinical isolate associated with the outbreak in Massachusetts) was diluted in phosphate-buffered water and inoculated into raw milk to yield 105CFU/ mL. Portions of 1.5 mL were dispensed into 13 X 100-mm borosilicate glass tubes, which

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were sealed and immersed in a water bath at temperatures ranging between 52.2 and 71.7"C. Inoculated samples of raw milk also were heated in a slug flow heat exchanger at 7 1.7 and 74.4"C. Thermal processing of inoculated milk samples at seven temperatures between 52.2 and 74.4"C led to D-values ranging between 28. I min and 0.7 s, respectively, including a I)717°C of 0.9 s and a Dh33oc of 19.9 s. These investigators also noted that the thermal resistance of strain Scott A remained unchanged over a 2-year period. Survivors from some heating trials also were tested and were no more heat resistant than the parent culture, which suggests that the extensive tailing observed by Bearns and Girard [28] cannot be explained on the basis of heat-resistant spontaneous mutants. Working in France in 1988, Lemaire et al. [227] used open vessels and sealed capillary tubes to assess resistance of L. monocytogenes strains to vat and HTST pasteurization, respectively. When samples of inoculated milk were held at 60°C in open vessels, DhOoC values for 38 different L. monocytogenes strains ranged from 1.3 to 6.5 s. In contrast, D7yC values of 0.06-1.5 s were obtained when L. monocytogenes was heated in sealed capillary tubes, with strains of serotype 1 being generally more heat resistant than those of serotype 4. These findings along with those of Bradshaw et al. [42] indicate that current minimum vat (61.7"C/30 min) and HTST (7 1.6"C/ 15 s) pasteurization requirements established by the FDA are probably adequate to destroy expected levels of L. monocytogenes in raw milk. In 1986, Donnelly and Briggs [92) reported results of a study that examined the influence of milk composition and incubation temperature on thermal resistance of L. monocytogenes. The researchers inoculated five L. monocytogenes strains into sterile whole milk, skim milk, and reconstituted nonfat milk containing 11% solids. Following incubation, they heat-treated milk containing I Ox L. monocytogenes CFU/mL in sealed glass vials at temperatures between 55 and 65°C. Thermal resistance of L. monocytogenes was not significantly affected by prior growth in skim, whole, or reconstituted nonfat milk with I I % solids. Additionally, thermal inactivation experiments using the most heatresistant strain resulted in D550C and D6,0cvalues of 24.0 and 0.1 min, respectively, and a z-value of 4.3"C. After extrapolating the linear thermal inactivation plot through 7 I .7"C, the authors concluded that the most heat-resistant strain used in their study would be unable to survive in whole milk during HTST pasteurization. Going one step further, Bradshaw et al. [43], in 1987, examined the thermal resistance of L. monocytogenes strain Scott A in raw, autoclaved, and commercially sterile whole milk (-3.25% milk fat) and raw and autoclaved skim milk (<0.5% milk fat). Products were inoculated at 105L. monocytogenes CFU/mL, and thermal resistance was determined by the sealed-tube method. Listeria-inactivation studies done with raw, autoclaved, and commercially sterile whole milk yielded I371 7 c cvalues of 0.9, 2.0, and 2.7 s, respectively, indicating significantly ( P 5 .05) greater survival in presterilized than in other samples of whole milk. When heated in presterilized skim milk, the D7,70cvalue for strain Scott A was 1.7 s. Although their data indicate that L. monocytogenes should not survive in properly pasteurized raw whole milk, their other findings raise questions concerning the adequacy of pasteurization to inactivate L. monocytogenes in reprocessed products. Working in Canada, Farber et al. [ I361 inoculated 1200 L of raw whole milk to contain 105L. monocytogenes (a mixture of 10 strains including Scott A) CFU/mL. After heating milk at 60-72°C for 16.2 s in a pilot plant-sized regenerative plate pasteurizer, L. monocytogenes was recovered from milk heated up to 67.5"C but was not recovered from milk processed at 69 or 72°C. Scientists from the FDA [240] also found that L.

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monocytogenes cells (2.6 X 105CFU/mL) freely suspended in raw milk were inactivated by the minimum HTST pasteurization process. Thermal inactivation of L. monocytogenes in reconstituted nonfat dry milk (NFDM) was investigated by El-Shenawy et al. [122] in 1989. Suspensions of L. monocytogenes cells in reconstituted NFDM (10% solids) were placed in capillary tubes which were heated in a water bath at 50, 55, 60, 65, 70, and 75°C for various times. Overall, thermal inactivation rates for L. monocytogenes were linear throughout the entire course of heating with estimated D62.80Cand D7,,70C-values of 20 and 0.94 s, thus reaffirming that pasteurization as defined by the FDA should inactivate freely suspended cells of L. monocytogenes. In 1991, Bradshaw et al. [44] reported that, in raw and sterile milk, other Listeria species were no more heat resistant than L. monocytogenes. Therefore, properly pasteurized milk should be free of all Listeria spp. Although L. monocytogenes was more resistant in presterilized or repasteurized milk than in raw milk, all investigations described thus far showed that HTST pasteurization will inactivate L. monocytogenes when the pathogen is freely suspended in milk at levels up to 105CFU/mL. In contrast, Fernandez Garayzabal et al. [141] reported, in 1987, that the pathogen, when inoculated at high levels into raw whole milk, could survive minimum pasteurization. The Spanish researchers inoculated milk to contain 3 X 106, 1 X 107,or 2 X 10’ L. monocytogenes CFU/mL and pasteurized the milk at 72 or 73°C for 15 s (HTST method) in a pilot plant-sized pasteurizer. Using cold enrichment, Listeria was detected in five of seven batches of milk treated at 72°C for 15 s, with an estimated D720c-valueof 1.8-2.1 s. Listeria, however, was not detected in the three pasteurization trials at 73°C for 15 s. This experiment was criticized by Lovett et al. [240] for possible overloading of the pasteurizer. Large initial populations may have been a factor in promoting Listeria survival during pasteurization in the study of Fernandez Garayzabal et al. [ 1411. Additionally, since numbers of L. monocytogenes in naturally contaminated raw milk are typically very low, these findings do not discount the adequacy of minimum HTST pasteurization. The heat resistance of L. monocytogenes Scott A in heavy cream (38% milk fat) and pasteurized ice cream mix (-10.6% milk fat) was investigated by Bradshaw et al. [43] using the sealed tube method. The organism had D6x,90c-values of 6.0 and 7.8 s in raw and autoclaved cream, respectively. Thermal processing of pasteurized ice cream mix at 68.3, 73.9, and 79.4”C resulted in D-values of 231.0, 31.5, and 2.6 s, respectively. Again their data indicate that L. monocytogenes should not survive in properly pasteurized ice cream mix and that the pathogen had increased heat resistance in reprocessed products. Holsinger et al. [173] investigated the effect of components of ice cream mix on thermal resistance of L. monocytogenes. The D600C of L. monocytogenes in the mix correlated more closely with the level of high-fructose corn syrup solids (HFCSS) or stabilizer (guar gum and carrageenan) than with that of milk fat. Therefore, higher thermal resistance was conferred by higher levels of HFCSS or stabilizer. Although the aforementioned studies were all done with cow’s milk, MacDonald and Sutherland [243] compared the thermal resistance of L. monocytogenes in sheep’s and cow’s milk using the sealed-tube method. The authors found that sheep’s milk had a protective effect on L. monocytogenes during heating at 65°C when compared with cow’s milk. When Listeria was initially present in milk at 106-107 CFU/mL, a count of s103 Listeria CFU/mL was observed after heating the sheep’s milk ( 5 and 10% fat) for 45 min at 65°C; however, when cow’s milk and sheep’s skim milk were treated similarly, no survivors were detected by direct plating. Thus milk fat in sheep’s milk protected Listeria

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during heating, whereas milk fat in cow's milk did not provide similar protection. When whole sheep's milk was inoculated to contain 1O6 L. monocytogenes CFU/mL and pasteurized at 68, 70, 72, and 74°C for 15 s in an APV plate pasteurizer, the pathogen was only detected in milk processed at 68OC, which indicates the adequacy of minimum HTST milk pasteurization. However, caution should be exercised when interpreting these data, since detection of Listeria in this study was done by direct plating of pasteurized milk on a selective agar rather than using a preenrichment procedure for enhanced detection of sublethally injured cells.

Therma I Inactivat ion of Intrace1Iular L. rnonocytogenes Studies discussed thus far have dealt with thermal inactivation of freely suspended cells of L. rnonocytogenes in milk and other fluid dairy products. However, in cases of naturally acquired listerial mastitis, the pathogen is normally not freely suspended in milk but rather exists as a facultative intracellular bacterium within phagocytic leukocytes (neutrophils and macrophages) typically present in milk. The facultative intracellular nature of L. rnonocytogenes has led some investigators to speculate that cells of the pathogen inside leukocytes may be partially protected from thermal inactivation and thus are more able to survive pasteurization than are freely suspended cells of the bacterium in milk. Intracellular L. rnonocytogenes cells induced by in vitro methods [45,93] were used in early studies [61,64]. In 1986, Bunning et al. [61] determined thermal resistance of L. rnonocytogenes in parallel experiments using freely suspended bacteria in raw milk as well as L. rnonocytogenes cells that were inside of mouse peritonea1 phagocytes. Phagocytes were elicited in mice by injecting 107heat-killed L. monocytogenes (strain Scott A) cells into the peritoneum and then were harvested by peritonea1 lavage. Differential staining indicated that the cell preparation was made up of 70% macrophages, 25% neutrophils, and 5% lymphocytes. Opsonized cells of L. monocytogenes (i.e., incubated in normal mouse serum at 37°C for 30 min) were incubated in the phagocytic suspension for 60 min to allow phagocytosis. Phagocytes containing listeriae (average of 2.7-19.1 organisms/cell) were washed thrice by centrifugation and suspended in raw milk to obtain l O5 intracellular ListerialmL. Thermal resistance determinations were done using the sealed-tube method of Bradshaw et al. [42,43] described earlier in this chapter. Mean D-values for suspensions of intracellular L. rnonocytogenes in raw milk held at 52.2, 57.8, 63.3, and 68.9"C were 3170.0, 490.0, 33.3, and 7.0 s, respectively, as compared with D-values of 2290.0, 445.0, 33.4, and 7.2 s when freely suspended cells were heat treated at the same temperatures. Extrapolation of the data led to 1 ~ 7 1 . 7 0 C -of~ a1.9 l ~and e ~ 1.6 s for phagocytized and freely suspended listeriae, respectively. Under these experimental conditions, the intracellular position did not appreciably protect L. rnonocytogenes from thermal inactivation during pasteurization. Subsequently, several methods were developed to obtain bovine phagocytes containing internalized cells of L. monocytogenes, and such phagocytes have proven useful in evaluating thermal resistance of intracellular listeriae. Briggs et al. [45] enhanced production of bovine phagocytes (93% neutrophils, 5% macrophages, and 2% lymphocytes) by infusing Escherichia coli endotoxin into the mammary gland. This procedure produced an average of 2.4 X 106phagocytes/mL of milk, of which 89% were viable. Although only 39% of the endotoxin-induced phagocytes ingested 1,. monocytogenes (average of 27 listeriadphagocyte) as compared with 64% of normal bovine phagocytes, no difference in bactericidal activity was observed between endotoxin-induced and normal phagocytes. In another study, Donnelly et al. [93] developed an in vitro assay to analyze uptake of

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L. monocytogenes cells by bovine phagocytes. Somatic cells harvested from fresh mastitic milk were composed of 6 1% neutrophils, 20% macrophages, and 19% lymphocytes. Although 75% of the neutrophils ingested opsonized L. monocytogenes cells as compared with only 41% of the macrophages, both cell types contained an average of 19 listeriae per phagocyte. Maximum Listeria uptake by phagocytes occurred within 30 min of incubation at 37°C. Following ingestion, listeriae were resistant to the bactericidal activity of phagocytes. In 1988, Bunning et al. [64] reported results of a study comparing thermal resistance of freely suspended and phagocytized cells of L. monocytogenes, the latter having been prepared as previously described using endotoxin-induced bovine phagocytes [45]. Sterile whole milk was inoculated to contain 1 Oh intracellular (average of 26 bacterialphagocyte) or freely suspended (obtained by sonicating phagocytes) L. monocytogenes cells/ mL and heated at 57.8, 62.8, 66.1, and 68.9"C using the sealed-tube method or at 66.1, 68.9, 7 1.7, and 74.4"C using a slug flow heat exchanger. Using the sealed-tube method, the predicted D,,,80c-valuefor intracellular L. monocytogenes was 53.8 s, indicating a safe 33.4-D margin of inactivation for vat pasteurization (62.8"C/30 min). Using the slug flow heat exchanger, D 7 1 , 7 0 C - ~ apredicted lue~ from linear regression analysis were 4.1 s for intracellular and 2.7 s for freely suspended listeriae. Hence, the intracellular position of L. monocytogenes did not significantly (statistically) increase heat resistance under the defined parameters of this study. More important, these results indicate potentially unsafe 3.7- and 5.6-D margins of inactivation for intracellular and freely suspended listeriae, respectively, using the present minimum HTST pasteurization requirements (7 1.7"C/ I5 s). The aforementioned data on heat resistance of intracellular L. monocytogenes were obtained using phagocytes that were artificially induced to engulf listeriae. Heat resistance of intracellular L. monncytogenes cells in milk from naturally or artificially infected cows was investigated by several groups of researchers [9 1,96,136,240].A study that examined heat resistance of L. rnonocytogenes in milk from a naturally infected cow was reported in 1962 by Donker-Voet [91]. Milk from this cow contained 2 X 103-2X 104extracellular listeriae and >I O6 leukocytes/mL but otherwise appeared completely normal. Although no attempt was made to examine bovine phagocytes for intracellular listeriae, the organism was presumably present in some of the leukocytes. After pooling the milk for a week and holding it at 4"C, milk was heated in a plate-pasteurizer at 54-76.5"C for 15 s and then examined for surviving Listeria cells. Unfortunately, by the time enough milk was obtained for a pasteurization trial, the milk was heavily contaminated with other microorganisms, making isolation of listeriae from milk extremely difficult. Furthermore, leukocytes may have disintegrated, and the bacterial cells they may have contained were liberated and became freely suspended cells in the milk. I n this study, L. monocytogenes survived a heat treatment of 59.0"C for 15 s but did not survive in milk heated at 262.3"C for 15 s. This experiment was repeated using naturally contaminated milk from the same cow that was held for only 2 days at 4°C. Pasteurized milk was added to the contaminated milk to increase the volume of milk available for pasteurization. Although the initial Listeria population was not determined in the diluted milk before heating, L. monocytogenes was detected in milk processed at 63.7"C for 15 s. However, L. monocytogenes was not found in milk heated at 66.3, 68.0, 70.0, or 72.8"C for 15 s. Pasteurization studies using L. monocytogenes-contaminated milk obtained from cows artificially infected with the bacterium were conducted in 1987 by Doyle et al. 1961. A laboratory culture of L. monocytogenes strain Scott A was inoculated into the udder of each of four Holstein cows. Once listerial mastitis had developed, milk from these animals

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was pooled and held 2 days (and in one instance 4 days) at 4°C until sufficient quantities were available to process in a pilot-scale plate pasteurizer (Cherry Burrell, model 217SB1) at 71.7-73.9"C for 16.4 s (nine trials) or 76.4-77.8"C for 15.4 s (three trials). Before pasteurization, milk contained < 102- 1.9 X 10'' free Listeria cells and 4.5 X 105-2.4 X 1O6 somatic cells/mL. In addition, the milk generally contained 103- 1O4 L. monocytogenes cells within polymorphonuclear leukocytes (PMNLs) per milliliter (average of 1.5-9.2 listeriae/PMNL). During pasteurization, 1 00-mL samples of milk were taken after 2, 4, and 6 min of operation and analyzed for L. monocytogenes using two direct-plating and three enrichrnent procedures. L. monocytogenes was isolated from milk in six of nine trials in which the milk was heated to 71.7-73.9"C for 16.4 s. In contrast, L. morzocvtogenes was not detected in milk from the remaining three trials in which the milk was processed at 76.4-773°C for 15.4 s. Additional studies on the fate of L. monocytogenes within PMNLs indicated that the organism was no longer detectable in PMNLs after 3 days of storage at 4°C. Disappearance of listeriae after 3 days was accompanied by partial degradation of PMNLs, with complete breakdown occurring after 4 days. These findings suggest that holding raw milk at 4°C for 4 or more days would elirninate any thermoprotective effect for listeriae that might result from their engulfment by PMNLs. In the study just described, Doyle et al. [96] contended that phagocytized L. monocytogenes (
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heat-injured cells remaining after minimum HTST milk pasteurization may grow under anaerobic conditions that may exist in phagocytes. The investigators suggested that previous studies included (a) sample preparation practices such as sonication or mechanical agitation in the presence of glass beads that may have disrupted phagocytes in milk, and (b) aerobic plating may not have permitted recovery of heat-injured cells, thus D-values for such studies were underestimated. Bunning et al. [63] argued that the homogenization of raw milk at all milk processing plants will disrupt the phagocytes and that anaerobic conditions did not exist in heat-treated milk as suggested by Knabel et al. [208]. In response to the previous study [208], Farber et al. [ 1331 investigated the impact of growth temperature (30, 39, and 43°C) and anaerobic incubation on recovery of L. monocytogenes (a mixture of 10 strains) during milk pasteurization in a regenerative plate pasteurizer at 63, 66, 69, and 72°C for a minimum holding time of 16.3 s. The milk was preheated at 85°C for 1 h and cooled before inoculation and then held at 4°C overnight to simulate commercial holding practice. Four detection procedures, direct plating, a threetube most probable number (MPN) method, cold enrichment, and a warm enrichment procedure were used and combined with both aerobic and anaerobic incubation. The milk was inoculated to contain 5.0 X 104L. rnonocytogenes CFU/mL, which possibly represents a worst-case situation. Consistent with the study of Knabel et al. [208], Listeria grown at higher temperatures were more heat resistant. When the milk was pasteurized at 72"C, L. monocytogenes was detected in four of four, two of five, one of four, and zero of four trials when the cells were grown at 43, 39 (with 3 days of holding at 4"C), and 3OoC, respectively. Therefore, L. monocytogenes cells grown at higher temperatures can survive the minimum HTST milk pasteurization process. Although anaerobic incubation did not appreciably enhance recovery of Listeria by direct plating, survivors in five of seven trails at 72°C were only detected under anaerobic conditions. An approximate D720c-valueof 8.1 s was calculated for Listeria grown at 43°C. Increasing the holding time at 4°C from overnight to 3 days decreased the heat resistance of this pathogen. Lovett et al. [240] investigated inactivation of both freely suspended and intracellular L. monocytogenes Scott A during the minimum HTST pasteurization (71.7"C, 15 s) in a two-phase slug flow heat exchanger. Freely suspended listeriae were obtained by inoculating raw milk to contain 2.6 X 105L. rnonocytogenes CFU/mL. Raw milk was also inoculated to contain 5 X 104Listeria CFU/mL, of which 3-91% (average of 54%) were intracellular, obtained through an in vitro internalization process. Raw milk for heat treatment was also obtained from experimentally infected cows; the milk contained 3.4 X 103L. monocytogenes CFU/mL, with 53% being internalized. Three different enrichment procedures were followed for detection of L. monocytogenes in pasteurized milks. The researchers did not detect L. monocytogenes in any of the 23 minimum HTST milk pasteurization trials. Recognizing the impact of heat shock on thermotolerance, Bunning and coworkers [63] studied the effect of heat shock on inactivation of L. monocytogenes during minimum HTST pasteurization of whole milk. Heat shocking (48"C, 15 min) of Listeria in milk increased the D71.70C from 3.0 1.0 to 4.6 2 0.5 s; the latter value is comparable to that for intracellular L. monocytogenes as measured in a previous study [64]. The authors considered D71.70c-values after heat shock and those obtained with intracellular Listeria as representing the upper limit of heat resistance in Listeria. However, after assessing the data through risk analysis, Bunning et al. [63] believed that this increase in D71.7"C is not a convincing reason to raise the minimum HTST milk pasteurization temperature (71.7"C, 15 s). _+

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Pasteurization Efficacy: Concluding Remarks In the past, effectiveness of pasteurization was measured by the ability of this treatment to rid milk of Mycobacterium bovis and Coxiella burnetti, the most heat-resistant nonspore-forming human pathogens known at that time. After L. rnonocytogenes was confirmed as a serious food borne pathogen, adequacy and safety of heat treatments commonly used in food processing, particularly the pasteurization process, were questioned. At least three research groups [96,133,141] found that L. monocytogenes survived the minimum HTST milk pasteurization process if the pathogen was present in sufficiently high numbers. However, numerous studies with freely suspended, intercellular, or heat-shocked L. monocytogenes cells showed that the minimum HTST pasteurization is a safe process. Alarmed by a few reports on unusually high heat resistance of L. monocytogenes, some food processing authorities gage effectiveness of pasteurization by ability of the treatment to inactivate L. monocytogenes. These authorities also consider pasteurization-equivalent treatments adequate when such treatments eliminate at least 6 logs of L. monocytogenes. These are the arguments that one must consider when making a conclusion about adequacy of pasteurization: (a) the safety margin of minimum pasteurization (under conditions, however, not commonly encountered in commercial processing) is not as great as many scientists originally believed, (b) contamination levels of L. rnonocytogenes in commingled commercial raw milk are much lower than those used in most thermal-inactivation studies, (c) recovery of a few injured Listeria cells in pasteurized milk, if they exist, is doubtful, (d) homogenization of milk destroys macrophages, thus protection of Listeria against heat by cellular internalization is unlikely, and (d) the thermoduric microflora in pasteurized milk is likely to compete with any surviving L. monocytogenes. Therefore, we along with Ryser and Marth [333], the CDC [13,14,18], FDA scientists [44,63,240], and the WHO [393] conclude that “pasteurization is a safe process which reduces the number of I,. monocytogenes occurring in raw milk to levels that do not pose an appreciable risk to human health.’’ Although the minimum HTST milk pasteurization is considered a safe process, most raw milk processing facilities have wisely adopted pasteurization temperatures well above the minimum legal limit.

Thermotolerance Induced by Stress Adaptation In nature, L. monocytogenes may be subjected to various environmental stresses, such as high and low temperature, acidic and oxidative conditions, and starvation [ 145,2611. Environmental stresses can induce stress-adaptative or stress-protective responses. For example, incubating a microorganism at a high but sublethal temperature will induce the so-called heat-shock response. Stress adaptation occurs in all bacteria, including L. monocytogenes. Resistance of L. rnonocytogenes to heat or other lethal factors can be greatly increased by heat shock or adaptation to other stresses. In this section, we will discuss heat resistance of L. monocytogenes in relation to stress adaptation and implications of the adaptive response in food processing. Bacteria respond to heat shocking by synthesizing new proteins, termed heat-shock proteins (HSPs) [3,76]. Induction of the heat-shock response or HSPs usually increases the thermotolerance of microorganisms. As opposed to the intrinsic or basic thermotolerance of microorganisms, heat-shock-induced thermotolerance is transient and nonheritable and thus is called acquired or adaptive thermotolerance [388]. Temperatures at which microorganisms are heat shocked affect the magnitude of the acquired (i.e., induced) ther-

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motolerance. Optimal heat-shock temperatures for maximal thermotolerance in mesophilic organisms with a wide range of growth temperatures are usually between 45 to 50°C; that is, 10- 15°C above the microbe's optimal growth temperature [232]. L. monocytogenes has optimal heat-shock temperatures in this range [ 13 11. The magnitude of heat-shockrelated thermotolerance is also affected by the length of exposure to heat shocking, heating menstruum, heating rates, physiological state of Listeria cells, and the method used to recover injured cells. Heat shocking L. monocytogenes in laboratory broth under optimal or near optimal heat-shock conditions increased the heat resistance, expressed as D-values, by severalfold [63,139,189,233]. Bunning et al. [63] found that heat shocking L. monocytogenes at 48°C for 15 rnin before heating in sterile, whole bovine milk increased the D,, 7uc-valuefrom 3.0 k 1.0 s (control) to 4.6 t 0.5 s. Feido and Jackson I1391 reported a heat-shockinduced increase at D60oC from 3.9 min (control) to 17. I min. Linton et al. [233] observed that DssoC-valuesof logarithmic-phase L. monocytogenes, after heat shock at 48°C for 20 min, increased 2.3-fold; however, heat shocking as such failed to change the z-value, the temperature change required to cause a 90% change in the D-value. Quintavalla and Campanini [309] found that thermotolerance of L. monocytogenes in a pork emulsion was two to three times more than it was in broth. Heat resistance of microorganisms also can be greatly increased by the presence of NaCl and sucrose in the heating menstruum. Curing salts increased heat resistance of the pathogen in beef samples [2451. The physiological state of the organism affects the magnitude of heat-shock-induced thermotolerance. Heat shocking log-phase L. monocytogenes at 48°C for 10-20 rnin increased the DSsoc-value by more than twofold [233,234]. In contrast, heat shocking stationary-phase cells of L. monocytogenes in a sausage mix at 48°C for 30 or 60 rnin did not significantly increase thermotolerance, although heat shocking for 120 rnin increased the D640C-value by 2.4-fold [ 1311. Growth temperature affects the thermotolerance of L. monocytogenes. Heat resistance of Listeria cultures usually increases as the growth temperature increases; however, growing cells in the range between refrigeration and their optimum growth temperature had no or only a slight effect on heat resistance of the bacterium [92,189,290,353,354]. Growth at temperatures above 37°C mimics heat-shock conditions. When heated at 52°C for 1 h, L. monocytogenes cultures that were grown at 37 and 42°C showed 3-4 logs more survivors than did the cultures incubated at lower temperatures (5, 10, 19, and 28°C) [354]. Other studies proved that growing L. monocytogenes at an elevated temperature (43°C) significantly increased the resistance of the pathogen to heat [208,133]. Patchett et al. [290] found that heat resistance (Dssoc-values)of L. monocytogenes growing at 10 or 30°C in continuous cultures was not significantly different. Recovery of heat-injured cells of L. monocytogenes or E. coli 0 157:H7 by anaerobic incubation or adding exogenous oxygen scavengers, catalase, or superoxide dismutase (SOD) to the plating medium increased measured D-values [203,207,208,234,27 1,2911. Heating can completely inactivate catalase and SOD [79], and thus makes the heated organism an obligate anaerobe. Anaerobic incubation or adding catalase or SOD probably prevents formation of oxygen-derived compounds (such as superoxide and H202)which are toxic to injured cells. Knabel et al. [208] found that growth at 43°C before heat inactivation in combination with anaerobic recovery of the injured cells resulted in heat resistance (D6280C) that was at least sixfold greater than when the pathogen was grown at 37°C and enumerated under aerobic conditions. Heat-shocked (42"C, 10 min) logarithmic-phase

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L. monocytogenes cells had a D550c-valueof 18.7 min and 26.4 min when the enumeration plates were incubated aerobically and anaerobically, respectively [234]. Incubation at 25°C improved recovery of injured L. monocytogenes cells [64]. Conditions similar to heat shock exist in food processing. Slow heating or cooking, preheating, hot water washing, mild thermal processes, and holding food in warm trays (as occurs in food service establishments) are examples of heat shock that may happen during food processing and handling. Heat shock may result, as suggested by Farber and Brown [ 131 1, when foods are minimally processed or when the food is too bulky to allow rapid heating. Heat shock may occur during vat pasteurization of dairy products or production of "sous-vide" processed refrigerated foods, both of which involve a long-time temperature coming-up and low-temperature heating/cooking [ 2331. The thermotolerance of L. monocytogenes, Salmonella typhimurium, and Enterococcus faecium was increased by low heating rates [203,244,309,3 10,3651. Quintavalla and Campanini [309] found that L. monocytogenes became more heat resistant during slow (OS"C/min) rather than fast heating. A nearly twofold increase in the D-value for L. monocytogenes was noted by Kim et al. [203] when the pathogen was heated in ground pork at I.3"C/min compared with 8.OoC/min. Stephens et al. [365] investigated heat inactivation of a 17-h-old culture of L. monocytogenes (Scott A) in Tryptic Phosphate Broth at 50--64"C by both instantaneous heating (adding a small portion of concentrated cells into a large volume of preheated medium) and slow heating (0.7- 1 1 "C/min). Compared with instantaneous heating, slow heating at a rate between 0.7 and 5"C/min significantly increased the heat resistance of L. monocytogenes. This increase was maximal at a heating rate of I0.7"Clmin with a population 1.7 X 105-foldhigher than that after instantaneous heating. The heat-shock-induced thermotolerance of L. monocytogenes persists for a variable time. Acquired thermotolerance of stationary-phase L. monocytogenes lasted at least 24 h at 4°C in a sausage mix [131], < 1 h at 35"C, and 2 4 h at the heat-shock temperature (42°C) [62]. Besides heat shock, adaptation to other environmental stresses may also increase the thermotolerance of pathogens. Farber and Pagotto [ 1341 found that exposing a stationary-phase culture of L. monocytogenes to a laboratory broth at pH 4.0 for 1 h rather than 2 or 4 h increased the DSROc-value in sterile whole milk from 2.75 to 3.90 min. A gradual decrease of pH to 4 during 4 or 24 h also significantly increased heat resistance. Replacing HC1 with acetic acid failed to increase heat resistance [ 1341. Lou and Yousef [238] found that starvation and adaptation of L. monocytogenes to sublethal levels of HCl, ethanol, and hydrogen peroxide significantly increased the thermotolerance. Maximum thermotolerance was observed in cells exposed to 4-8% (v/v) ethanol, pH 4.5, and 500 ppm hydrogen peroxide; the corresponding averages of Ds60Cin a phosphate buffer (pH 7.0) were 4.1, 8.8, and 2.9 min, whereas nonadapted L. monocytogenes cells had a D560C of 1.O min. In phosphate buffer, starvation at 30°C for up to 163 h increased the D-value of the remaining viable cells to 13.6 min (2381. Sudden osmotic shock (holding cells in a Tryptic Phosphate Broth with 3.0-9.0% [w/v] NaC1) and osmotic adaptation (growth at high NaCl concentrations) significantly increased the thermotolerance of L. monocytogenes Scott A [ 1901. Thermotolerance of L. monocytogenes at 60°C increased during osmotic up-shift until the cells became almost as heat resistant as the culture grown for 48 h at the same high osmotic conditions. Increased thermotolerance was rapidly lost (<5 min) during an osmotic down-shock [ 1901. Heat-shock and other environmental stresses also affect the virulence of L. monocytogenes. Environmental stresses are sensed by pathogens as signals for expression of viru-

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lence factors to enhance survival [20,235,257,28 I]. Heat shocking may increase the virulence of L. rnonocytogenes. When L. rnonocytogenes was heat shocked at 48°C for 2 h, listeriolysin 0 was almost totally lost; however, subsequent growth of the heat-shocked cells at 37°C resulted in a 40-fold increase in production of the listeriolysin, whereas unshocked cells exhibited only a 2-fold increase [202].

ACIDITY Growth a t Low pH According to Bergey 's Manual of Systematic Bacteriology [34 11, L. monocytogenes can only grow at pH values from 5.6 to 9.6, with optimal growth occurring at neutral to slightly alkaline pH values; the latter was verified by Petran and Zottola [300]. The minimum pH value for growth is based on the work of Seeliger [340], who, in 1961, reported that L. monocytogenes failed to grow in dextrose (glucose) broth at pH <5.6 after 2-3 days of incubation at 37°C. In addition, subcultures from the medium were no longer routinely successful. Listeriosis outbreaks linked to consumption of fermented dairy products have reopened the issue of a minimum pH requirement for growth which has now been revised downward. During 1987, Lang et al. [219] examined growth at 13°C of L. monocytogenes (strain Ohio, isolated from recalled Liederkranz cheese) in TB adjusted to pH 5.0 and 5.6. Following lag periods of 2.0 days at pH 5.0 and 0.5 day at pH 5.6, the pathogen grew and reached maximum populations of 1.5 X 108and 4 X 10' CFU/mL in TB adjusted to pH 5.0 and 5.6, respectively. During logarithmic growth, the organism exhibited generation times of 13.1 and 4.4 h in media adjusted to pH 5.0 and 5.6, respectively. Thus, although Listeria failed to grow in TB at pH 5.0 during the initial 2 days of incubation, further incubation led to growth of the organism with maximum populations being reached after approximately 21 days at 13°C. Subsequent investigations have shown that L. rnonocytogenes can proliferate in laboratory media adjusted to even lower pH values. When inoculated into Trypticase Soy Broth acidified with hydrochloric acid, according to George et al. [153], all 16 L. monocytogenes strains tested initiated growth at pH values as low as 4.39-4.63 during extended incubation at 20 or 30°C. Although results from other independent studies [40,15 1,288,3591confirm the ability of L. monocytogenes to multiply in similar laboratory media adjusted to pH 4.4-4.6 with hydrochloric, citric, or malic acid, Farber et al. [135] observed growth of L. monocytogenes at 30°C in double-strength BHI broth acidified with hydrochloric acid to a pH value as low as 4.3. Furthermore, L. innocua, L. seeligeri, and L. ivanovii also were reported to grow in BHI broth acidified with hydrochloric acid to pH values as low as 4.53, 4.88, and 5.16, respectively [151]. Thus the minimum pH at which L. monocytogenes and most other Listeria spp. can grow is well below pH 5 provided that these organisms are incubated at near-optimum temperatures and allowed sufficient time to overcome an extended lag phase. As might be expected, growth of Listeria at low pH values is markedly influenced by incubation temperature and the type of acid added to the medium; the latter will be discussed in some detail later in this chapter. In one study [397], TB previously adjusted to pH 5.0 and 5.6 was inoculated to contain -1 X 103L. monocytogenes CFU/mL and then incubated at 4 and 13°C. Listeriae not only failed to grow when incubated in TB

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(pH 5) at 4"C, but populations of the bacterium decreased clO-fold during 67 days of storage. In contrast, increasing the incubation temperature to 13°C led to growth at pH 5, with the organism attaining a final population of -1 X 10' CFU/mL. George et al. [ 1531 found that minimum pH values for growth of 16 L. monocytogenes strains in Trypticase Soy Broth increased in the range of 4.39-5.45 as the temperature of incubation decreased from 30 to 4°C. Sorrells et al. [359] reported that four different L. monocytogenes strains grew in TB acidified to pH values as low as 4.40 following 7-28 days of incubation at 10°C, whereas growth of this organism at pH 4.4 was previously only observed at 220°C. Hence, some L. monocytogenes strains may be able to grow, albeit slowly, in laboratory media adjusted to pH 4.4 and incubated at near-refrigeration temperatures. Buchanan and Klawitter [54] also reported a similar effect of incubation temperature on growth of Listeria in TPB acidified to pH 4.5 with HCl. At 37"C, L. monocytogenes Scott A was completely inactivated after 50 h of incubation, with populations, remaining stable at 10 and 5°C. However, at 28 and 19"C, the organism grew to -107 and -10' CFU/mL in -100 and -500 h, respectively. Growth of L. monocytogenes in acid or acidified foods confirms the findings in laboratory media. In a study prompted by the listeriosis outbreak in Canada linked to consumption of contaminated coleslaw [337], Conner et al. [74] demonstrated that L. monocytogenes can tolerate and, in some instances, grow in cabbage juice at pH values <5.6. Juice expressed from fresh cabbage was adjusted with lactic acid to pH values of 3.8-5.6, inoculated with L. rnonocytogenes at 104CFU/mL, and incubated at either 5 or 30°C. After 3 days at 3OoC, Listeria reached maximum populations of -109 CFU/mL in cabbage juice which had an initial pH 2 5.2. Rapid growth of listeriae during this period was followed by equally rapid destruction, with the organism being no longer detectable after -15 days at 30°C. In cabbage juice adjusted to pH 5 and incubated at 3OoC, Listeria exhibited a 3-day lag period and then grew to maximum populations 2 10' CFU/mL after 7 days of incubation before numbers decreased. At pH 5 4.8, L. monocytogenes was inactivated in samples incubated at 30°C. Although incubation at 5°C prevented growth of L. monocytogenes in cabbage juice adjusted to pH 5 5.6, listeriae populations remained constant in samples at pH 2 5.2 during 22 days of storage. Interesting findings were reported by Parrish and Higgins [288] on potential growth of' L. monocytogenes in orange juice with modified pH. Initial Listeria populations of 106 CFU/mL increased approximately 1 and 2 logs in orange serum adjusted to pH values of 4.8 and 5.0, respectively, during the first 2 days of incubation at 30°C before decreasing to nondetectable levels 6 days later. In 1988, Ryser and Marth [331J examined growth of L. monocytogenes at different pH values in whey collected during manufacture of Camembert cheese. Samples of whey were adjusted to pH values between 5.0 and 6.8, filter sterilized, inoculated to contain 5 X 10'-1 X 103L. monocytogens (four strains) CFU/mL, and incubated at 6°C. Although no growth occurred in whey at pH 5 5.4, small numbers of the organism survived during the entire 35-day storage period. In contrast to the study involving cabbage juice [74], all four strains grew in whey at pH 5.6 after 3 days of incubation at 6°C. Under these conditions, the four Listeria strains had generation times ranging between 25.3 and 3 1.6 h and attained maximum populations of 1 X 107CFU/mL after 24 days at 6°C. As expected, L. monocytogenes had significantly (P < .OS) shorter generation times in whey samples at pH 6.2 (14.8-21.1 h) and pH 6.8 (14.0-19.4 h) than at pH 5.6. The organism also attained higher final populations in whey at pH 6.2 and 6.8 than at pH 5.6.

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Survival at Low pH Although growth of L. monocytogenes at pH < 4.3 has not yet been documented, this organism appears to be fairly acid tolerant. According to Reimer et al. [314], L. monocytogenes was recovered from inoculated samples of citrate/phosphate buffer that were acidified to pH 3.3 and held 4 h at 37°C. However, the pathogen survived < I h in a similar buffer adjusted to pH 1.4. Resistance of L. monocytogenes to acid was measured in Dvalues by Ahamad and Marth (41. The bacterium exhibited average D-values of 13.3 and 11.3 days when held at 7°C in TB previously adjusted with citric acid to pH values of 4.0-4.1 and 3.6-3.7, respectively. Since these authors obtained average D-values of only 2.2 and 1.4 days for corresponding cultures incubated at 35°C L. monocytogenes can clearly tolerate exposure to acid far better at near-refrigeration than at ambient temperatures. Such behavior raises concerns about the safety of certain refrigerated acid and lowacid foods that are often subjected to postprocessing contamination. Growth temperature and growth rate before acid challenge also affect acid resistance of L. rnonocytogenes. Patchett et al. [290] measured the acid tolerance of continuous cultures of L. monocytogenes that were grown at different growth rates or temperatures (10 and 30°C). At the same growth rate, L. monocytogenes grown at the higher temperature was more acid resistant, whereas at the same temperature (30"C), cultures grown at a slower growth rate were more acid tolerant. Survival of Listeria in acid foods also varies with pH and temperature of storage, as previously observed with laboratory media. Conner et al. [74) demonstrated that inactivation rates for L. monocytogenes in acidified cabbage juice were inversely related to pH with the organism surviving 49 days at pH 5.0-4.8 as compared with <2 I days in samples of cabbage juice adjusted to pH 4.6 and 4.4. Parrish and Higgins [288] investigated behavior of L. monocytogenes at 4°C in inoculated (- 10' CFU/mL) samples of orange serum adjusted to pH values of 3.6-5.0. Survival of the pathogen ranged between 21 days at pH 3.6 and >90 days at pH 4.8 and 5.0 with slight growth of listeriae during storage limited to orange serum adjusted to pH 5.0. Listeria was inactivated faster at higher rather than lower incubation temperatures, with the pathogen being eliminated after 5 and 8 days from orange serum at 30°C and adjusted to pH values of 3.6-4.0 and 4.2-5.0, respectively. Although acidic fruit juices appear to be unlikely sources of L. monocytogenes, the fact that this pathogen survived well beyond the normal shelf life of nonsterile orange juice (orange serum) suggests that such products should not automatically be eliminated as possible vehicles of infection in future epidemiological investigations of human listeriosis. Proper acid development is critical to the safety and quality of fermented foods. Behavior of L. monocytogenes in these foods depends on numerous extrinsic and intrinsic factors, including the pH. Camembert [329] (a mold-ripened cheese), Brick cheese [332], and white pickled cheese [ 1) supported growth of L. monocytogenes, with the pH of these cheeses being 5.9-7.2, 6.9-7.3, and >6.0, respectively. In contrast, the bacterium was inactivated rapidly in Parmesan [403], mozzarella [50], and water-buffalo mozzarella cheese [378], with final pH values of these cheeses being 5.0-5.1 , 5.2-5.3, and 4.0, respectively. In most other cheeses investigated, L. monocytogenes survived to various degrees. The bacterium persisted at least 28 days in creamed and uncreamed cottage cheese at pH 5.02-5.68 [327], 70 to 2434 days in Cheddar cheese at pH 5.0-5.15 13281, > 1 15 days in Colby cheese at pH 5.0-5.18 [401], 270 days in semihard Manchego-type cheese at pH 5.10-5.80 [90], 290 days in Trappist cheese at pH 4.70-5.42 [214] and feta cheese at pH 4.6 [287], <66-80 days in Swiss cheese [49], >50 days in blue cheese [286], and

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2180 days in cold-pack cheese food without preservatives at pH 5.21-5.45 [330]. Viable counts of L. monocytogenes decreased in cottage cheese stored at 4- 12°C [ 170,3021.Similar studies concerned with behavior of L. monocytogenes in fermented meats have shown that this bacterium can survive in hard salami at pH 4.3-4.5 during refrigerated storage [ 1881. When cow’s milk was inoculated to contain 103and 107Listeria CFU/mL, made into yogurt, and stored at 4OC, the pathogen survived for 2 and 7 days, respectively, at pH 4.2-5.0 [25 I]. Other investigators, however, reported that L. monocytogenes remained viable 13-27 days in yogurt stored at 4°C [70]. Survival of the pathogen in yogurt was reduced when milk was fermented at 42°C with thermophilic starters compared with fermentations that were done at 37°C with mesophilic starters [335].Although these and other studies will be discussed in greater detail in later chapters of this book dealing with behavior of Listeria in dairy and meat products, it may be concluded that L. monocytogenes is unlikely to initiate growth in food products which have a pH 5 5.2.

Acid Adaptation and Acidoduric Properties Acid adaptation can enhance survival of many microorganisms, including L. monocytogenes, when exposed to lethal acidic conditions. Extensive investigations on acid adaptation have been done with Salmonella typhimuriurn and Escherichiu coli 1145,3251, but fewer reports have dealt with L. rnonocytogenes [ 147,216,239,2751. Kroll and Patchett [216] investigated the effect of acid shocking on growth and survival of L. monocytogenes in Yeast Dextrose Broth at 37°C. Acid shocking at pH 3.0 or 3.5 for 20 min or preincubation at pH 5.0 did not affect the growth rate of L. monocytogenes at pH 7.0, but the lagphase was prolonged by acid shocking at pH 3.0. Prior incubation at pH 5 rather than pH 7, increased survival of L. monocytogenes by 3 logs during acid shock at pH 3 for 40 min. Adaptation of exponentially growing L. monocytogenes for 1 h at 35°C to three acidic conditions, (a) pH 5.0, (b) pH 4.5, or (c) pH 5.0, followed by additional incubation at pH 4.5 significantly (P < .OS) increased survival at pH 3.5 in a citrate/phosphate buffer [239]. Acid resistance of the pathogen was significantly greater after adaptation to the mild acidic conditions (a) or after stepwise increase to the high acid-condition (b) than to the highacid conditions (c) alone. The authors suggested that food fermentations, which involve a gradual lowering of pH, could lead to acid adaptation of L. monocytogenes. O’Driscoll et al. [275]obtained acid-adapted L. monocytogenes by incubating exponentially growing cells for 1 11 at 37°C in Tryptic Soy Yeast Extract Broth (TSYEB) acidified to pH 5.5 with lactic acid. This treatment markedly decreased inactivation of L. monocytogenes when the bacterium was inoculated into the same medium at pH 3.5. Exposure to pH 3.5 for 1 h reduced the population of unadapted cells by 3 logs, whereas numbers of acid-adapted cells decreased <1 log. The authors found that lactic and acetic acid were more effective than hydrochloric acid in inducing acid-adaptive responses in L. monocytogenes. De novo synthesis of ‘‘acid stress proteins” is presumably required for induction of the acid-tolerance response [275]. Acid adaption also cross protects L. monocytogenes against a variety of deleterious factors such as lethal doses of hydrogen peroxide, heat, NaC1, ethanol, and certain surface active hydrophobic compounds [ 134,238,239,2751. Since acid adaptation increases the general resistance, including acid tolerance, it is not surprising that acid-adapted cells of L. monocytogenes, like those of S.typhimurium [228] and E. coli 0157:H7 [229], survive better in both acidic and fermented foods than do unadapted cultures [ 1471. Compared with unadapted cultures, acid-adapted (at pH 5.5 with lactic acid) L. monocytogenes cul-

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tures and unadapted cultures of an acid-tolerant mutant showed enhanced survival during storage of cottage cheese (pH 4.71) for 15 days at 4OC, ripening of Cheddar cheese (pH 5.16-5.25) for 70 days at 8OC, storage of yogurt (pH 3.9) for 48 h at 4OC, active milk fermentation (pH <4.8 or <5.5), and storage in acidic foods such as salad dressing (pH 3.0) and orange juice (pH 3.76) for 7 h at 4°C. However, no significant differences were seen in survival of both types of cells in mozzarella cheese (pH 5.6). The acid-adapted L. monocytogenes culture and its acid-tolerant mutant ( 105CFU/mL) were partially inactivated in yogurt during 48 h of storage at 4°C with -3 and -5 log reductions for these two types of cells, respectively, whereas the unadapted control was inactivated within 24 h. When salad dressing (pH 3, attained by adding acetic acid) was inoculated to contain 106L. monocytogenes CFU/ml and stored at 4OC, the unadapted cells were completely inactivated in 15 min, whereas both types of acid-adapted cells survived up to 90 min. L. monocytogenes (105 CFU/mL) also was added to milk being actively fermented with S. thermophilus at 37°C when the pH decreased to 4.8 or 5.5. During an additional 7 h of fermentation (pH was reduced to 4.15), populations of unadapted L. monocytogenes cells decreased >3 logs, whereas numbers of adapted and mutant cells decreased 5 1 log [147]. O'Driscoll et al. [275] found that long-time acid challenge selected for acid-tolerant mutants of L. monocytogenes. This is contrary to findings of Buchanan et al. [53]that no subpopulation of acid-tolerant L. monocytogenes developed after treatment with a combination of 1.0% lactic acid, 6.3% NaCl, and 100 pg/mL NaN02at 19OC, a condition known to cause tailing of inactivation curves. This discrepancy may have resulted from differences in methods used in these two studies to select mutants. According to O'Driscoll et al. [275], acid-tolerant mutants have increased virulence compared with that of the parental cells. Intraperitoneal injection of 105CFU mutant cells/ mL into mice resulted in death of three of four infected mice, whereas none of the mice infected with parental cells showed any signs of infection. Additionally, higher counts of mutant rather than parental L. monocytogenes were found in the spleen after injection [275]. Virulent and avirulent Listeria strains also respond differently to stress [268]. Avirulent strains of L. monocytogenes did not multiply [78] or were completely inactivated [ 1691 inside macrophages, whereas virulent strains survived and multiplied inside the macrophage [78,169]. Therefore, considering that the increased acid tolerance of acid-adapted or acid-tolerant mutants of L. monocytogenes may help the pathogen to survive inside macrophages, and that L. monocytogenes can produce hemolysin over a wide range of pH values (55-29) [200], it would not be surprising to observe the increased virulence of acid-adapted or constitutively acid-tolerant cells as demonstrated by O'Driscoll et al. [275]. Environmental stresses presumably are used by L. monocytogenes and other pathogens as signals for expressing virulence factors and enhancing survival [20,211,212,235]. Although weak organic acids and their salts inhibit growth of L. monocytogenes, these compounds may enhance the virulence of this pathogen, and so should not be overlooked in assessing the safety of preserved foods. Kouassi and Shelef [211] tested the effect of salts of five weak acids on growth of L. monocytogenes and associated secretion of listeriolysin 0, the exotoxin important for the spread of the pathogen, in TSB (pH 7.2-7.4) at 35 and 20°C. Citrate, acetate, lactate, and propionate increased secretion of listeriolysin 0, with only sorbate inhibiting secretion of this toxin. The inhibitory effect of sorbate was later confirmed by the same authors [212]. McKellar [255] found that

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listeriolysin 0 is stable at >pH 5.3 (lactic acid), and has maximum activity at pH 4.0-5.0.

WATER ACTIVITY The moisture requirement for microbial growth can best be expressed in terms of water activity (a,), 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. Like most bacterial species, L. monocytogenes grows optimally at a, -0.97 [300]. However, when compared with most foodborne pathogens, this bacterium has a rather unique ability to multiply at a, values as low as 0.90. L. monocytogenes can grow in complex laboratory media containing up to 10% NaCl [341]. Skovgaard [351] estimated the a, of such a medium at -0.93 and therefore predicted that L. monocytogenes would not grow at a, < 0.93. The minimum a, for growth of L. monocytogenes estimated by Skovgaard was confirmed by Sperber [360]. Using liquid laboratory media adjusted with NaCl to various a, values, growth of L. monocytogenes at 35°C was observed at an a, of 0.943 but not at 0.935. Similarly, adjustment of a, using sucrose and glycerol allowed growth of L. monocytogenes at minimum a, values of 0.941 and 0.932, respectively. More recently, growth of L. monocytogenes at lower a, was observed by Petran and Zottola [300], Sorrells and Enigl [358], and Miller [260]. The bacterium grew in TSB containing 39.4% sucrose (a, = 0.92) when incubated at 30°C for 24 h [300] or in BHI broth containing 12% NaCl (a, -0.92) during incubation at 10 and 25°C [358]. Tapia de Daza et al. [370] found that when the a, of TSB was adjusted with glycerol, sucrose, or NaCl, two strains of L. monocytogenes grew minimally (determined as detectable turbidity of the culture in 20 days) at a, values of 0.90, 0.92, and 0.93 at 3OoC, and 0.92, 0.93, and 0.94 at 4"C, respectively. Nolan et al. [274] found the minimum growth (at least 1 log increase in 22 days at 2 1 "C) a, of L. monocytogenes in TSBYE to be 0.90, 0.92, and 0.92 when water activity was adjusted with glycerol, NaC1, and sucrose, respectively. When L. monocytogenes Scott A was grown at 28°C in BHI broth adjusted to different a, values (0.99-0.80) with glycerol, NaC1, or propylene glycol; the minimum a, values for growth at 28°C were 0.90, 0.92, and 0.97, respectively [260]. Although L. monocytogenes does not appear to grow at a, < 0.90, the bacterium can survive for extended periods at lower a, values. Shaharnat et al. [343] reported that the bacterium survived at least 132 days at 4°C in Trypticase Soy Broth containing 25.5% NaCl, which would be expected to have an a, of -0.83. Survival of L. monocytogenes under reduced moisture depends on both the a, and the dominant solute in the medium. In BHI broth adjusted to the same a, values, the pathogen survived longest with glycerol and shortest with propylene glycol and NaCl yielded intermediate survival [260]. Nolan et al. [274] also reported generally shorter survival of L. monocytogenes in NaC1-adjusted than in sucrose-or glycerol-adjusted TSYEB. Survival of L. monocytogenes during processing and storage of food may depend on a, of the medium. When sucrose/phosphate buffer solutions were inoculated to contain 104-105 L. monocytogenes CFU/mL and held at 140"C, Sumner et al. [369] found that the pathogen was about four times more heat resistant in buffer having an a, value of 0.90 as compared with 0.98. Thus, given the inverse relationship between a, and thermal resistance along with the ability of L. monocytogenes to grow at an a, value of 0.92 and

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ferment concentrated sucrose solutions, this organism also may be important to companies that manufacture foods containing high levels of sugar, as has already been demonstrated for Karo corn syrup stored at refrigeration temperatures 12841. Of more practical importance to food processors, Johnson et al. [ 1881 found that L. monocytogenes survived at least 84 days at 4°C in fermented hard salami which had an a, between 0.79 and 0.86. Extended survival of listeriae in sausage occurred despite the presence of 5.0-7.8% NaCI, 156 ppm sodium nitrite, and a pH of 4.3-4.5. The authors suggested that L. monocytogenes might survive longer at an a, of 0.9 1, which is occasionally found in commercial hard salami. However, they also predicted that growth of the bacterium in such sausage would be unlikely given the combination of salt, sodium nitrite, low pH, and low storage temperature. Additional information concerning the relationship between a, and growth/survival of L. monocytogenes can be obtained from several dairy-related studies. Using pasteurized whole milk inoculated to contain -500 L. monocytogenes CFU/mL, Ryser and Marth [328] manufactured Cheddar cheese which, according to Marcos and Esteban [250], had a, values between 0.972 and 0.979. The organism survived as long as 224 and >434 days in Cheddar cheese (pH 5.0-5.1) ripened at 13 and 6OC, respectively. Since Listeria reportedly grows well within this a, range, the combined effects of low pH and lowripening temperature probably played a dominant role in preventing growth of listeriae. Camembert cheese, also prepared by Ryser and Marth [329], had a, values between 0.959 and 0.984 [250], which should have allowed growth of L. monocytogenes. However, listeriae populations remained constant or decreased in cheese at pH 4.6 to -5.5 during the first 20-30 days of ripening. Initiation of rapid Listeria growth in cheese at a pH between -5.5 and 6.0 illustrates that pH rather than a, is primarily responsible for determining growth characteristics of listeriae in Camembert cheese. Parmesan cheese was made of pasteurized milk inoculated with 104- 10sListeria CFU/mL [4031. Listera was inactivated rapidly in this cheese and was not detectable after 2- 16 weeks of ripening. A combination of low moisture (30.1-3 1.4%), low pH (5.0-5. I), and heat treatment during curd cooking (51°C for -45 min) likely contributed to the rapid demise of Listera in this cheese.

ANTIMICROBIAL COMPONENTS IN FOOD Some food components that are either naturally present or added during formulation and processing have antimicrobial activity and thus contribute to food preservation. Of these antimicrobial components, some are applied mainly to control foodborne microflora, whereas others have dual or multiple functions. Control of L. monosytogenes by these components has been heavily investigated during the past decade. The following is an account of selected food components with antimicrobial activity in relation to control of L. monocytogenes in food.

Salt (i.e., sodium chloride or NaCl), an important ingredient in defining the water activity (a,) of many foods, affects microbial growth and survival in such foods. Salt, however, also exerts antimicrobial effects that can not be explained by its ability to lower a food's a,. Therefore, resistance of L. monocytogenes to salt and interaction of this important ingredient with the pathogen are described here in some detail.

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Salt Tolerance According to Bergey 's Manual of Systematic Bacteriology [34 11, L. monocytogenes can grow in Nutrient Broth (NB) supplemented with up to 10% !w/v) NaCl. Although this viewpoint concerning tolerance of Listeriu to NaCl is apparently based on results from Larsen [224], preliminary data from one investigative team [lSl], reported in 1988, indicate that one strain of L. monocytogenes grew at 8-30°C during extended incubation in BHI broth (pH 5.0) that contained up to 12% NaCl. Under identical conditions, single strains of L. ivanovii, L. seeligeri, and L. innocua were only slightly less halotolerant, with growth ceasing in the presence of >10% NaCl. In agreement with these findings, Sorrells and Enigl [358] found that two strains of L. monocytogenes grew in TSB containing 10% NaCl at 35°C or 12% NaCl at 10 and 25°C. Listeria may grow to high numbers in the presence of moderate amounts of salt. Hudson [ 1771 inoculated BHI broth, which contained 6.5% NaCl, with 106 CFU L. monocytogeizeslml. The bacterium increased by at least 3 logs after 15 and 26 days at 10 and 0-4"C, respectively. Lang et al. [219] found that growth of L. monocytogenes in TB containing 6% NaCl was markedly influenced by pH. In their study, TB containing either 0 or 6% NaCl (w/ v) was adjusted to pH 5.0, 5.6, 6.2, and 6.8 with HCI, inoculated to contain -5 X 102 L. monocytogenes CFU/mL, and incubated at 13°C. When grown in salt-free media at pH 5.0, 5.6, 6.2, and 6.8, this organism had generation times of 13.1, 4.4, 3.5, and 2.9 h, as compared with 77.8, 7.2, 5.0, and 6.3 h in the same medium containing 6% NaCl, respectively. Thus the combination of pH 5 and 6% NaCl was most effective in inhibiting growth of Listeria. Borovian 1401 also reported that L. monocytogenes grew at 10°C in culture media adjusted to pH 4.5 and 6.0 and containing 5 4 and 5 7 % NaCl, respectively. These findings agree with those of Lang et al. [219]. Extended survival of listeriae occurs at a wide range of salt concentrations. Studies at ambient temperatures demonstrated that L. monocytogenes can persist at least 150 days in pure salt 13481 and 545 days in 0.85% NaCl 13051. In 1955, Stenberg and Hammainen [364] reported that 10 L. monocytogenes strains survived > 1 year at 20-24°C in NB containing I % glucose and 10% NaCl. Listeriae also survived 34-68 days and 24 days in the same medium containing 12 and 24% NaCI, respectively. When Stenberg and Hammainen [364] stored organs (liver, heart, kidney) from Listeria-infected mice in salt solutions at 4"C, L. monocytogenes remained viable for 238-246, 88-1 12, and 27 days in solutions containing 3, 6, and 12% NaCI, respectively. Survival of Listeria in the presence of salt varies with the storage temperature. In experiments by Shahamat et al. [343], L. monocytogenes was inactivated in Trypticase Soy Broth containing 10.5, 13.0, and 25.5% NaCl after 14, 9, and 4 days of incubation at 37"C, respectively. Survival times in media containing 25.5% NaCl increased from 3 days at 37°C to 24 days at 22°C and to > 132 days at 4°C. Sorrells and Enigl [358] found that -106 CFU/mL of L. monocytogenes (two strains) in TSB, which contained 12 and 14% NaCI, decreased to a nondetectable level in 14-21, and 36 days at 35 and 25"C, respectively, whereas reductions of only -2 logs occurred when listeriae were kept in 14% NaCl for 36 days at 10°C. In a study by Hudson [ 1771, L.monocytogenes populations (106 CFU/mL) in BHI broth containing 26.5% NaCI, decreased 4, 2, and 0 logs at 10, 0-4, and -- 18"C, respectively, after 33 days of storage with D-values of 6 and 19 days at 10 and 0-4"C, respectively. Presence of 16.5% NaCl in the same medium did not affect the Listeriii count after 33 days of storage at all three temperatures. These data indicate

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that survival by Listeria in concentrated salt solutions can be increased dramatically by lowering the incubation temperature. Several studies have examined the fate of L. rnonocytogenes in salted foods and food-related products. Conner et al. [74] determined growth patterns of L. rnonocytogenes in cabbage juice supplemented with 1 5 % NaCI. Two Listeria strains grew at 30°C and pH 6.1 in cabbage juice containing 1% NaCl but failed to grow in the presence of 11.5% NaC1. In another study, Kukharkova et al. [218] demonstrated that L. rnonocytogenes survived >60 days in meat stored at 4°C in a 30% NaCl brine solution which also contained nitrate. According to Sielaff [348], L. rnonocytogenes was detected in infected beef that was immersed in a solution of 22% NaCl and stored 100 days at 15-20°C. Results just described indicate that this pathogen is likely to survive for long periods in salted foods, particularly meat. The high osmotic tolerance of L. rnonocytogenes indicates that immersing products such as cheese and salmon in brine solution (6-26% NaCl) is not a reliable preservation method to control L. rnonocytogenes.

Physiological Response t o Salt Studies by Brzin [47,48] during the mid 1970s demonstrated that L. rnonocytogenes undergoes various morphological changes when grown in media containing high levels of NaCl. Listeria cells were elongated (maximum length 55 pm) and filamentous when incubated at 37 or 30°C for 24 h on 5% human serum agar containing 8-9% NaCl and 0.4-0.6% agar. Under these conditions, cell multiplication was inhibited without simultaneous inhibition of cell growth (elongation). Attempts to grow listeriae on the same medium containing 9% NaCl led to complete inhibition of cell division and either partial or total cessation of cell growth, which ultimately led to fewer elongated and deformed cells. Microscopic changes in Listeria cells grown on salt agar also were associated with changes in colonial morphology. When incubated at 30 or 37°C in the presence of 8-9% NaC1, L. monocytogenes produced star-like colonies characterized by a rough surface, irregular border, and longer than usual straight or coiled protrusions. Such colonies contained large numbers of elongated filamentous cells. In addition to long twisted filamentous forms, occasional fusiform and spheroplast-like forms also were observed, particularly for cells from small colonies. In contrast, when grown on the same medium and incubated at 22 or 10°C, colonies became progressively smoother and tended to develop regular borders, Cells from these colonies were less elongated and filamentous with microscopic changes being most pronounced in cells from small rather than large colonies. Altered morphological forms of L. rnonocytogenes persisted only as long as the bacterium was grown on a medium containing 8-9% NaCl. Listeriae reverted back to their typical nonfilamentous, nonelongated form after 24 h of incubation on salt-free media. These results were recently verified by Isom et al. [ 1831, who found that when L. rnonocytogenes was grown in TSB, filament formation started above 1000 mM NaCl and peaked at 1200- 1500 mM. Interestingly, elongated cells also developed when L. rnonocytogenes was grown in media that were (a) adjusted to pH 5-6 with citric acid, (b) adjusted to pH > 9, or (c) supplemented to contain 11.75 mM H2O2. Osmoprotectants or osmolytes, which are involved in osmotic shock response, are required for growth and survival of L. rnonocytogenes in high-osmotic feeds. L. rnonocytogenes primarily utilizes glycine betaine (trimethylglycine), carnitine (P-hydroxy-y-N-trimethyl aminobutyrate), proline, and K+ as osrnoprotectants [289] with glycine being the most effective and preferred [35]. Addition of 1 mM glycine betaine, 1 mM carnitine, or 10 mM proline significantly stimulated growth of L. rnonocytogenes at 10 and 37°C in a

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minimal medium containing 3% NaCl, with final numbers reaching 109CFU/mL. A population of only 10' CFU/mL was reached in the absence of osmoprotectants [35]. In accordance with these findings, KO et al. [209] and Smith [352] reported that exogenous addition of betaine stimulated growth of L. monocytogenes in a defined medium with a high content of NaC1. Foods usually contain enough osmoprotectants to support microbial growth. Plant foods are rich in betaine, whereas foods of animal origin are high in choline (the precursor of betaine) and carnitine [35]. Processed meats (bologna, frankfurters, wieners, ham, bratwurst, salami) contain betaine at 0.34-0.48 nmol/mg and carnitine at 0.23-0.95 nmol/ mg [352]. Processed and ready-to-eat meats, which are high in salt and low in a,, can contain L. monocytogenes. The capacity of L. rnonocytogenes to grow on the surface of processed meats is reportedly related to the organism's ability to accumulate high levels of betaine and carnitine (200- 1000 nmol/mg cell protein), with salami supporting neither good growth nor the accumulation of betaine or carnitine [352].

Organic Acids and Their Salts Growth and inactivation rates for L. monocytogenes vary markedly in the presence of different acids. Most organic acids permitted in food are applied as acidulants (e.g., acetic and lactic acids), whereas others, particularly their salt forms, are used as preservatives (e.g., potassium sorbate and sodium benzoate). Effectiveness of these weak organic acids as antimicrobial agents is related to the amount of the undissociated form present. Concentration of the undissociated form of a weak organic acid which is related to pH of the medium and the pK, of the acid can be calculated using the Henderson-Hasselbalch equation. For example, at pH 5,35.5% of acetic (pK, 4.74) and 5.8% of L-lactic acid (pK, 3.79) will be undissociated. Undissociated organic acids can pass through the cell membrane, dissociate inside the cytoplasm, and interfere with metabolic processes of the microbial cell. The antimicrobial action of these acids is attributed to cytoplasm acidification, as well as the specific antimicrobial effect of the particular anionic species. A selection of organic acids and their salts will be addressed in relation to control of L. monocytogenes in food. A more comprehensive account of these acids and their role in food preservation can be found elsewhere [95]. The mechanism of microbial inhibition just discussed appears applicable to some of the acids that will be discussed in this chapter. However, it is not clear how some of the organic acid derivatives (e.g., parabens) inactivate microorganisms or inhibit growth.

Acidifying Agents Behavior of L. monocytogenes in media containing organic acids is affected by both pH and the incubation temperature. Experiments to determine the effect of lactic acid on growth of L. rnonocytogenes at different pH values and incubation temperatures were conducted by Bojsen-Mgller [39]. Polymyxin-Tryptose Phosphate Broth containing 0, 0.003, 0.03, and 0.3 M lactic acid was adjusted to pH 5, 6, and 7 using HCl or NaOH, inoculated to contain l O3 L. monocytogenes CFU/mL, and incubated at 35 or 4°C. When incubated at 35°C and pH 5, Listeria populations decreased in broth that contained 0.3 and 0.03 M lactic acid, whereas rapid growth occurred with 0 and 0.003 M lactic acid after a prolonged lag period. At pH 5 and 4"C, Listeria was eliminated after 27-42 days from broth containing 0.3 M lactic acid; populations of the pathogen remained unchanged in the same medium containing the three lower concentrations of lactic acid. At pH 6 and in the presence of 0.3 M lactic acid, the listeriae population increased 2 logs at 35°C and

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did not grow at 4°C. However, at pH 6 and 4°C Listeria growth occurred at the three lower concentrations (0,0.03, and 0.003 M) of lactic acid. At pH 7, listeriae grew at 35"C, regardless of lactic acid concentration; however, at 4°C and the same pH, the pathogen only grew in media containing 10.03 M lactic acid with a 7-day lag time. Using an experimental design similar to that of Bojsen-MQIler [39], Ahamad and Marth [4] examined the ability of acetic, citric, and lactic acid to prevent growth of L. monocytogenes in TB during extended incubation at 7-35°C. As expected, the pathogen was markedly affected by type and concentration of acid as well as incubation temperature. The presence of as little as 0.05% acetic acid (pH 5.8-5.9) caused noticeable inhibition of Listeria, with deleterious effects of acetic as well as citric and lactic acid being more evident at low rather than high incubation temperatures. Increasing the concentration of acetic, citric, or lactic acid to 0.2% (pH 4.4-4.6) completely suppressed growth of the organism at all incubation temperatures, with death of the pathogen occurring in the presence of 10.3% (pH < 4.2-4.3) acetic, citric, or lactic acid. In contrast, citric acid was less inhibitory than acetic acid, with growth of listeriae occurring in all samples with 0.1% citric acid regardless of temperature. The relationship between incubation temperature and inhibition of L. monocytogenes was most pronounced with lactic acid; the pathogen proliferated in the presence of 0.1% lactic acid at all temperatures except 7°C. Results from a follow-up study [ 5 ] showed that during extended incubation at both 13 and 35"C, the presence of 0.3 and 0.5% citric acid in TB was most injurious to L. monocytogenes followed in order by similar concentrations of lactic and acetic acid. Acid-injured listeriae survived approximately nine times longer at 13" than 35°C. In 1989, Sorrells et al. [359] published results of a study that examined the effect of pH, acidulant, time, and temperature on growth and survival of L. monocytogenes in TSB acidified to pH values of 4.4-5.2 with hydrochloric, acetic, lactic, malic, or citric acid. Based on average minimum pH values permitting growth of four L. monocytogenes strains at 10, 20, and 35"C, acetic acid was again most inhibitory (pH 5.04) followed by lactic (pH 4.73), citric (pH 4.53), malic (pH 4.46), and hydrochloric acids (pH 4.46). These findings generally agree with those of Ahamad and Marth [4] and several other investigators [33,116,135]. As in the previous study by Ahamad and Marth [4], longest survival of listeriae occurred at lower rather than higher incubation temperatures. However, since the inhibitory activity of the various acids tested was markedly different when based on equal molar concentrations of acid rather than pH, these data again indicate that differences in antilisterial activity of acidulants depend on both type and concentration of acid rather than on pH alone. According to results just described and those from subsequent studies [4,184,213, 359,4001, acetic acid is most listericidal and listeriostatic followed by citric acid when used on an equal weight (% w/w) or molar concentration basis at the same pH. However, based on equal molar concentrations of undissociated organic acid at the same pH, the order was reversed (i.e., citric >lactic ?acetic acid) for growth inhibition at 2 5 3 7 ° C [51,400]. Differences in listericidal activity among the three acids were much greater at low rather than at high concentrations, and diminished as the concentration of undissociated acid increased to 3 mM [5I]. Citric and lactic acids have different effects on survival of listeriae. Although high levels of both acids inactivated listeriae in a similar pattern, low levels (0.1-0.5 M) of citric acid, especially at pH 5-6, protected listeriae from death [51,521. Low concentrations (50 mmol/mL) of citric acid were also noted by Young and Foegeding [400] to enhance growth of L. monocytogenes at pH 4.7-5.0.

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Besides cytoplasm acidification caused by undissociated organic acids, specific inhibitory effects of these undissociated species on metabolic processes of L. monocytogenes were also noted [ 184,4001. Ita and Hutkins [ 1841 grew L. monocytogenes in TSB (with 0.6% yeast extract), adjusted the pH of the culture to 6.5-3.5 using acetic, lactic, or citric acid and held the culture for 4-6 h at 37°C. Although at low external pH (pH,) values, citric and lactic acids were more effective than acetic acid in lowering the intracellular pH (pH,), acetic acid was the most bactericidal. After 24 h of incubation at pH 3.5, acetic acid produced a pH, near S and decreased the population by -4 logs, whereas citric acid decreased the pH, to <4 and only caused < I log reduction. Young and Foegeding [400] also demonstrated that at an equal pH,, growth of Listeria was in this order: acetic >lactic >citric acid. In a more recent study, Kouassi and Shelef [213] investigated the influence of different salts of weak acids (0-5% sodium propionate, acetate, lactate, and citrate) on metabolism of L. monocytogenes in a defined medium at pH 6.7-6.8 during incubation at 35°C. Cell growth was inhibited by 2 1 % propionate, 2 3 % acetate, and 2 5 % lactate, with the relative inhibitory activity in this order: propionate > acetate > lactate > citrate. Citrate at 5% only slightly inhibited growth. Of these salts, only lactate (1 -5%) supported growth [213]

Lactates Antimicrobial effects of sodium and potassium lactates were reviewed by Shelef [345]. These organic salts are used at 1-4% as additives in baked goods and meat and poultry products. The mechanism of antimicrobial activity of lactates is not well understood; however, cytoplasmic acidification, specific anionic effect, a,-lowering and chelating action may all contribute to the inhibitory properties. Generally, 2-4% of sodium or potassium lactate is listeriostatic [345]. Combinations of lactate with NaC1, nitrate, or low temperature potentiated the overall antibacterial effect against L. monocytogenes [68,298,389]. Lactates, which do not change the pH of foods, were more effective in inhibiting microorganisms including L. rnonocytogenes ( I O3 CFU/g) in meats than in laboratory broths 13451. Shelef and Yang [347] found that >5% lactate was required to inhibit L. monocytogenes in TSB whereas 2.6% lactate in meat inhibited growth at refrigeration temperatures and 4% lactate did so at all temperatures tested. These authors also noted that lactate was more effective in comminuted beef than in comminuted chicken. In a subsequent study, Chen and Shelef [68] examined the antimicrobial activity of three lactates (sodium, potassium, and calcium) in cooked strained beef, which contained different moisture contents and was stored at 20°C. They found that all three lactates were equally effective in inhibiting growth of L. rnonocytogenes. In cooked strained beef prepared without lactates, 125% moisture (a, = 0.932) was required for complete growth inhibition, whereas at 55% moisture (a, = 0.963), growth of L. monocytogenes (Scott A) was completely inhibited by 2 4 % sodium lactate or a combination of 2-3% lactate and 2% NaCI. When 103--104L. monocytogenes CFU/g were inoculated into pork liver sausage containing 55% moisture and 2% NaC1, incubation at 5°C but not 20°C increased the inhibitory effect of the three lactate salts [389]. Although L. rnonocytogenes populations increased, 5 and 4.5 logs in pork liver sausage at 20 and 5°C after 10 and 50 days, respectively, the count of the organism in the presence of 4% lactate changed by - 1.33 to 1.4 logs at 20°C and by - 1.49 to 0.88 log at 5OC, with calcium lactate being the most antilisterial. Results of Pelroy et al. [298] are in accord with these findings. The investigators found that L. monocytogenes (- 10 CFU/g) was completely inhibited in vacuum-packaged, cold-processed, and smoked comminuted salmon containing 2% sodium lactate and 3% water-phase NaCl

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and stored for 40-50 days at 5°C. Similar inhibition for up to 35 days at 10°C was observed in the presence of a combination of 3% lactate and 3% water-phase NaCl or of 2% lactate, 3% NaC1, and 125 ppm NaN02. L. monocytogenes grew appreciably in control samples that contained 3% water-phase NaCl or 3% NaCl plus 125 ppm NaNO, [298]. The antilisterial activity of lactate in broth and bologna-type sausages was modeled recently by Houtsma et al. [175]; the model can be applied to predict Listeria growth in the presence of lactates in this product. In 1995, Buncic et al. [60] reported on the antilisterial activity of sodium lactate and other antimicrobial agents in a buffered BHI broth (pH 5.5) held at 4°C. When used alone or in combination with sodium nitrite ( I 25 ppm) and/or polyphosphate (0.5%) sodium lactate (4%) prevented growth of L. monocytogenes (initial population 107CFUI mL) during 7 weeks of incubation at 4°C; however, no bactericidal effect was observed. The antilisterial activity of sodium lactate was improved by addition of nisin (400 IU/ mL) but not 0.3% sorbate. Lactate and nisin had a synergistic effect against L. monocytogenes which was further enhanced by addition of polyphosphate (0.5%). Combinations of lactatehisin and lactate/nisin/polyphosphatedecreased Listeria population by 2.2-2.4 and 4.2 logs after 28 and 20 days, respectively. In contrast, nisin alone only resulted in an initial 1.1 -log decrease, but listeriae grew during prolonged refrigerated storage.

Sodium Diacetate Sodium diacetate (CH,COOH CH,COONa), which contains acetic acid (about 40%) and sodium acetate, is considered a “generally recognized as safe (GRAS)” additive by the FDA. It is used as an acidulant, flavoring agent, and antimicrobial agent in foods [67,346]. Shelef and Addala [346] investigated the antilisterial activity of sodium diacetate in BHI broth at 35, 20, and 5°C. After adding sodium diacetate (18-35 mM) to BHI broth, the resulting mixture of pH 6.3-5.25 was inoculated to contain about 103L. monocytogens CFU/mL. Inhibition of L. monocytogenes increased with increasing diacetate levels and decreasing incubation temperatures. The minimum inhibitory concentrations (MICs) of diacetate in BHI broth were 35, 32, and 28 mM at 35, 20, and 5OC, respectively. Based on equal levels of undissociated acetic acid at different pH values, sodium diacetate was more effective and had lower MICs at 35°C than did acetic acid; the MICs 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. The same study also assessed the antimicrobial activity of diacetate in meat [346]. Sodium diacetate added to ground beef (pH 5.6) or beef slurry (pH 5.6) greatly inhibited growth of aerobic microflora during storage at 5°C. Addition of 21 and 28 mM sodium diacetate decreased the pH of ground beef from 5.6 to 5.17 and 5.10, respectively. Growth of aerobes was measured with and without adjusting the sodium diacetate-containing ground beef to pH 5.6. Populations of aerobes, after storage at 5°C for 8 days, reached 7.12 and 6.21 logs and 5.98 and 5.1 1 logs in pH-adjusted and pH-unadjusted ground beef containing 2 1 and 28 mM sodium diacetate, respectively. However, these organism, also increased from 3.38 to 9.72 logs in the sodium acetate-free control. Similar trends were seen in beef slurry. When 15 different aerobic bacteria were tested, sodium diacetate generally was more inhibitory to gram-negative than to gram-positive bacteria, although some exceptions were noted [3461. Schlyter et al. [338] investigated the antibacterial activity of sodium diacetate alone or in combination with the commercial shelf-life extender, ATLA 2341 (a fermentation product from lactic acid bacteria, Quest International Bioproducts, Sarasota, FL) in turkey

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slurry (pH 6.2) at 25°C. During 7 days of incubation, the L. monocytogenes population, initially present at 3.7 logloCFU/g, increased to 8.6 logs in the control and 6.8 logs in the slurry, which was treated with 0.3% (21 mM) sodium diacetate. Additionally, sodium diacetate at 05% (35 mM) inhibited growth and caused a slight decrease in the viable Listeria population. The presence of ATLA 2341 at 0.25-0.75% did not appreciably affect growth of listeriae; however, a synergistic inhibitory effect against Listeria was observed when the additive, at the levels just indicated, was used in combination with 0.3 and 0.5% diacetate. In a subsequent study by the same group [339], adding 0.3 and 0.5% sodium diacetate to turkey slurry made the product listericidal when held at 4 and 2S°C, respectively. Antilisterial activity of sodium diacetate was synergistically enhanced by 2.5% sodium lactate or 5000 units pediocin/mL but not by 30 ppm of sodium nitrite. The count of Listeria in turkey slurry, which contained pediocin only, decreased by 0.9 log and then increased and reached a final population (8.0 logs) similar to that of the control. Combined use of pediocin and 0.3% diacetate at 4°C or pediocin and 0.5% diacetate at 25°C gave counts of Listeria in the product that were -7 logs lower than those in the additive-free controls.

Sodium Propionate In 1987, Lang et al. [219] found that L. monocytogenes grew at 13°C in TB at pH 5 supplemented with 0 and 6% NaC1; however, growth was prevented by addition of 5000 ppm propionic acid at both salt concentrations as well as by the combination of 6% NaCl and 0.1% propionic acid. Using TB at pH 5.6 and containing 0, 1000, or 5000 ppm propionic acid, L. monocytogenes grew to final populations of 107--10*and 104- 105CFU/mL in the presence of 0 and 6% NaC1, respectively. Generation times calculated for listeriae in the salt-free medium at pH 5.6 and containing 0, 1000, and 5000 ppm propionic acid were 4.4, 10.3, and 16.1 h, respectively, rather than 7.2, 18.1, and 42.1 h, respectively, in the same medium containing 6% NaCl. Similar behavior by L. monocytogenes was subsequently noted during extended incubation at 4-30°C in BHI broth (pH 5.9) containing 4.0% NaCl and 0.15% potassium sorbate [151]. El-Shenawy and Marth [ 1 171 demonstrated that >2000 ppm sodium propionate can inhibit growth of L. monocytogenes in TB at pH 5. Generation times for L. monocytogenes in TB at pH 5.6 and without sodium propionate decreased from 68 to 49 min as the incubation temperature increased from 4 to 35°C. In TB at pH 5.6 and containing 3000 ppm sodium propionate, generation times decreased from 3.0 days to 4.5 h as the incubation temperature increased from 4 to 35°C. Using TB at pH 5 and containing 3000 ppm sodium propionate, Listeria populations decreased 1 log during 67 days of incubation at 4°C. When the same medium was incubated at 35"C, numbers of L. monocytogenes decreased -3 logs, with the organism no longer being detected after 78 days, In a follow-up study, El-Shenawy and Marth [ 1201 investigated the antilisterial activity at 13 and 35°C of sodium propionate in combination with common organic acids. TB was prepared to contain 0, 500, 1500, or 3000 ppm sodium propionate and the pH of the medium was adjusted to 5.0 or 5.6 with HC1 or one of four common organic acids (acetic, tartaric, lactic, and citric). Decreasing the pH from 5.6 to 5.0 enhanced the antilisterial activity of propionate. Organic acids, when compared with HCl, greatly enhanced the antilisterial activity of propionate, with acetic acid being the most effective followed by tartaric, lactic, and citric acids. Lowering the incubation temperature from 35 to 13°C not only diminished the growth rate of L. rnonocytogenes, but also decreased the maximum population of the bacterium for all combinations of propionate and organic acids.

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When used at 3000 ppm, Ryser and Marth [330] found that sodium propionate was less effective than sorbic acid in eliminating four strains of L. monocytogenes from coldpack cheese food at pH 5.20-5.45. Cheese food was inoculated to contain -5 X 102L. monocytogenes CFU/g and stored at 4°C; the pathogen survived an average of 142 and 130 days in product that contained sodium propionate and sorbic acid, respectively. In contrast, the pathogen was present in cheese food made without preservatives at levels of I X 102CFU/g after 6 months of refrigerated storage.

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Potassium Sorbate Since receiving GRAS status in the United States during the 1950s, potassium sorbate and sorbic acid have been widely used to extend the shelf life of many foods, including butter, cheese, meat, cereals, and bakery items. Although most effective against yeasts and molds, these antimicrobial agents also inhibit a wide range of bacteria, particularly aerobic catalase-positive organisms. Consequently, the ability of potassium sorbate and sorbic acid to inhibit L. monocytogenes has been assessed in laboratory media and several foods. Moir and Eyles [265]measured the minimum inhibitory concentrations (MICs) of sorbate against L. monocytogenes Scott A in buffered BHI broth. These authors reported MICs of 400-600 and >5000 mg/L at pH 5 and 6, respectively, when the culture was incubated at 35°C and 1500 mg/L in broth of pH 6 which was refrigerated at 5°C. According to data collected by El-Shenawy and Marth [ 1 1.51, the ability of potassium sorbate to prevent growth of L. monocytogenes is related to temperature and pH. In the absence of potassium sorbate, generation times for L. monocytogenes in TB at pH 5.6 decreased from I . 13 days to 49 min as the incubation temperature increased from 4 to 35°C. Addition of 2500 ppm potassium sorbate prevented growth of Listeria at 4°C and led to complete demise of the organism after 66 days, whereas listeriae grew with a generation time of 9 h in the same sorbate-containing medium incubated at 35°C. The lower the storage temperature and pH of the medium, the greater was the effectiveness of sorbates against L. monocytogenes. In a subsequent study, El-Shenawy and Marth [ 1 191 investigated the antibacterial activity of sorbate in the presence of other organic acids. TB was prepared to contain 500, 1500, and 3000 ppm potassium sorbate and pH of the medium was adjusted to 5.0 or 5.6 using HCI or organic acids (acetic, tartaric, lactic, or citric), inoculated with L. monocytogenes, and incubated at 13 or 35°C. When compared with HCl as an acidulant, the antilisterial activity of sorbate was enhanced more by organic acids, with acetic and tartaric acids being more effective than lactic and citric acids. Working with food, Ryser and Marth [330]found that four strains of L. monocytogenes were eliminated faster from cold-pack cheese food at pH 5.45 that contained 3000 ppm sorbic acid (4100 ppm potassium sorbate) rather than from the same product at pH 5.2 manufactured without preservative. After inoculating cheese food containing sorbic acid with one of four L. monocytogenes strains at a level of -5 X 1O2 CFU/g, the pathogen survived an average of 142 days at 4°C. Although L. monocytogenes failed to grow in cheese food with a pH of 5.21 prepared without sorbic acid, the pathogen survived during the normal 6-month shelf life of the product at potentially hazardous levels of 1 X 1 O2 CFU/g. Since potassium sorbate works best at pH < 6.0 and is generally ineffective at pH > 6.5, it is not surprising that Dje et al. [SS] found potassium sorbate to be of little use in inactivating L. monocytogenes in samples of reconstituted nonfat dry milk. More recently, the antilisterial activity of potassium sorbate and other antimicrobial agents was investigated by Buncic et al. [60] in buffered BHI broth (pH 5.5) at 4°C. Potassium sorbate (0.3%) prevented growth of L. monocytogenes (initially 1O7 CFU/mL)

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during 6 weeks of incubation; however, no bactericidal effect was observed. Strong listericidal effects were observed when sorbate was used in combination with sodium nitrite (125 ppm), polyphosphate (0.5%),or nisin (400 IU/mL). Nitrite alone was only inhibitory to L. monocytogenes, whereas nisin alone only caused an initial 1.1 -log decrease, with Listeria survivors subsequently growing during prolonged incubation. Combined use of sorbate and nitrite caused a 6.7-log reduction in 6 weeks. Adding polyphosphate to this sorbatehitrite combination resulted in 6.4-log reduction in about 4 weeks. The combination of sorbate and nisin caused a 4.5-log reduction in 5 weeks, and this synergistic effect was further increased by addition of nitrite (125 ppm). No antilisterial interaction was observed between sorbate and 4% sodium lactate. A combination of lactate, sorbate, and nisin prevented growth of Listeria but did not cause significant inactivation. Adding nitrite to the three agent combination reduced populations by 3.7 logs after 37 days, with the same reduction being achieved in I2 days by further incorporation of 0.5% polyphosphate.

Sodium Benzoate El-Shenawy and Marth [ 1 141 reported that sodium benzoate is more inhibitory to L. monocytogenes than is either potassium sorbate or sodium propionate. In this study, TB was supplemented with 0-3000 ppm sodium benzoate in increments of 500 ppm, adjusted to pH 5.0 and 5.6 with hydrochloric acid, inoculated to contain 103L. monocytogenes CFU/ mL, and incubated at 4, 13, 21, or 35°C. At pH 5.6, L. monocytogenes was inactivated in the presence of 22000 ppm sodium benzoate after 60 days of incubation at 4°C. At pH 5 , the organism was completely nonviable in TB containing 2 1500 ppm sodium benzoate after 24-30 days of incubation at 4"C, whereas lower concentrations of sodium benzoate led to gradual decreases in numbers of listeriae during 66 days at 4°C. Inhibition of Listeria by benzoate decreased at incubation temperatures above 4°C and at pH values higher than 5. Inhibition and inactivation of L. monocytogenes in the presence of sodium benzoate is affected by (a) temperature (i.e., more rapid at higher than lower incubation temperatures), (b) concentration of benzoic acid (i.e., more rapid at higher than lower concentrations), and (c) pH (i.e., more rapid at lower than higher pH values) as well as the type of acid used to adjust the growth medium. When TB was acidified to pH values of 5.0 and 5.6 with acetic, tartaric, lactic, or citric acid rather than hydrochloric acid, El-Shenawy and Marth [ I 161 found that the antilisterial activity of sodium benzoate was greatly enhanced. For example, 1500 ppm sodium benzoate led to complete inactivation of L. monocytogenes after 96 h at 35°C when acetic or tartaric acid was used to adjust the pH of the medium to 5 ; under the same conditions, the pathogen remained viable at least 78 h longer when the pH of the medium was adjusted with hydrochloric acid. The authors concluded that acetic and tartaric acid were most effective in enhancing the antilisterial effects of sodium benzoate followed by lactic and citric acid. Using a minimal glucose-citrate medium (lacking a nitrogen source) adjusted to pH 5.5 with sodium hydroxide, Yousef et al. [404] demonstrated that death rates for L. monocytogenes were affected far more by incubation temperature than by the presence of 1000-3000 ppm benzoic acid in the medium with D-values decreasing - 100-fold as the incubation temperature was increased from 4 to 35°C. Injured listeriae also were detected after plating samples on restrictive and nonrestrictive media. The extent of cell injury was somewhat greater at lower than higher incubation temperatures. These data along with the isolation of an apparent sodium benzoate-resistant strain of L. monocytogenes from an animal-based dairy ingredient [ 1021 suggest that use of benzoic acid alone

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to control Listeria in food is questionable. However, Emme et al. [ 1251 found that repeated exposure of L. monocytogenes to 1000 ppm benzoic acid did not increase resistance of the organism to this widely used food additive.

Parabens and Other Benzoic Acid Derivatives Parabens are esters of p-hydroxybenzoic acids. Of these esters, methyl, propyl, and heptyl parabens are approved in several countries for direct addition to food. Since the pK, of these derivatives is higher than that of benzoic acid, the molecules remain undissociated at pH values up to 8.5. Although benzoic acid is effective as an antimicrobial agent only in acidic foods, parabens retain activity over a wide range of pH values [81]. Payne et al. [294] examined the ability of methyl and propyl paraben to inhibit growth of L. monocytogenes on Tryptose Phosphate Agar plates during 18 h of incubation at 35°C. Using eight strains of the pathogen, propyl and methyl paraben yielded minimum inhibitory concentrations of 5 12 and >5 12 ppm, respectively. Moir and Eyles [265] treated a culture of L. monocytogenes at 35 and 5°C with methyl paraben in buffered BHI broth and measured the MICs. Overall, L monocytogenes was more resistant to this paraben than were other psychrotrophic bacteria, namely, Pseudomonas putida, Yersinia enterocolitica, and Aeromonas hydrophila. When Listeria in broth of pH 6 was incubated at 5°C in the presence of 1000 ppm methyl paraben, the count decreased only -2 logs in 4 months, with 86-99.9% of the viable cells being injured after 2 months. The MIC, defined as the lowest concentration preventing visible growth in buffered BHI broth after 10 days of incubation at 30°C or 3 months at 5"C, was 300-700 ppm at pH 5 and 3OoC, 13001600 ppm at pH 6 and 3OoC,and 600 ppm, at pH 6 and 5°C. Therefore, the MIC decreased as incubation temperature or pH decreased. Parabens also exhibited antilisterial activity against Listeria when the additive was tested in food. Using reconstituted nonfat dry milk (10% solids) inoculated to contain 10' or 103L. monocytogenes CFU/mL, Payne et al. [294] found that populations of listeriae were approximately three to four orders of magnitude lower in samples containing 1000 rather than 0 ppm propyl paraben following 24 h of incubation at 35°C. When these experiments were repeated at refrigeration temperatures [881, Listeria counts in milk samples containing 1000 ppm propyl paraben remained constant during 10 days at 4°C. Dje et al. [88] also investigated behavior of L. monocytogenes in 10% (w/v) aqueous suspensions of raw chicken meat and frankfurters to which 1000 ppm propyl paraben was added. Although L. monocytogenes attained maximum populations of 1O8 CFU/mL in propyl paraben-free chicken suspensions following 24 h at 35"C, numbers of listeriae increased only approximately 10-fold to a maximum of 105CFU/mL in similar suspensions containing 1000 ppm propyl paraben. However, addition of 1000 ppm propyl paraben to frankfurter suspensions failed to prevent growth of L. monocytogenes, with similar growth rates and maximum populations of 108CFU/mL appearing in samples prepared both with and without propyl paraben after 24 h of incubation at 35°C. Since L. monocytogenes and propyl paraben are primarily present in the water and lipid phases of these suspensions, respectively, the higher percentage of fat in frankfurter than in chicken suspensions likely accounts for increased growth of listeriae in the former. Antilisterial activity of p-aminobenzoic acid (pK, of 4.8) relative to common organic acids (formic, propionic, acetic, lactic, and citric) was investigated by Richards et al. [3 181. The authors reported that p-aminobenzoic acid had greater inhibitory activity against L. monocytogenes, S. enteritidis, and E. coli than did other organic acids. The MIC of p-aminobenzoic acid, measured against L. monocytogenes in BHI broth after 24

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h of incubation at 37OC, was 9-12 mmol/L at pH 6.7-6.8 compared with MICs of 1820 mmol/L for formic acid at pH 6.1-6.2, 18-25 mmol/L for propionic acid at pH 6.06.3, 18-30 mmol/L for acetic acid at pH 5.5-6.2, 35-40 mmol/L for lactic acid at pH 5.2-5.4, and 12-14 mmol/L for citric acid at pH 5.2-5.4. When BHI broth was adjusted to pH 6.5 with the acids included in the study, their antilisterial potency was as follows: p-aminobenzoic >propionic >formic >acetic >citric >lactic acid. Antilisterial activity of p-aminobenzoic acid was pH dependent, with higher activity being observed at lower pH values; however, this acid showed some inhibitory activity even at near neutral pH values near neutral.

Fatty Acids and Related Compounds Interest in the potential food applications for fatty acids and related compounds that possess antimicrobial properties also continues to increase with potential applications in food. Antilisterial activity of free fatty acids, particularly those of medium chain length, has been demonstrated. In addition to their primary function as food emulsifiers, some fatty acid esters, particularly monoacylglycerols (monoglycerides) and esters of sucrose, inhibit a wide spectrum of microorganisms, including L. monocytogenes.

Free Fatty Acids Pfeiffer et al. [301] investigated the individual effects of 100 ppm butyric, caprylic, and caproic acids on growth of four L. monocytogenes strains in TB at pH 5.6 during incubation at 13°C. Butyric and caproic acid failed to inhibit growth of L. nzonocytogenes. In contrast, the average generation time for L. monocytogenes was about twice as long (9.40 h) in TB containing caprylic rather than caproic or butyric acid or in the control without fatty acids. Along with the slower growth rate, slightly lower Listeria populations were observed after 14 rather than 7 days for butyric and caproic acid. Wang and Johnson [385] tested the antilisterial activity of fatty acids which are naturally present in milk and suggested that some of these compounds could be effectively used as antilisterial agents in dairy products. According to results of this study, L. monocytogenes Scott A, grown in BHI broth, at 35OC, was inactivated by 10, 20, 100, 200, and 200 pg/mL of glycerol monolaurate, lauric (C 12:0), linolenic (C 18:3), linoleic (C18:2), and potassium salts of conjugated isomers of linoleic acids (K-CLA), respectively, with corresponding concentrations of 10, 10, 20, 50, and 100 pg/mL inactivating the pathogen at pH 6. In contrast, 200 pg/mL of myristic (C14:0), palmitic (C16:0), or stearic (C18: 0) acid were not inhibitory. In skim and whole milk, K-CLA showed strong listeriostatic activity, which was enhanced by the presence of 2000 pg/niL citrate, 100-200 pg/mL butylated hy droxyanisol (BHA), ascorbate, or a-tocopherol. Kinderlerer and Lund [205] examined the MICs of hexanoic and octanoic acids, both of which have a pK, of 4.85, against 10 L. monocytogenes strains and 2 L. innocua strains growing in TSB supplemented with yeast extract and glucose (TSYGB) and adjusted to pH 5.5 and 5.0. Octanoic acid exhibited lower MICs against listeriae than hexanoic acid, and MICs varied among different strains of L. monocytogenes. After 162 h of incubation, MICs for octanoic acid were <1.41-3.49 and 1.41-3.49 mmol/L at pH 5.0 and c3.18--7.86 and 3.18-7.86 mmol/L at pH 5.5 for L. munocytogenes and L. innocua strains, respectively. Hexaonic acid MICs were 6.89-8.61 mmol/L after 138 h of incubation at pH 5.0 and > 10.33 mmol/L after 89 h of incubation at pH 5.5 for L. monocytogenes and L. innocua strains except for one L. rnonocytogenes strain with MICs of <6.89 and

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<0.861 mmol/L at pH 5.0 and 5.5, respectively. Therefore, some strains of L. innocua appear to be more resistant to hexanoic acid than L. rnonocytogenes. Free fatty acids are present in certain cheeses, with blue cheese being reported to contain 4.6 and 1.9 mmol/ kg of hexanoic and octanoic acids, respectively [246]. Therefore, concentrations of these two fatty acids comparable to those found in cheese may have marked antilisterial activity in acidic foods. However, the antilisterial activity of these two fatty acids would be expected to decrease dramatically in blue cheese during ripening as the pH increases from about 4.6 to 6.2 [286].

Fatty Acid Monoesters Some fatty acid monoesters of glycerol (i.e., monoacylglycerols, which are commonly known as monoglycerides) and sucrose are potentially useful as antimicrobial food additives. Monolaurin, the lauryl glycerol monoester, and sucrose laureate were studied extensively and will be discussed in some detail below. The antilisterial activity of monolaurin was first reported in 1992 by Oh and Marshall [276] and Wang and Johnson [385]. Oh and Marshall [276] noted that monolaurin was more antilisterial than other common antimicrobial agents (sorbate, propyl paraben, tertiary butyl hydroxyquione [TBHQ], propyl gallate, and butylated hydroxyanisol [BHA]) and inhibited four L. rnonocytogenes strains (- 10' CFU/mL) in a laboratory broth at 35°C with MICs of 3-4, 7, 9, and 10 pg/mL at pH 5.0, 5.5, 6.0, and 7.0, respectively. Thus different L. rnonocytogenes strains had similar sensitivity to monolaurin. Wang and Johnson [385] found that L. rnonocytogenes was inhibited by 10 pg/mL monolaurin in BHI broth at pH 5 and 6. When L. monocytogenes was cultured on BHI agar plates, the MICs for monolaurin were 96, 14, 7, and 5 pg/mL at pH 7.0, 6.0, 5.5, and 5.0, respectively [3 121. A monolaurin MIC of 16 pg/mL for L. monocytogenes on Tryptic Soy Agar (TSA) also was reported by Bal'a and Marshall [25]. Inhibition of microorganisms, including L. rnonocytogenes, is more pronounced using monolaurin than other fatty acid monoesters. The minimum bactericidal concentrations at which 103-10" L. rnonocytogenes CFU/mL was completely inactivated in BHI broth (pH 6) after 24 h at 37°C were 25,50, and 75 yg/mL for monolaurin (MC,,), monocaprin (MC,,), and monomyristin (MC,,), respectively, whereas a concentration of 300 pg/mL monocaprylin (MC,) was only inhibitory [387]. According to these authors, L. rnonocytogenes was not inhibited by 300 pg/mL of monopalmitin (MC,,), monostearin (MC,,), monoolein (MC18:,),or monolinolein (MCIX:?). Gram-positive bacteria are more sensitive to monolaurin than are gram-negative organisms. L. monocytogenes was the most resistant among the gram-positive bacteria that were tested by Razavi-Rohani and Griffiths [3 121. These investigators used a spiral gradient method and found that growth of L. monocytogenes on BHI agar was completely inhibited by a minimum of 96 pg/mL monolaurin, whereas complete inhibition of six other gram-positive bacteria (Bacillus, Stuphylococcus, and Lactococcus) required a minimum of 8-24 lg/mL monolaurin. In contrast, concentrations as high as 3170 pg/mL failed to inhibit nine gram-negative bacteria [3 121. Monolaurin in combination with other treatments or antimicrobial agents, such as low temperature, low pH, organic acids, chelating agents, and antioxidants, exhibited enhanced antimicrobial activity. Gram-negative bacteria were inhibited by some of these combinations. In the presence of 5 4 % NaCl, growth of gram-negative bacteria on BHI agar was inhibited by <2 pg/mL monolaurin [312]. As described previously, the MICs of monolaurin decreased as the pH decreased [276]. Oh and Marshall [277] investigated

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the antilisterial activity of monolaurin (5-9 pg/mL) at different temperatures (7, 15, and 35°C) and pH values (5.0,5.5, and 7.0) in TSBYE. Although monolaurin was listeriostatic at most temperature/pH combinations, listericidal activity was detected using 8-9 pg/mL of monolaurin at pH 5.0 with L. monocytogenes strain Scott A (initially inoculated at 1O3 CFU/mL) reportedly undetectable at this concentration after 20.0-22.0, 6.0, and 0.5 days at 7, 15, and 35"C, respectively. The study just described provides evidence that inhibition of L, monocytogenes by monolaurin is highly temperature-dependent. Although Wang and Johnson [385] found that monolaurin at 200 pg/mL was listericidal in skim milk at 4"C, no antilisterial activity was observed at 30°C. Oh and Marshall [278] reported a greater listeriostatic effect in TSBYE at 35°C when 18 pM monolaurin was used in combination with sublethal concentrations of organic acids (acetic, benzoic, lactic, or citric) than when monolaurin or the acids were used separately. When tested in crawfish tail meat at 4OC, 336 mM lactic acid decreased L. rnonocytogenes populations from about 103CFU/g to nondetectable levels in 10 days; however, similar inactivation could be achieved using a combination of 0.72 mM monolaurin and 224 mM lactic acid. When used alone, 224 mM lactic acid resulted in complete inhibition 12781. Bal'a and Marshall [25] investigated the combined effect of NaCl (2.5-7.8%), pH (5.4-7.8), temperature ( 5 , 15, 25, and 35"C), and sublethal levels of monolaurin (2-8 pg/ L) on growth of L. monocytogenes on double (salt-pH) gradient plates and found significant interactions between these factors. More recently, Razavi-Rohani and Griffiths [3 I2,3 131 detected enhanced antimicrobial activity of monolaurin when this compound was used in combination with ethylenediaminetetraacetic acid (EDTA), BHA, low pH, or NaC1; however, no marked enhancement was observed in the presence of lysozyme. The presence of EDTA not only reduced the MICs for all gram-positive bacteria (70 pg/ mL for L. rnonocytogenes), but also sensitized gram-negative bacteria to monolaurin (MICs of 90-1500 pg/mL). However, no decrease in monolaurin MICs was caused by the presence of EDTA at pH 1 6 . Sodium citrate and monoglyceride citrate, two other chelating agents tested, decreased the antimicrobial effect of monolaurin [3 121. Antilisterial activity of monolaurin increased in the presence of organic acids and antioxidants [386]. According to Wang and Johnson, inhibitory activity of monolaurin ( 5 pg/g) against L. monocytogenes in BHI broth was enhanced by other antilisterial agents such as BHA (100 pg/g), TBHQ (30 pg/g), propyl gallate (200 p/g), acidulants (0.1% acetic or lactic acid), and potassium sorbate (0.1%) [386]. Propyl gallate (200 p/g) and lactic acid ( 0.2%) enhanced the bacteriostatic activity of monolaurin and monocaprin against L. rnonocytogenes in seafood (imitation crab meat and cooked shrimp) and Camembert cheese, respectively, which were stored at 4°C. However, when tested in foods, 0.1% glycine, sodium citrate, propylene glycol, or Tween 20,O. 1-0.2% potassium sorbate, 200 pg/mL lysozyrne, or 200 pg/mL BHA did not enhance activity of monoacylglycerols [386]. Oh and Marshall [280] reported that combined use of 200 pg/g rnonolaurin and 0.5% lactic acid significantly inhibited growth of Listeriu in crawfish meat homogenate stored at 4"C, whereas these additives had little or no effect on growth of Listeria when used separately. Antilisterial interactions occur not only between monolaurin and other factors but also among different monoacylglycerols as well. Wang et al. [387] reported an additive effect between monolaurin ( 100 pg/mL) and monocaprin ( 100 pg/mL) and a synergistic effect between monomyristin (200 pg/mL) and monocaprin (200 pg/mL) when these compounds were tested in skim milk at 4°C. The authors also noted strong antilisterial activities by monoacylglycerols synthesized from coconut oil. These monoacylglycerols had a MIC

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(10 pg/mL), which was lower than that of monolaurin (25 pg/mL) when both agents were tested in BHI broth (pH 6) with incubation at 37°C. Compared to milk fat-derived monoacylglycerols, which were not inhibitory to L. monocytogenes at 300 pg/mL, coconut monoacylglycerols are rich in lauric (C12), myristic (C14), and capric (C10) acids, and thus may contain higher levels of monolaurin, monomyristin, and monocaprin. Demonstrated antilisterial activity of these three dominant monoacylglycerols and the synergistic interaction between them may account for the high antilisterial activity of coconut monoacylglycerols. When tested in refrigerated skim milk, 2% milk, and whole milk, monocaprin, monolaurin, and coconut oil-derived monoacylglycerols showed strong antilisterial activity. At >200 pg/mL, monocaprin was more effective than monolaurin in inactivating L. monocytogenes in all three refrigerated milks, possibly because of the higher water solubility of monocaprin. High temperatures of incubation and high fat content of the growth medium decreased the effectiveness of monoacylglycerols. In skim milk containing 250 pg/mL of the coconut-derived monoacylglycerols, L. monocytogenes (- 1O3 CFU/mL) was inactivated after storage at 4°C for 7 days but grew to 107- 1 Ox CFU/mL at I 3 and 23°C. Concentrations of 250-400,500-750, and 750- 1000 pg/mL of coconut-derived monoacylglycer01s were required to inactivate L. monocytogenes in refrigerated skim milk, 2% milk, and whole milk, respectively [387]. Compared with 1000 pg/g monolaurin or monocaprin, coconut monoacylglycerols ( I000 pg/g) and a combination of monolaurin (500 pg/g) plus monocaprin (500 pg/g) exhibited greater listericidal activity in beef frankfurter slurries (pH 5.0 and 5.5) and seafood salad (pH 4.9) stored at 4°C. However, at 12OC, Listeria grew rapidly in beef frankfurter slurries (pH 5.5) with or without such levels of these monoacylglycerols. When present in turkey frankfurters (pH 5.6 and 6.1) stored at 4"C, the monoacylglycerols ( 1000 pg/g) were bacteriostatic, with half this concentration being listeriostatic in cooked shrimp (pH 7.1), imitation crab meat (pH 6.5), and Camembert cheese (pH 6.2) [386]. Although 10-96 pg of monolaurin/mL can inhibit Listeria growth in culture media [25,276,277,312,385,3871, much higher concentrations are required to elicit similar levels of inhibition in foods. Monoacylglycerols may interact with food components such as lipids and protein, and thus become less available for contact with microorganisms and so less capable of exerting an antimicrobial effect. Wang and Johnson [385] found that antilisterial activity of monolaurin decreased dramatically as the fat content of milk increased. Although 100 pg/mL and 2200 pg/mL monolaurin was listeriostatic and listericidal, respectively, in skim milk at 4OC, 200 pg/mL monolaurin showed no such activity in whole milk. Levels of monolaurin required to inhibit L. monocytogenes vary with the type of food. Oh and Marshall [280] found that 200 pg/g monolaurin did not significantly inhibit growth of L. monocytogenes in refrigerated crawfish tail meat homogenate packaged in air or a modified atmosphere. However, this level of monolaurin significantly decreased the growth of Listeria in vacuum-packaged meat samples. Earlier these authors [278] found that 0.72 and 1.44 pM monolaurin extended the generation times of L. monocytogenes in crawfish tail meat stored at 4°C from 16.7 (for untreated control) to 28 and 48 h, respectively. In 1997, Wang and Johnson [386] reported that antilisterial activity of monoacylglycerols was much greater in beef frankfurter slurries and seafood salad than in turkey frankfurter slurries, summer sausage, cooked shrimp, imitation crabmeat, yogurt, and cottage and Camembert cheeses. Monolaurin had greater antilisterial activity in 2% chocolate milk than in 2% milk, possibly because of the enhancing effect of cocoa. Since monolaurin is poorly soluble in water at high concentrations and has a soapy

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flavor, the antimicrobial activity of more water-soluble monoacylglycerols and water-soluble derivatives of monolaurin were investigated. Although less inhibitory than monolaurin in broth cultures, the more water-soluble monocaprin at >200 pg/mL showed higher antilisterial activity than similar concentrations of monolaurin in refrigerated milk [387]. However, monocaprylin (MC,) was only inhibitory at 300 pg/mL when tested in broth cultures [387]. When the water-soluble triglycerol 1,2-laureate was used alone, it did not inhibit L. monocytogenes; however, in the presence of 380 pg/mL EDTA, the pathogen was inhibited by this monolaurin derivative at a MIC of 380 pg/mL on agar plates [312,313]. Beside monoacylglycerols, fatty acid esters of sucrose, which are commonly used as food emulsifiers, exhibit antimicrobial activity against a wide range of spoilage and pathogenic bacteria. Monk et al. [267] found that 400 pg/mL of sucrose monolaurate was lethal to L. monocytogenes (Scott A) in TPB at 3OoC, whereas lower concentrations (100200 pg/mL) exhibited a strong listeriostatic effect. L. monocytogenes was generally more sensitive than S. aureus to sucrose monolaurate alone or in combination with various enhancers, such as EDTA and organic acids. As with monolaurin, EDTA (50-200 pg/ mL) synergistically enhanced the antilisterial effect of sucrose monolaurate; this enhanced inhibition was more pronounced as the incubation temperature decreased from 30 to 15 or 5°C. A similar effect of EDTA on sucrose monolaurate activity was previously reported by Sikes and Whitfield [349]. However, addition of 0.1%acetic or lactic acid to the growth medium decreased inhibition during the initial 32 h of incubation but resulted in significantly lower final populations during subsequent incubation. Sucrose monolaurate alone or in combination with EDTA synergistically increased the thermal inactivation of L. monocytogenes 12671. Monolaurin was reported by Oh and Marshal1 [279] to enhance destruction of L. monocytogenes in biofilms on stainless steel. Similar to the findings for monolaurin, higher concentrations of fatty acid esters of sucrose were required to inhibit L. monocytogenes in foods, particularly those with high fat contents, than in laboratory media. Sikes and Whitfield [349] reported decreased antilisterial activity of sucrose monolaurate, present alone or in Combination with EDTA and BHA, when the fat content of their model food system increased. Monk and Beuchat [266] found that when added to uncooked ground beef, 0.30% sucrose laureate or 0.32% sucrose palmitate sigriificantly inhibited growth of L. monocytogenes (Scott A) at 5"C, whereas 0.32% sucrose stearate or 0.15% sucrose oleate showed no antilisterial activity. Such levels of these esters had little or no effect on S. aureus and psychrotrophic bacteria in ground beef.

Sodium Nitrite Studies undertaken by Shahamat et al. [342] during the 1970s examined effects of various concentrations of sodium nitrite and sodium chloride on growth of L. monocytogenes in TSB at different temperatures and pH values. When incubated at 37, 22, and 4°C in broth at pH 7.4, L. rnonocytogenes grew at nitrite concentrations as high as 25,000, 30,000, and 10,000ppm, respectively. Inhibitory effects of nitrite were enhanced at pH 6.5, particularly at lower incubation temperatures, with complete inhibition being caused by 1500 ppm nitrite at 4°C. MICs of nitrite were further reduced at all three incubation temperatures and at pH 5.5, with 600 ppm nitrite being sufficient to inhibit growth at 4°C. The bacteriostatic activity of nitrite was greatest at pH 5 and at 22 and 3 7 T , with no growth reported at nitrite concentrations >800 and 400 ppm, respectively. Addition of 3% sodium chloride to TSB failed to increase the bacteriostatic action of sodium nitrite. Although MICs for

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nitrite were only slightly lower with 5.5 or 8.0% sodium chloride at pH 7.4 or 6.5, the combination of 5.5 or 8.0% sodium chloride and pH values of 5.0 or 5.5 led to MICs for nitrite that were generally 8- to 20-fold lower than controls prepared without sodium chloride. Inhibitory effects of nitrite were again most pronounced at 4°C when the chemical was combined with sodium chloride. The study by Shahamat et al. [342] was criticized by McClure et al. [254], who found that autoclaving caused nitrite to decompose. After autoclaving nitrite-containing TSB at pH 5, no nitrite was detected. Since only 10-72% nitrite was detected after autoclaving nitrite-containing TSB at pH 5.3-6.7 [254], McClure et al. [254], together with Buchanan et al. [%I, believed that the antimicrobial activity of nitrite was seriously underestimated by Shahamat et al. [342]. Using filter-sterilized rather than autoclaved sodium nitrite, McClure et al. [254] reported much greater antilisterial activity of nitrite in TSB. Filter-sterilized nitrite, at 50 pg/mL and pH 5 , prevented visible growth for 48 h; however, 200 pg/mL autoclaved nitrite under the same conditions did not retard Listeria growth. As mentioned earlier, behavior of L. monocytogenes in food and culture media depends on the interactive effects of temperature, pH, type of acidulant, salt content, a,, and types and concentrations of food additives that may be present in the system. The effectiveness of sodium nitrite as an antilisterial agent also is strongly influenced by these same factors. Buchanan et al. (581 used a factorial design to determine the effect of sodium nitrite (0- 1000 ppm) in combination with incubation temperature (5-37"C), initial pH (4.5-7.5), sodium chloride (0.5-4.5%), and atmosphere (aerobic vs anaerobic) on growth of L. monocytogenes in TPB. Although lag periods, generation times, and maximum populations were all affected by these five interacting variables, sodium nitrite was most listeriostatic when used in conjunction with low pH, increased sodium chloride, refrigeration temperatures, and anaerobic conditions that simulated vacuum packaging. McClure et al. [254] reported that antilisterial activity of nitrite strongly depended on pH. At pH 2 6, nitrite, even at 400 pg/mL, had little antilisterial activity. However, below pH 6, nitrite, even at 50 pg/mL, exhibited antilisterial activity. Visible growth (defined as 0.03unit increase in ODhO0 in 21 days at 5-30°C) of this pathogen in TSB at 20°C was prevented by 50 pg/mL sodium nitrite and pH 5 5.3. Autoclaving nitrite dramatically decreased its antilisterial activity. Buchanan et al. [53] found that inactivation of L. monocytogenes by NaNO, was affected by several factors, with lactic acid being the most influential. At high levels of lactic acid (low pH), the listericidal action of nitrite increased, whereas low levels of lactic acid had little effect on nitrite activity. The antilisterial activity of sodium nitrite and other antimicrobial agents also was studied by Buncic et al. [60]. Growth of L. monocytogenes (initially 107CFU/mL) in buffered BHI broth (pH 5.5) incubated at 4°C for up to 7 weeks was prevented by addition of 125 ppm sodium nitrite or a combination of 125 ppm sodium nitrite and 0.5% polyphosphate. Polyphosphate (0.5%) alone did not prevent growth of Listeria. This is consistent with results from several research groups [58,254,342], who noted that nitrite inhibited L. monocytogenes at low pH values. Nitrite and nisin also had a synergistic effect against L. monocytogenes. When used alone, nisin (400 IU/mL), resulted in an initial 1.1 -log reduction, with Listeria survivors eventually growing during extended incubation. However, a combination of nisin and nitrite (125 ppm) not only prevented Listeria regrowth but caused a further (1.4 log) reduction when compared with nisin alone. Working with meat products, Johnson et al. [ 1881 found that growth of L. monocytogenes was suppressed at 4°C in hard salami (pH 4.3-4.5) that contained 5.0-7.8% NaCl

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plus 156 ppm sodium nitrite. Although their findings agree with those of Shahamat et al. [342], the combination of low water activity (a, 0.79-0.86) and low pH were probably more important in preventing growth of listeriae than was the addition of 156 ppm sodium nitrite. Findings of Glass and Doyle [ 1561 also indicate that 3.5% sodium chloride plus 156 ppm sodium nitrite in sausage batter at pH 6.2 controlled growth of L. monocytogenes in the product during fermentation at 90°F. Under these conditions, additional acid development through fermentation is essential to prevent growth of listeriae.

Antioxidants Antioxidants such as BHA, butylated hydroxytoluene (BHT), TBHQ, and propyl gallate comprise an important category of food additives. Although primarily used to prevent oxidation of fat, some of these antioxidants also possess antimicrobial activity. Limited trials on destruction of L. monocytogenes by BHA were initiated by AlIssa et al. 161 during the early 1980s. Tryptone Soy Broth containing 50 ppm BHA was inoculated to contain 105L. monocytogenes CFU/mL and examined for numbers of the bacterium. The Listeria population decreased -3 logs during the first 12 h at 37°C and remained at a level of -102 CFU/mL after 24 h of incubation. The same authors also found that successive subculturing of L. monocytogenes in a medium containing glycerol followed by inoculation into Tryptone Soy Broth containing 50 ppm BHA led to rapid Listeria growth with populations reaching 10'- 1O9 CFU/mL after 24 h at 37°C. Development of BHA resistance correlated with a high lipid content in the cell wall and membrane from prior growth in a medium containing glycerol. Payne et al. [294] investigated the potential of BHA, BHT, TBHQ, and propyl gallate to inhibit growth of L. monocytogenes on Tryptose Phosphate Agar during 18 h of incubation at 35°C. Using an agar dilution method, TBHQ was the most effective antioxidant tested with a MIC of 64 ppm followed by BHA, propyl gallate, and BHT with MICs of 128, 256, and 5 12 ppm, respectively. Although these findings may at first appear promising for the food industry, L. monocytogenes is far more likely to encounter sublethal levels rather than MICs of antioxidants in food. Consequently, Yousef et al. [405] examined the growth kinetics of L. monocytogenes strain Scott A in TB containing BHA (100-300 ppm), BHT (300-700 ppm), and TBHQ (10-30 ppm) during 54 h of incubation at 35°C. Overall, these findings agreed with those of Payne et al. [294] in that TBHQ again was most inhibitory to L. monocytogenes followed by BHA and BHT. According to the authors, L. monocytogenes exhibited increasingly longer lag periods and generation times as well as lower maximum populations in the presence of BHA at 100-200 ppm, with concentrations 2300 ppm proving to be lethal. Since all three growth parameters were increasingly affected as the sublethal concentrations of BHA increased, the organism was probably unable to detoxify this antioxidant metabolically. Therefore, addition of up to 200 ppm BHA to food, as permitted by the FDA, will likely contribute to overall keeping quality and safety of some products. Unlike BHA, L. monocytogenes was unaffected by (-300 ppm BHT; however, poor solubility of BHT in TB prevented critical analysis of this antioxidant at concentrations >300 ppm. Interestingly, increasing the concentration of TBHQ from 10 to 30 ppm led to an exponentially longer lag period for L. monocytogenes, but it did not appreciably affect generation times or maximum populations. Hence, unlike BHA, these observations suggest that L. monocytogenes can metabolically detoxify sublethal concentrations of TBHQ to safe levels and initiate growth thereafter. From this, it appears that BHA may be of greater benefit than TBHQ for inhibiting growth of listeriae in food.

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Payne et al. [294] examined the antilisterial activity of TBHQ in reconstituted NFDM (1 0% solids) that was inoculated to contain 10 L. monocytogenes CFU/mL; addition of 150 ppm TBHQ prevented growth of the pathogen and variably inactivated it during 24 h of incubation at 35°C. Although numbers of listeriae increased nearly 100fold in similar samples inoculated to contain - 10' L. monocytogenes CFU/mL, maximum populations were still approximately three orders of magnitude lower than those observed in TBHQ-free control samples. Results obtained by repeating these experiments at refrigeration temperatures were more encouraging [88], with original Listeria inocula of 10' and 1O3 CFU/mL being reduced to nondetectable levels or remaining constant, respectively, during 10 days at 4°C. Although these preliminary findings suggest that addition of TBHQ to foods at FDA-permissible levels of 1200 ppm may be of some benefit in inhibiting and/or inactivating L. monocytogenes, present FDA regulations [ 151 stipulate that TBHQ and all other such additives can only be used as antioxidants and cannot be added indiscriminately to foods for other purposes. Chung et al. [7 I ] investigated the antimicrobial activity of propyl gallate, another antioxidant food additive. The authors used a well diffusion method and measured the zone of inhibition after 48 h of incubation at 32°C. Propyl gallate prevented growth of two strains of L. monocytogenes, but ellagic acid, the hydrolytic product of propyl gallate, failed to inhibit the bacterium. The authors further examined the effect of this antioxidant against L. monocytogenes growing in cabbage juice at room temperature. Results were consistent with that of the well diffusion method; propyl gallate but not ellagic acid exhibited antilisterial activity. Propyl gallate at 500 pg/mL prevented growth of L. monocytogenes (initially 6.3 X 10' CFU/mL) in cabbage juice and 250 pg/mL allowed -2-log increase after 4 days; however, a final population of 108 CFU/mL was attained in the control.

Liquid Smoke Many commercially available liquid smoke products used in processed meats and sausages can inactivate common foodborne organisms, including E. coli, S. aureus, and Lactobacillus viridescens. These artificial liquid smoke flavorants owe their activity to the presence of phenolic compounds and acetic acid, both of which are bactericidal at relatively low concentrations. After L. monocytogenes was recognized as a possible health hazard in ready-to-eat meat and sausage products, several investigators examined the potential of various liquid smoke compounds to inactivate L. monocytogenes in phosphate buffer and culture media commonly used to isolate this pathogen from meat products. Using sterile phosphate buffer at pH 5.64 and inoculated to contain 1 X 105L. monocytogenes CFU/mL [259], three of five liquid smoke compounds (Charsol- 10, Aro-Smoke P-50, and CharDex Hickory, Red Arrow Products, Manitowoc, WI) tested at a concentration of 0.5% reduced numbers of listeriae to nondetectable levels after 4 h at ambient temperature. When the concentration of liquid smoke products was decreased to 0.25%, numbers of listeriae were still reduced to nondetectable levels within 4 h using either CharSol- I0 or Aro-Smoke P-50. However, CharDex Hickory was far less effective at the lower concentration with 24 h of incubation required to inactivate the pathogen completely. Listericidal activity of these liquid smoke compounds also appeared to be at least partially related to levels of acetic acid present in the various preparations. Subsequently, Wendorff 13921 found that the same liquid smoke compounds were

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far less listericidal when added to USDA-recommended Listeria Enrichment Broth rather than phosphate buffer. Interactions between liquid smoke constituents and protein in the enrichment broth were probably at least partially responsible for the observed decrease in listericidal activity. Although three of five liquid smoke compounds were effective against L. monocytogenes in buffer and to a lesser extent in culture media, later work by Wendorff [392] showed that concentrations of liquid sinoke needed to inactivate L. monocytogenes in processed meats were well above organoleptically acceptable limits. In 1992, Faith et al. [128] reported that adding 0.2 and 0.6% (v/v) of liquid smoke (CharSol Supreme, Red Arrow Products, Manitowoc, WI) to wiener exudate inactivated L. monocytogenes with D-values of 36 and 4.5 h, respectively, whereas Listeria in the smoke-free exudate grew from initial levels of 10sto 108CFU/mL after 3 days at 25°C. The authors further investigated the antilisterial activity of selected smoke components in TB at pM 7. When the culture was incubated at 37"C, they found that among 11 phenol compounds tested, only isoeugenol retarded growth of Listeria. Although final maximum populations were similar, lag-phase duration increased linearly from -3 to 21 h as isoeugenol levels increased from 0 to 200 ppm. It was estimated that an isoeugenol concentration of 236 ppm was required to increase the lag phase by one order of magnitude. L. monocytogenes in the presence of 100 ppm isoeugenol L. monocytogenes also was inhibited to a greater extent when the pH of TB was adjusted to 5.8 compared with 7.0.

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Spices, Herbs, and Plant Extracts Although primarily used as flavoring and seasoning agents, many spices contain specific chemicals and/or essential oils that can inactivate or inhibit various pathogenic and spoilage organisms. Consequently, surveys were made to identify spices that might be useful in inhibiting growth of L. monocytogenes in food. Ting and Deibel [375] reported results from one such survey in which a concentration gradient plate method was used to study the effect of 13 different spices on three L. monocytogenes strains. Although the pathogen remained completely viable in the presence of 3% (w/v) black pepper, chili pepper, cinnamon, garlic, mustard, paprika, parsley, and red pepper during extended storage at 4 and 24"C, Listeria was sensitive at 24°C to cloves, oregano, sage, rosemary, and nutmeg, with calculated MICs of 0.60-0.70, 0.50-0.70, 0.70-0.90, 0.90-1 .O, and 1.1- I .4%, respectively. The latter five spices also were inhibitory to L. monocytogenes at 4°C. When added to TSB, growth of the pathogen at both incubation temperatures was prevented by as little as O S % cloves, oregano, or sage. Listericidal effects were observed with 10.5% clove at 4 and 24°C and 10.5% sage at 4°C. When exposed to 0.5% clove, the Listeria population decreased >2 logs after 24 days at 4°C or after 24 h at 24°C. A reduction in L. monocytogenes population of >5 logs was observed with 1% cloves in 7 days at 4°C or 3 h at 24°C. Sage levels 0.5 and 1.0% resulted in :.3- and >5-log decreases in Listeria populations, respectively, after 14 days of incubation at 4°C. Unfortunately, further experiments showed that cloves and oregano were both no longer active against listeriae when present in a meat slurry at a level of 1%. The ability of commercially available spices to prevent growth of L. monocytogenes in TB also was investigated by Bahk et al. [24]. With the exception of an increased lag phase, behavior of listeriae during extended incubation at 35 and 4°C was relatively unaffected by 0.5% ginger, onion, garlic, or mustard as well as ginseng, saponin, or mulberry extract at concentrations 50.3%. However, unlike the previous study, addition of 0.5%

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cinnamon was somewhat inhibitory to L. monocytogenes, with the pathogen attaining maximum populations that were 1.5-2.6 orders of magnitude lower than in controls. Growth of listeriae at 35°C also was partly suppressed by 0.5%cloves, with the pathogen attaining a maximum population 1.6 logs lower than that observed in controls. However, Listeria populations decreased steadily in TB containing 0.5% cloves during extended incubation at 4°C. Working in Thailand, Stonsaovapak and Chareonthamawat [367a] used a similar experimental design to test nine dried native Thai spices, namely, cinnamon, black pepper, white pepper, cloves, cardamom, coriander, star anise, nutmeg, and cumin seed at concentrations of 1,3, and 5% for activity against L. rnonocytogenes in TSB containing 10' CFU/ mL. As in the previous two studies [24,375], cloves exhibited strong listeriocidal activity with concentrations 2 1%, reducing Listeria populations >8 logs and >6 logs after 7 days of incubation at 35 and 4OC, respectively. When exposed to concentrations 2 1 % for 7 days, nutmeg and star anise also reduced numbers of listeriae 5-8 logs at 35°C and 2-6 logs at 4°C. Using 2 1 % white pepper, black pepper, or coriander, Listeria population decreased 2-5 logs and 1-2 logs at 35 and 4OC, respectively, with these and the other spices being most effective at concentrations of 5%. However, little if any inhibition was observed using cardamom regardless of concentration or incubation temperature. Antilisterial activity of 32 plant essential oils was investigated by Aureli et al. [22] in Italy. The essential oils were dissolved in ethanol at a concentration of 1 5 (v/v) with antilisterial activity assessed on TSA plates using the disc diffusion method. Five essential oils showed activity against four strains of L. rnonocytogenes. Essential oils of origanum and thyme were most active against Listeria, followed by oils of cinnamon, clove, and pimento. Further tests with thyme, origanum, and cloves showed that the three oils maintained antilisterial activity at a 150 (v/v) dilution. Although nutmeg, rosemary, and sage were found to the antimicrobially active by Ting and Deibel [375], the essential oils of the three spices failed to inhibit listeriae. Essential oils that did not inhibit listeriae were those of basil, camomile, celery, coriander, cumin, estragon, fennel, garlic, ginger, laurel, lemon, mandarin, marjoram, neroly, onion, orange, parsley, pepper, peppermint, pettigrain, saffron, and vanilla. Survival of L. rnonocytogenes ( IO5-1O6 CFU/mL) in a saline solution containing 0.1% (v/v) of essential oils of origanum, thyme, cinnamon, cloves, or pimento was similar among five strains of the pathogen. Oil of pimento had the greatest activity and that of cinnamon the least. Essential oil of pimento decreased the population of Listeria from > 103CFU/mL to an undetectable level in 1 h. All five oils reduced the viable count to undetectable levels after 4 h of incubation at room temperature. In minced pork stored for 8 days at 4 and 8OC, adding 100 pL of the diluted (15, vol/vol) essential oil of thyme to 25 g of product, decreased the population of L. rnonocytogenes by -1 log, whereas the organism grew from 1 X 107to 6 X 107and 2 X 108CFU/g in untreated controls at 4 and 8OC, respectively. In 1993, Hefnawy et al. [ 1661 reported on the sensitivity of L. rnonocytogenes strains Scott A and V7 to 10 spices in TSB at 4°C and found the latter strain to be generally more resistant. L. monocytogenes Scott A decreased from an initial population of 105to <1 CFU/mL by 1% sage in 1 day; I % allspice in 4 days; 1% red pepper, paprika, garlic powder, or cumin in 7 days; 5% black pepper in 4 days; or 5% mace in 7 days, whereas 5% nutmeg partially inactivated the organism and 1-5% white pepper enhanced growth. For strain V7, only 1, 3, and 5% sage reduced the initial population (-107 CFU/mL) to < I CFU/mL in 7, 4, and 4 days, respectively, whereas 3-5% allspice, mace, or nutmeg showed partial inactivation.

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Pandit and Shelef [285] investigated the antilisterial activity of 18 spices and herbs by measuring growth of L. monocytogenes on BHI agar with (0.1%) and without spices/ herbs. The authors found that only rosemary and cloves showed antilisterial activity, and contrary to previous studies [22,375], cinnamon, nutmeg, oregano, and sage had no activity. Other noninhibitory spices and herbs were ajowan, allspice, asafoetida, cardamom, cumin, fenugreek, ginger, marjoram, black mustard, red hot pepper, and turmeric. Growth of Listeria on agar plates was completely inhibited by 0.5% rosemary or 1.0% cloves. An aqueous extract of rosemary only prevented growth of Listeria in BHI broth at 35°C for the first 24 h, whereas the same levels of rosemary or its ethanol extract had significant listericidal activity. The authors further investigated the antilisterial activity of rosemary oil and its four major components (cineole, borneole, camphor, and pinene), rosemary oleoresin, rosemary oil encapsulated in modified starch, and an antioxidant fraction extracted from rosemary by liquid carbon dioxide. Rosemary oil ( 10 pL/ 100 mL) was listeriostatic during 48 h of incubation, whereas oleoresin (up to 100 mg/ 100 mL) did not inhibit listeriae. Encapsulated oil was listeriostatic at 1 pg/lOO mL, and listericidal at 5 pL/lOO mL. The antioxidant extract at 0.02 g/lOO mL was listericidal. Of the four rosemary oil components, only pinene at 0.1 pL/ 100 mL suppressed growth of Listeria, whereas the other three components had no activity at concentrations up to 1 pL/lOO mL. When added to refrigerated (5°C) pork liver sausage, 1% rosemary, 0.5% rosemary oil, 0.3% antioxidant extract, or 5% encapsulated oil suppressed growth of Listeria, with maximal populations of -log, -108, -105, and -105 CFU/mL being attained after 25, 25, 50, and 50 days, respectively, whereas 1O3 CFU/mL of Listeria in the control reached 109CFU/ mL after 25 days. Tassou et al. [372], working in Greece, investigated the antimicrobial effect of the essential oil of mint on L. monocytogenes and S. enteritidis in a broth culture and in three foods; tzatzilu (cucumber and yogurt salad, pH 4.3), taramosalata (fish roe salad, pH 4.9), and piit6 (pH 6.8). Concentrations of mint oil tested in this study were 0.5, 1.0, 1.5, and 2.0%, v/w, and incubation temperatures were 4 and 10°C. Although gram-positive bacteria generally are more sensitive to essential oils than gram-negative microbes, the authors found that L. monocytogenes was more resistant to mint essential oil than S. enteritidis. Antibacterial activity varied between foods and pH values. The presence of 1-2% essential oil in tzatziki decreased salmonellae numbers to undetectable levels after 3-6 h, whereas >2 logs of the organism remained viable after 6 days in the untreated control that initially contained l O7 L. monocytogenes CFU/g. However, in the same treated food, populations of L. rnonocytogenes remained unchanged for the first 4 days and decreased gradually during subsequent storage, with >4 logs being detected after 8 days at both temperatures. Compared with the control, the presence of mint oil decreased the rate of death of L. monocytogvnes in the product. Populations of L. rnonocytogenes and S. enteritidis decreased slightly in taramosalata made with and without mint essential oil during 9 days of incubation at 4 and 10°C. The essential oil was without antimicrobial activity in pit&, which had an almost neutral pH. In all pgt6 samples, salmonellae populations decreased at 4°C or decreased and then increased at 10°C, whereas listeriae increased > 1 and >3 logs at 4 and IOOC, respectively, after 6 days of incubation. Greater antimicrobial activity of mint oil was observed in broth than in food, with both organisms being inhibited by 0.5-2.0% mint oil in broth at pH 6.6. Because of the potential link between consumption of chocolate milk and listeriosis, Pearson and Marth [297] studied the effect of caffeine and theobromine, two methylxanthine compounds in cocoa, against L. monocytogenes strain V7 (initially 103CFU/rnL)

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in a modified TPB and skim milk at 30°C. L. monocytogenes grew similarly in both media. Although limited antilisterial activity was observed with 2.5% theobromine, the authors found that 0.5% caffeine had antilisterial activity in both substrates and increased the lag phase from <3 (control) to 6-9 h, increased generation times from 1.2 to 2.17 h, and decreased the final maximum population from 8.6 to 7.2 logs. A combination of 2.5% theobromine and 0.5% caffeine had slightly more antilisterial activity than did 0.5% caffeine alone. In conclusion, essential oils are more effective in broth than in foods. Because of their hydrophobic nature, antilisterial activity of essential oils is adversely affected by high fat and protein contents in food. Antimicrobial activity of essential oils is enhanced by low pH, high salt content, and low storage temperatures.

Lysozyme Lysozyme is an important natural enzyme which prevents bacterial growth (particularly gram-positive organisms) in foods of animal origin, including hen’s eggs and milk. Lysozyme is particularly attractive as a food preservative, since the enzyme is active between 4 and 95OC, stable over a wide range of pH values, specific for bacterial cell walls, and is not harmful to humans. Although not yet approved as a food additive in the United States, lysozyme has been used successfully in Europe to prevent “blowing” caused by Clostridium spp. in Gouda, Edam, and other brine-salted cheeses. In 1987, Hughey and Johnson [ 1791evaluated four L. monocytogenes strains isolated during foodborne listeriosis outbreaks for their susceptibility to lysozyme. After nongrowing cells of L. monocytogenes in the stationary growth phase were suspended in phosphate buffer containing 10 mg of lysozyme/L, 70-80% of the cells were lysed after 6 h, as determined by optical density measurement. In 1994, Johansen et al. [ 1871 tested the ability of egg white lysozyme to lyse five L. monocytogenes strains, which were suspended in phosphate buffer of pH 7. At 200 U/mL, lysozyme caused lysis of the five strains, and the rate of lysis was maximum at a lysozyme concentration of 1000 U/mL. Sensitivity of Listeria to lysozyme varied with the temperature at which the bacterium was grown before the treatment. Cells grown at 5°C were the most and those grown at 37°C the least sensitive, with cells grown at 25°C showing intermediate sensitivity. A similar effect of Listeria growth temperature on lysozyme sensitivity was noted by Smith et al. [355]; the authors found that L. rnonocytogenes grown at 37°C was 1.8-2.5 times more resistant to lysis by lysozyme than were cells grown at 5, 12, and 19°C. In contrast to the listericidal effect of lysozyme in nutrient-poor buffers, Listeria can grow in nutrient-rich media containing lysozyme. Growth inhibition detected in such media depends on the concentration of lysozyme, pH of the medium, incubation temperature, and the presence of various growth modifiers. According to Hughey and Johnson [179], four strains of Listeria grew in BHI broth containing 20-200 mg of lysozyme/L. Similarly, cells of L. monocytogenes in the logarithmic growth phase were not lysed after 12 h of exposure to 100 mg of lysozyme/L. In a more recent investigation [187], the presence of 10,000 U/mL lysozyme in TSB at 5°C extended the lag phase from 0 to 8 days at pH 7.0, and from 9 to 60-70 days at pH 5.5. A similar pattern was seen at 25°C with a lag phase at pH 5.5 of 4 h without lysozyme and 37 h with 50,000 U/mL lysozyme. The authors attributed the pH-dependent increase in bacteriostatic activity of lysozyme to the growth-retarding effect of low pH, which allowed cell lysis to proceed faster than

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cell multiplication. The lytic action of lysozyme was not affected as the pH was lowered from 7.0 to 5.5. Various chemical treatments also have been examined for their ability to potentiate lysis of growing cells of L. monocytogenes in the presence of lysozyme [ 1791. Addition of 0.85% lactic acid stopped growth of L. monocytogenes and led to gradual lysis of cells by lysozyme. EDTA was the most effective potentiator of cell lysis, whereas 0.05% potassium sorbate, 0.25% glycine, 5 mM sodium acetate, 0.95% ethanol, 0.01% sodium dodecyl sulfate, 5 mM thioglycolate, 5 mM dithiothreitol, and 10 mM ascorbic acid were relatively ineffective. The inhibitory action of EDTA and lysozyme toward Listeria also was tested using BHI agar. Although 100 mg of lysozyme/L or 1 mM EDTA failed to inhibit growth of two of four L. monocytogenes strains, the combination of EDTA and lysozyme led to substantial growth inhibition as compared with the additive-free control [ 1791. The antimicrobial activity of lysozyme against L. monocytogenes suspended in water was enhanced by prior treatment with lipase, an enzyme naturally existing in milk, or frozen storage for up to 6 weeks [ I 10,1111. Enhancement of lysozyme activity against L. monocytogenes suspended in buffer or broth by lipase also was observed by Liberti et al. [23 11. When compared with results using laboratory media, the antilisterial activity of lysozyme is variably reduced in foods. Hughey et al. [I801 found that lysozyme was more effective in controlling L. monocytogenes in vegetables than in meat products. A combination of lysozyme and EDTA inactivated L. monocytogenes (at 10" CFU/g) in fresh green beans, fresh corn, shredded cabbage, shredded lettuce, and carrots during storage at 5OC, whereas Listeria in control samples grew to 106-107CFU/g. In fresh pork sausage (bratwurst), lysozyme was only listeriostatic for 2-3 weeks and did not prevent growth during extended storage of the food. During ripening of Camembert cheese, lysozyme alone or in combination with EDTA decreased listeriae by about 1 log during the first 3-4 weeks, and then the Listeria count increased slowly and reached 106- 10' CFU/g after 55 days of ripening. Carminati and Carini [66] found that lysozyme at 25 and 1000 ppm only caused 22 and 57% reductions in count, respectively, for two of four L. monocytogenes strains inoculated into sterile skim milk. Addition of lysozyme to heat-treated skim milk that contained Listeria did not inhibit outgrowth of survivors. Kihm et al. [201] found that minerals in milk protected L. monocytogenes against lysozyme. Hen's egg white lysozyme (100 mg/L) had no antilisterial activity in whole milk. However, removal of minerals from milk by cation exchange slightly enhanced activity of lysozyme at 4°C. Prior heating (62.5"C for 15 s) of L. monocytogenes in a phosphate buffer sensitized the pathogen to lysozyme in MES (2-[N-morpholino] ethanesulfonic acid) buffer or demineralized milk. Furthermore, heating the pathogen at 55°C in the presence of lysozyme greatly increased inactivation of the pathogen in demineralized rather than undemineralized milk. Although lysozyme alone did not inhibit Listeria growth in milk [20 I ] , Payne et al. [295] found that lysozyme plus EDTA had an interactive antimicrobial effect against L. monocytogenes growing in ultrahigh-temperature (UHT) milk. Although lysozyme activity is affected by many food components, adding lysozyme to foods is potentially beneficial to control this pathogen. As discussed previously, lysozyme alone or in combination with EDTA inactivated Listeria in vegetables and retarded growth of the pathogen in fresh pork sausage and Camembert cheese [ 1SO]. Lysozyme (500 ppm), when added to acidified sterile skim milk (pH 5.3) that was stored at 4°C for 6 weeks (i.e., simulation of cheese making), enhanced inhibition of Listeria [66]. Egg white lysozyme contributed to the high antilisterial activity of some mayonnaise products

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[126]. Consistent with this finding, Wang and Shelef [383] found that raw egg albumin at levels >15% was bactericidal to L. monocytogenes in TSB at 35"C, with this activity being primarily attributed to lysozyme. Wang and Shelef [384] later found that L. monocytogenes growth in raw cod fish fillets could be retarded by lysozyme alone or in combination with EDTA. The fish fillets were dipped for 10 min at 20°C in solutions of lysozyme (3 mg/mL), EDTA (5-25 mM), or a combination of lysozyme (3 mg/mL) and EDTA (25 mM), inoculated with about 103 CFU/g L. monocytogenes, and then monitored for Listeria growth during storage at 20°C for 3 days or at 5°C for I7 days. The authors found that lysozyme plus EDTA had substantial antilisterial activity at both storage temperatures. Listeria populations in the control and in samples pretreated with 5-10 mM EDTA increased to about 108CFU/g after storage at 2OoC, whereas the pathogen only increased <2 logs CFU/g in samples pretreated with 15 and 25 mM EDTA. In lysozyme-treated samples stored at 2OoC, final Listeria populations were about 10-fold lower than in the control. Lysozyme and EDTA (25 mM) interacted synergistically and resulted in > 1-log decrease of listeriae in the first 18 h; the pathogen never grew to a level exceeding the initial population during the entire storage period. When control and treated samples were stored at 5°C for 17 days, Listeria populations increased 1 log in the control, remained almost unchanged in EDTA-treated samples, and decreased up to 1 log in samples treated with lysozyme or with the combination of lysozyme and EDTA.

Hydrogen Peroxide Although hydrogen peroxide is used as a preservative, particularly for raw milk in some parts of the world, use of this antimicrobial agent in the United States is very limited. The FDA permits adding up to 0.05% (w/w) hydrogen peroxide to raw milk intended to be made into certain kinds of cheese. It has also been approved by the FDA for sterilizing multilayer packaging materials used in aseptic processing systems. The antilisterial effect of hydrogen peroxide at levels permitted by the FDA was investigated in milk by Dominguez et al. [89]. These investigators found that L. monocytogenes was eliminated from autoclaved milk that had been inoculated to contain 9.5 X 107 L. monocytogenes CFU/mL, treated with 0.0495% hydrogen peroxide, and held for 24 h at 15°C. However, when raw milk was treated with 0.0495% hydrogen peroxide, inoculated to contain -2 X 105L. monocytogenes CFU/mL, and incubated at 4OC, numbers of listeriae increased slightly as compared with the natural microflora. In another experiment by the same researchers, samples of autoclaved milk containing 50.0495% hydrogen peroxide were inoculated to contain a mixed culture of L. monocytogenes, S. aureus, and Enterococcus faecalis (each organism at --I X 107CFU/mL) and incubated for 48 h at 4, 15, and 22°C. Although L. monocytogenes populations decreased approximately 10-, 16-, and 40-fold during the first 24 h of incubation at 4, 15, and 22OC, respectively, the organism grew during the second 24 h and reached populations 2 1 X 107CFU/mL. In a subsequent study, Kamau et al. [ 1951 observed that 0.6 mM (i.e., 0.002%) H202slightly inhibited growth of L. monocytogenes in bovine milk that was mildly heated (57"C, 20 min), cooled, and stored at 10°C compared with the control without H202.Thus hydrogen peroxide was relatively ineffective in decreasing numbers of listeriae in raw milk or milk containing equal numbers of S. aureus and E. faecalis. The impact of hydrogen peroxide on destruction of L. monocytogenes by heat was investigated by two research groups. Kamau et al. [ 1961 reported that the presence of 0.6

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mM H202in milk did not enhance inactivation of L. monocytogenes by heat. Lou and Yousef [238] found that adaptation of L. monocytogenes to H202increased the resistance of this pathogen to heat. The authors added 500 ppm hydrogen peroxide into a culture of L. monocytogenes in the exponential growth phase, incubated the culture for an additional 1-2 h at 35"'-,, and then determined heat resistance of treated Listeria cells in a H20,-free phosptidte buffer. Compared with the unadapted culture, H202adaptation increased DS(,oC-values by 2.9-fold. The same authors 12391 also reported an increase in resistance of L. monocytogenes to a lethal level (i.e., 0.1%, w/w) of H202after the bacterium was adapted to pH 4.5-5.0, 500 ppm H202,5% ethanol, 7% NaCI, or heat shocked at 45°C for 1 h.

Lactoperoxidase System The lactoperoxidase (LP) system, a naturally occurring antimicrobial system in milk, has been proposed as a means for extending the shelf life of raw milk when extended refrigerated storage is not possible, as in certain developing countries. Proper functioning of this system depends on adequate levels of lactoperoxidase, thiocyanate, and hydrogen peroxide. Lactoperoxidase in milk represents 1 % of whey proteins [3 151, which is an adequate amount for functioning of the lactoperoxidase system. Thiocyanate, however, is present in bovine milk at only 1-7 pprn [36,37] and H202needs to be added exogenously or generated by exogenous enzymes, such as glucose oxidase. In the LP system, lactoperoxidase catalyzes the oxidation of thiocyanate (SCN-) by hydrogen peroxide to hypothiocyanous acid (HOSCN) and hypothiocyanate (OSCN-); these endproducts are responsible for inactivating the microflora common to milk, including S. aureus, Salmonella typhimurium, psychrotrophic pseudomonads, and some lactic acid bacteria. Siragusa and Johnson [350] reported results of a study which examined inhibition of L. monocytogenes by the LP system. Their model LP system contained equimolar concentrations (0.3 mM) of potassium thiocyanate and hydrogen peroxide in TSB fortified with 0.5% yeast extract. After addition of 0.37 U lactoperoxidase/mL, flasks were inoculated with 1,. monocytogenes in the late logarithmic growth phase. L. monocytogenes had lag periods of 147.3-159.6, 46.6-55.5, 16.4-17.1, and 7.1 h in the presence of the LP system and 61.4-77.4,23.5-32.5,7.5-10.3, and 4.3-5.7 h in the control (with or without 0.3 mM H202)when the pathogen was incubated at 5, 10, 20, and 3OoC, respectively. Although the LP system appreciably extended the lag phase, maximum specific growth rates were not affected. When the LP system was tested in sterile reconstituted skim milk at 2OoC, the lag phase of L. monocytogenes was extended from 9 h (control) to 12-36 h. Maximum Listeria populations also were lower with the LP system than in controls. Thus, in this particular study, the LP system was bacteriostatic rather than bactericidal to L. monocytogenes, and it was more effective at low than at high incubation temperatures. In a subsequent study, Kamau et al. [ 1951 activated the lactoperoxidase system by adding 2.4 mM SCN- and 0.6 mM H202to preheated (57"C, 20 min) bovine milk, which contained adequate residual lactoperoxidase (9.2 mg/mL). Concentrations of SCN- and H202used in this study did not have measurable antimicrobial activity against L. monocytogenes. When the LP system was activated at 35"C, L. monocytogenes (initially 104CFU/ mL,) decreased slightly in the first 2 h and began to grow after 8 h. At 10°C, the LP system inhibited Listeria for 96 h before appreciable growth was observed. The times required to achieve half of the maximum growth were 16.9, 11.7, and 10.6 h at 35°C and 436, 170, arid 137 h at 10°C in milk (a) with activated LP systems, (b) with 0.6 mM H202,

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and (c) without additives, respectively. At 35OC, Listeria grew in all milk samples at a similar maximum specific growth rate (0.162-0.221 h-I), whereas at IOOC, the pathogen had a lower specific growth rate (0.0047 h-I) in the LP system-activated milk than in that of the control (0.0103-0.0123 h-I). Although Kamau et al. [195] and Siragusa and Johnson [350] reported that the LP system was mainly bacteriostatic toward L. monocytogenes in a laboratory medium and preheated milk, several other research groups noted appreciable bactericidal activity of the system. El-Shenawy et al. [I211 found that initial L. monocytogenes populations of 30-50 CFU/mL decreased to nondetectable levels following 2 h of exposure to the LP system at 35°C. Using selective and nonselective plating media, these researchers also demonstrated that the pathogen was not sublethally injured during exposure to the LP system. Denis and Ramet [86] reported that the LP system completely eliminated L. monocytogenes (initial populations 10'-10' CFU/mL) from TSB with 0.65% yeast extract following 5 1 , 2-6, and 4-10 days of incubation at 30, 15, and 4OC, respectively, depending on the initial inoculum. However, unlike the previously described model broth systems, these authors added glucose oxidase to their LP system. Since this enzyme oxidizes glucose to gluconic acid, the resulting lowering of pH likely increased the bactericidal effect of the LP system beyond what would have been observed in similar model systems having pH values near neutrality. Furthermore, L. monocytogenes is also inhibited and/or inactivated in TB and milk containing 20.75% gluconic acid during extended incubation at 13 and 35°C [ 1181. Hence, these findings likely reflect the combined effects of the LP system, pH, and gluconic acid rather than that of the LP system alone. Additional investigations dealing with antilisterial activity of the LP system in UHT milk rather than in culture media appeared in the scientific literature. Using two different UHT milk-based LP systems containing lactoperoxidase (30 mg/L), potassium thiocyanate (84 mg/L), glucose (10 g/L), and glucose oxidase (2 mg/L) both with and without urea peroxide (376 mg/L) as a hydrogen peroxide-generating mechanism, Earnshaw and Banks [ 1011 found that initial L. monocytogenes populations of 104CFU/mL decreased to 102CFU/mL in both LP systems during 6 days of incubation at 10°C. Denis and Ramet [86] also found that L. monocytogenes populations decreased in a similar UHT milkbased LP system containing lactoperoxidase, potassium thiocyanate, and glucose oxidase. However, unlike the previous study, their LP system completely eliminated the pathogen (initial populations of 101-104CFU/mL) from UHT milk following 6-21 and 7-30 days of incubation at 15 and 4OC, respectively, with estimated D-values of approximately 5 and 8 days at these same temperatures. Thus, as expected, the LP system was more detrimental to listeriae at higher rather than lower temperatures. In contrast, without the LP system, the pathogen attained populations of 1OS and 1O4 CFU/mL following 7 days of incubation at 15 and 4OC, respectively. Several groups investigated antilisterial activity of the LP system in raw milk containing naturally occurring levels of lactoperoxidase. El-Shenawy et al. [121] used an LP system in raw milk containing naturally occurring levels of lactoperoxidase along with 0.25 mM thiocyanate anion and 0.25 mM hydrogen peroxide, and they found that L. monocytogenes was often only slightly inhibited. In samples inoculated to contain 104 and 107L. monocytogenes CFU/mL, the pathogen attained maximum populations of 2 108 CFU/mL after overcoming an extended lag phase. However, this LP system was far more effective in raw milk inoculated to contain Listeria populations (i.e., 1O2 CFU/mL) similar to those that have been observed in cases of naturally occurring listerial mastitis. Under these conditions, the pathogen was completely inactivated after 2-4 and 12-24 h of incu-

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bation at 35 and 4°C. Thus, as was true for microbiological media and UHT milk, the LP system was again more effective in raw milk stored at higher rather than lower temperatures. In a subsequent study, Gaya et al. [ 1501 investigated antilisterial activity of the LP system activated by adding equal concentrations (0.25 mM) of sodium thiocyanate and H 2 0 2to raw bovine milk stored at 4 and 8°C. The authors reported D-values of 4.1- 1 1.2 days at 4°C and 4.4-9.7 days at 8°C for four L. monocytogenes strains added to the LP system-activated milk. Lactoperoxidase activity decreased during incubation, with the loss being more rapid at 8°C than at 4°C. In a more recent study, Zapico et al. [407] reported that the activated LP system in goat's milk remained bactericidal against three L. monocytogenes strains for 3-9 days at 4°C and 1-7 days at 8°C. Bacteriostatic activity against Listeria was observed at 20°C. The LP system can be used in conjunction with thermal processing to increase destruction of listeriae in raw milk. Kamau et al. [ 1961 reported that the LP system (0.24 mM SCN- and 0.6 mM H202)enhanced thermal inactivation of L. monocytogenes in preheated (57"C, 20 min) bovine milk containing 9.2 pg/mL of lactoperoxidase. Biphasic heat inactivation curves were observed when the LP system was activated, with most of the population being heat sensitive and inactivated rapidly during heating. The D-values (based on the heat-resistant fraction of the population if biphasic inactivation curves occurred) in milk (a) with the activated LP system, (b) with 0.6 mM H202,and (c) without any additives (control) were 10.7, 29.4, and 30.2 min at 52.2"C, 1.6, 11.1, and 8.2 min at 55.2"C, and 0.5,2.6, and 2.3 min at 57.8"C, respectively. When the LP system-activated milk was held at 35°C for different periods before heating, thermotolerance of L. monocytogenes decreased as the holding time increased, with the D 55.2"C being only 6.8 min after 16 h of holding time. In summary, L. monocytogenes is susceptible to the LP system, especially at low incubation temperatures. The LP system also can be used in combination with other treatments, such as heat to increase inactivation of listeriae. This system will likely prove to be useful for decreasing numbers of naturally occurring listeriae in raw milk before milk processing facilities receive the product.

Lactoferr in The presence of iron in culture media stimulates growth of some microorganisms. Lactoferrin, a glycoprotein found in mammalian milk, exerts its antimicrobial activity through binding of iron. Thus the antimicrobial activity of lactoferrin is affected by its degree of iron saturation and iron availability in the medium. The degree of saturation of lactoferrin with iron can be reduced by dialysis, and the resulting product is known as apo-lactoferrin. Both lactoferrin and apo-lactoferrin exhibit antilisterial activity. Recently, lactoferrin was found to inhibit invasion of L. monocytogenes into cultured intestinal cells [ 191. Payne et al. [296] studied the effect of bovine lactoferrin and apo-lactoferrin, with 52% and 18% iron saturation, respectively, on growth of L. inonocytogenes in UHT milk with 2% fat. After 18 h of incubation at 35"C, two strains of L. monocytogenes grew in the presence or absence of lactoferrin (46 mg/mL), but the count of Listeria was 1.6- 1.8 logs lower in treated milk than in the control. Compared with lactoferrin, apo-lactoferrin had greater antilisterial activity. When added to milk incubated at 35"C, apo-lactoferrin was strongly listeriostatic at 15 mg/mL and listericidal at 30 mg/mL. Addition of 0.125 M ferric ammonium citrate eliminated the inhibitory effect of 30 mg/mL apo-lactoferrin against Listeriu.

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These researchers [295] subsequently investigated the antimicrobial activity of apolactoferrin, EDTA, lysozyme, or their combinations in UHT milk. When applied separately or in combinations, these substances did not inhibit P. Jluorescens and S. typhimurium. However, inhibition of L. monocytogenes and E. coli 0 157 :H7 was observed using these compounds, with a combination of 15 mg/mL apo-Iactoferrin and 150 mg/mL lysozyme retarding growth of L. monocytogenes. Lactoferricin, a small antimicrobial peptide (25 amino acid residues) resulting from hydrolysis of bovine lactoferrin by gastric pepsin, has strong antilisterial activity in culture media [381]. The MICs of lactoferricin for four L. monocytogenes strains ( 106CFU/mL) at 37°C were 0.3-0.6 pg/mL in 1% peptone and 1-3 pg/mL in Peptone-Yeast ExtractGlucose (PYG) broth. The presence of up to 10 mg/mL of various sugars or starch did not affect the antilisterial activity of lactoferricin. Addition of gelatin or bovine serum at 10 mg/mL slightly increased the MICs of lactoferricin. However, up to 100 mM NaC1, KC1, or NH4C1 and up to 5 mM of Mg,C12 or CaC1, increased the MICs to 6-9 pg/mL for one of the most resistant Listeria strains. Lactoferricin maintained its antilisterial activity over a pH range of 5.5-7.5. This peptide was bactericidal to L. monocytogenes growing in PYG broth at 37°C; treatment of 104-106 CFU/mL of L. monocytogenes with 31 pg lactoferricin/mL for 60 min reduced the viable population to below a detectable number (i.e., <100 CFU/mL) for three strains and to 300 CFU/mL for the fourth strain. In a subsequent study [30], in addition to L. monocytogenes, lactoferricin inhibited a wide range of pathogenic and spoilage bacteria: E. coli, Salmonella enteritidis, Yersinia enterocolitica, Campylobacter jejuni, S. aureus, Corynebacterium diphtheriae, Clostridium perfringens, Klebsiella pneumoniae, Proteus vulgaris, Streptococcus mutans, and Pseudomonas aeruginosa. Levels of 0.3- 120 pg/mL of lactoferricin were required to inhibit these organisms. As with L. monocytogenes, this peptide inhibited E. coli 0 1 11 over a pH range of 5.5-7.5, with the greatest activity occurring at slightly alkaline pH values.

BIOPRESERVATION The terms biological preservation, biopreservation, and biocontrol all refer to the use of microorganisms or their metabolic products to inhibit or inactivate undesired microorganisms in foods. Biopreservation, as a means of naturally controlling pathogens and spoilage microorganisms, especially in minimally processed foods, has been extensively studied and excellent reviews are available [2,162,174,256,272,31 1,3361. Lactic acid bacteria (LAB) and their metabolic products are commonly used in biopreservation, since these bacteria are used in many traditional foods and are GRAS. Biopreservation by LAB occurs because these bacteria compete with other microorganisms for nutrients and/or because they produce antimicrobial compounds, such as weak acids, hydrogen peroxide, diacetyl, and bacteriocins. The discussion in this section will focus on biopreservation with bacteriocins or bacteriocin-producing LAB which target L. monocytogenes in food. Bacteriocins are antimicrobial substances that have a peptide or protein component essential for their activity. Although most bacteriocins have a narrow spectrum of inhibition and only inhibit closely related species, some bacteriocins, such as nisin and pediocin, have a relatively broad spectrum and can inhibit some less closely related organisms. A large number of LAB bacteriocins are active against L. monocytogenes. Although their modes of action vary, bacteriocins usually destabilize the cytoplasmic membrane of sensitive cells, increase membrane permeability, and dissipate the proton motive force by form-

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ing water-filled transmembrane pores or channels [ 1851. Bacteriocins produced by LAB are grouped into four distinct classes [206]. Bacteriocins of class I are lantibiotics, lanthionine-containing peptides, such as nisin. Class I1 includes small (<10 kD), non-lanthionine-containing, relatively heat-stable bacteriocins, such as pediocin PA- 1, P02, or AcH. Class 111bacteriocins form large (>30 KD) heat-labile molecules. Class IV includes bacteriocins with nonpeptide moieties. Nisin-producing strains of Lactococcus lactis subsp. lactis have been used legally in the United States and elsewhere to manufacture certain cheeses and other dairy products that require a mesophilic fermentation. Although bacteriocin-producing LAB may be added as a starter culture or fermented ingredient to various foods, legal issues arise when foods are supplemented with purified bacteriocins, as discussed by Fields [ 1421 and Post [306]. Approval from US regulatory agencies, mainly the FDA and the USDA, is required for the application of purified bacteriocins [ 1421. According to the US Code of Federal Regulations [ 1381, 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 [ 142,2721. Of purified bacteriocins, only nisin has been approved by the FDA for use in pasteurized cheese spreads [ 1 1,1371. Approvals for use of nisin in other food products were subsequently granted by the FDA. Drawbacks of biopreservation are limiting large-scale application of this technology in the food industry. When bacteriocins are added to foods, they usually show only a modest antimicrobial effect, with the targeted organism often becoming bacteriocin-resistant by one or more mechanisms [237,262,263,272]. Additionally, common LAB bacteriocins are not active against gram-negative bacteria. Destabilization of the outer membrane of gram-negative bacteria by other factors or treatments is required for LAB bacteriocins to be active against these bacteria [367]. Therefore, a bacteriocin is normally used in food processing as a hurdle in combination with other treatments. Several research groups reported data indicating the value of bacteriocins in combined treatments. In these studies, bacteriocins were tested in combination with sublethal heat, acid, and freezing-thawing [ 193,249,2721, lysozyme and chelating agents [367], other bacteriocins [ 1611, high hydrostatic pressure [ 165,1941, and pulse electric field [ 1941. Biocontrol of L. monocytogenes in food can be achieved by (a) adding bacteriocinproducing microorganisms to foods, (b) fermenting foods with bacteriocin-producing LAB, (c) adding bacteriocin-containing fermentates, (d) adding bacteriocin crude extracts or purified bacteriocins, or (e) incorporating bacteriocin-containing food ingredients [272]. It should be cautioned, however, that in the United States, application of purified bacteriocins to food is subject to the legal restrictions discussed earlier. Most studies on biopreservation have dealt with nisin, pediocin, and the LAB producing these bacteriocins. Therefore, the following discussion will deal mainly with these two bacteriocins.

Nisin Nisin is a bacteriocin produced by certain strains of L. lactis subsp. lactis and has proven to be extremely useful in preventing outgrowth of Clostridium spp., including Clostridium botulinum, in fermented dairy and meat products. In 1980, Mohamed et al. [264] reported results from a series of experiments that were designed to determine the effectiveness of nisin against Listeria. When NB at pH 7.4 contained 4-16 International Units (IU) of nisin per milliliter, populations of L. monocytogenes decreased >5 logs during 28 h at 37°C. After this initial decrease, Listeria grew rapidly and attained final populations of

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108CFU/mL. Decreasing the pH of the medium from 7.4 to 5.5 led to a 16-fold decrease in the level of nisin required to inhibit the bacterium. Strains of L. monocytogenes may vary in resistance to nisin. Using Trypticase Soy Agar, Benkerroum and Sandine [311 found that six L. monocytogenes strains were variably resistant to nisin with MICs ranging from 1.4 X 102to 1.18 X 105IU/mL. Several additional studies also have demonstrated various degrees of nisin resistance for L. monocytogenes. Although Tatini [373] found that 512-1024 ppm nisin was required to inhibit growth of 12 L. rnonocytogenes strains in laboratory media, S. typhimuriurn and E. coli remained viable in the presence of up to 10,000ppm nisin. Although these findings suggest that L. monocytogenes may be less resistant to nisin than some other potentially hazardous microorganisms, one must keep in mind that some unusually resistant strains of L. monocytogenes do exist [ 163,262,263,2371.In 1989, Harris et al. [ 1631 examined sensitivity and resistance of L. monocytogenes to nisin. According to these authors, populations of listeriae decreased 6-7 logs when nisin levels in BHI agar were increased from 0 to 10 pg/ mL. However, a relatively stable population of nisin-resistant mutants (- 100- 1000 CFU/ mL) developed on agar plates containing 1-50 pg nisin/mL with nisin-resistant mutants occurring at a frequency of 10-6-10-x in media containing 50 pg nisin/mL. Although all nisin-resistant mutants selected from agar plates were more resistant than their parent strains, further testing revealed that nisin resistance was related to ability of nisin-resistant strains to bind nisin rather than to specific genes coding for nisin resistance in plasmid DNA. Similar nisin-resistant mutants also were obtained by Ming and Daeschel [262, 2631. Besides nisin resistance observed in spontaneous mutants, Lou [237] found that acid adaptation or starvation increased resistance of L. monocytogenes to nisin and pediocin. As indicated by the earlier findings of Mohamed et al. [264], antilisterial activity of nisin is strongly influenced by various environmental factors, including pH. Benkerroum and Sandine [3I] determined the sensitivity of one L. monocytogenes strain to nisin in Tryptose Soy Broth adjusted to pH values of 3.5-7.0. Populations increased -1 log in broth cultures at pH 7.0 and 6.48 during the first 12 h of incubation, but no increase in count was observed in similar samples adjusted to pH 5 5.94. Enhanced activity of nisin against Listeria at lower pH values also has been observed by Harris et al. [ 1631. Furthermore, data from Tatini [3731 indicate that average minimum nisin concentrations of 5 12, 1365,2560, and 2496 ppm were required to inhibit growth of several L. monocytogenes strains on Trypticase Soy-Yeast Extract Agar adjusted to pH values of 5.0, 5.5, 6.0 and 6.5, respectively. Thus increased susceptibility of L. monocytogenes to nisin at pH values <6 appears to be fairly well established. The antilisterial action of nisin is further complicated by incubation temperature and the presence of sodium chloride. According to Tatini [373], minimum concentrations of nisin necessary to inhibit growth of L. monocytogenes were typically two to four times greater at 35 than at 4°C. In addition, when L. monocytogenes was incubated at 4°C in broth containing 1400 pprn nisin, lag periods for the various strains tested increased from 16 to 69 days as the nisin concentration increased from 0 to 400 ppm. In 1989, Harris et al. [ 1631 also reported that addition of 2% sodium chloride enhanced the listericidal activity of nisin in laboratory media, particularly at levels of <10 pg/mL. Although many European countries have allowed direct addition of nisin to food for some time, this practice was not permitted in the United States until 1989, when FDA officials amended the food standard for pasteurized process cheese to allow addition of not more than 250 pprn nisin to the finished product [ 1 1,16,17]. However, since allowable

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levels of nisin may not completely inhibit L. monocytogenes in pasteurized process cheese spreads that have been subjected to postpasteurization contamination, addition of nisin to such products should not preclude use of proper sanitary practices. Nisin-producing starter cultures provide some protection against L. monocytogenes during cheese manufacture. Mainsnier-Patin et al. [248] inoculated L. monocytogenes into milk which was used to make Camembert cheese. Nisin-producing or nonproducing starter cultures were used in making the cheese. Counts of L. monocytogenes in the final product were 2.4 log lower in cheese made with the nisin-producer than that made with the nonproducing strain. In another study, Zottola et al. [409] made Cheddar cheese with a nisinproducing L. lactis starter and then prepared pasteurized processed cheese using this Cheddar cheese as an ingredient. Over 56 days of storage, populations of L. rnonocytogenes decreased more rapidly in processed cheese made with rather than without the nisin-producing culture. The psychrotrophic and facultative nature of L. monocytogenes makes this pathogen a potential safety hazard for many minimally processed and vacuum/modified atmosphere-packaged foods which require refrigeration. Incorporating bacteriocin or bacteriocin-producing LAB into these products could be an effective way of minimizing L. monocytogrnes growth and survival. However, sensory changes caused by addition of biopreservatives must also be considered to ensure consumer acceptance [256]. Adding 10,000IU/mL nisin to cooked pork tenderloin that was packaged in air and refrigerated prevented growth of inoculated L. monocytogenes but not Pseudomonas fragi. However, use of nisin (1000 and 10,000IU/mL) in combination with modified atmosphere packaging ( 100% CO2 or 80% CO2 + 20% air) inhibited growth of both bacteria, and this inhibition was more pronounced at refrigeration than at room temperature [ 1291. Lactic acid bacteria, such as Leuconostoc spp., often spoil minimally heat-processed vacuumpackaged meat products. Some of the spoilage LAB are sensitive to nisin and nisin-producing Lactococcus spp. Thus Yang and Ray [399] suggested that biocontrol of these spoilage LAB with nisin or nisin-producing strains could be an effective solution.

Pediocin Pediocin, a wide-spectrum bacteriocin produced by certain strains of Pedicoccus acidilactici, is a potent inhibitor of L. monocytogenes. Several research groups have reported that P. acidilactici strains H, PAC1.O, and PO2 produced pediocin AcH, PA-1, and P02, respectively. Luchansky et al. [24 I ] later reported that the restriction enzyme fragments generated from plasmids encoding for these three pediocins were identical, with the three producer strains also yielding identical genomic DNA fingerprints. These findings, in combination with the DNA sequences for pediocin AcH and PA-1 (250a, 270a) indicate that the three bacteriocins are similar in structure. Control of L. monocytogenes by pediocin in several foods was explored. In a series of studies on meat and meat products, Luchansky and his coworkers investigated the antilisterial effect of pediocin AcH in wiener exudate, wiener packages, and turkey summer sausage [8524 1,4061. In refrigerated exudate from beef wieners, pediocin AcH decreased L. nzonocytogenes numbers by 0.74 log within 2 h. When the exudate was inoculated with L,. nzonocytogenes and a pediocin-producing strain of P. acidilactici and kept at 25OC, the count of L. monocytogenes increased initially and then markedly decreased during extended incubation compared to the control. Pediocin activity in wiener exudate was detected during the late stages of P. acidilactici growth [406]. Degnan et al. [85]

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later investigated survival of L. monocytogenes in vacuum-packaged beef wieners which contained pediocin- or non-pediocin-producing P. acidilactici. Pediocin was produced during early to late logarithmic phase. When packages were temperature abused at 25°C for 8 days, listeriae populations increased by 3.2 log CFU/g in the absence of any Pediococcus strain, remained unchanged in the presence of the non-pediocin-producing strain, and decreased by 2.7 logs in the presence of the pediocin producer. Results from several other research groups are consistent with these findings. Berry et al. [32] found that the bacteriocin-producing strain P. acidilactici JD 1-23 provided more protection against L. monocytogenes during storage of vacuum-packaged frankfurters at 4°C compared with a plasmid-cured derivative of JDl-23. When either of these strains was used at a level of 107CFU/g, growth of Listeria was inhibited for up to 60 days. However, some antilisterial activity also was observed at low levels ( 103-104CFU/g) of the pediococcus strains. When pediocin was tested against L. monocytogenes attached to fresh beef muscle [241], pretreatment of the muscle with pediocin slightly decreased L. monocytogenes attachment [ 1131. According to Goff et al. [157], pediocin that was bound to heat-killed producer cells remained strongly active in irradiated raw chicken breast meat which was stored at 5°C. Bacteriocin activity is usually adversely affected by certain food components. Degnan et al. [83] improved pediocin activity in food slurries by using pediocin encapsulated in phosphatidylcholine-based liposomes or by combining pediocin with 0.1% Tween 80. Control of L. monocytogenes by pediocin-producing starter cultures was observed during the manufacture of dry or semidry sausage. Berry et al. [34] noted -2-log reduction in numbers of L. monocytogenes using a bacteriocin-producing strain of Pediococcus and < 1-log reduction with a nonbacteriocinogenic starter during fermentation of semidry sausage. Foegeding et al. [ 1441 compared the antilisterial activity of two starters, a pediocin-producer, P. acidilactici PAC 1.O, and its isogenic pediocin-negative derivative, during dry sausage production. The investigators observed that pediocin produced in situ was partially responsible for listeriae inactivation during the sausage fermentation and drying. Antilisterial activity from a pediocin-producing strain also was demonstrated during manufacture of turkey summer sausage [241]. During the 12-h fermentation, the presence of pediocin and non-pediocin-producing strains decreased L. monocytogenes populations by 3.4 and 0.9 log CFU/g, respectively. Pediocin (-5000 AU/g) was recovered from sausage even after 60 days of refrigerated storage. Besides use of bacteriocins and their producing bacteria, LAB fermentates can also be used to improve control of L. monocytogenes in food [84,338]. Several criteria for selection of suitable biocontrol microorganisms for use in meat or meat products were proposed by McMullen and Stiles [256]. Biopreservation microorganisms should be psychrotrophic, produce bacteriocins early in the growth cycle, and exhibit little negative effect on product quality. Bacteriocins produced by these bacteria should be active (bactericidal) and stable in the food environment. The authors concluded that nisin-producing lactococci are poor biocontrol organisms, since they do not grow well at chill temperatures or in meat products. Pediocin-producing pediococci are also poor meat biopreservatives, because antilisterial activity occurs only at abuse temperatures [85,241,406]. McMullen and Stiles [256] recognized the merit of using alternative bacteriocin producers in meat. Carnobacterium piscicola LV 17 and Leuconostoc gelidum UAL 187 grow well, produce broad-spectrum bacteriocins at refrigeration temperatures, and proved to be good biocontrol organisms in meats; their application in meats was reviewed by these authors. Potential benefits of pediocin to the dairy industry were explored by several research

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groups. Pucci et al. [308] inoculated commercial samples of cottage cheese (pH 5.1), cheese sauce (pH 6.0), and half-and-half (pH 6.6) to contain 102-104L. monocytogenes CFU/g and then added a crude extract of pediocin PA-1 to these products. According to these authors, viable numbers of L. monocytogenes decreased rapidly in all foods during the first day of refrigerated storage. Although the pathogen attained populations of 103- 105 CFU/mL or CFU/g in cheese sauce and half-and-half following 7- 14 days of refrigerated storage, these levels were still approximately 2-5 logs lower than those observed in corresponding samples prepared without PA-1 powder. Motlagh et al. [270] studied the effectiveness of pediocin AcH (up to 1350 AU/mL), produced by P. acidilactici H, in controlling L. monocytogenes in reconstituted dry milk, ice cream, and cottage cheese at 4 and 10°C. After 1 h of storage at 4"C, this treatment decreased numbers of L. monocytogenes by 1 log. When milk was inoculated to contain 102or 104L. monocytogenes CFU/ mL and incubated at 4°C for 28 days and at 10°C for 12 days, 1350 AU/mL pediocin reduced Listeria populations about 2- to 4-logs during the first day of storage but did not inhibit growth of Listeria survivors. Liao et al. 12301 prepared a pediocin P02-containing powder through fermentation of whey permeate with P. acidilactici P02. When the powder was added to Listeria-contaminated whole milk, the antilisterial activity of this preparation was clearly demonstrated. Addition of pediocin 5, produced by P. acidilactici UL5, to 1% fat milk reduced the viable L. monocytogenes by -3 logs after one day of storage at 4°C [176]. In addition to meat and dairy products, other foods may benefit from biopreservation by pediocin and pediocin-producing strains. Choi and Beuchat [69J added a crude bacteriocin extract from P. acidilactici M to kimchi during fermentation. This treatment immediately reduced numbers of L. monocytogenes in the inoculated product and inhibited growth by the organism during 16 days of fermentation. Adding pediocin PA- 1, curvaticin FS47, or lacticin FS56 to liquid whole egg dramatically reduced the heating time required to inactivate L. monocytogenes in this product [272].

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Other Bacteriocins In addition to nisin and pediocin, several other bacteriocins also are effective against L. monocytogenes. Lactobacillus bavaricus MN, a meat isolate, inhibited growth of L. monocytogenes in a model beef gravy at 10°C, with this inhibition being attributed to a bacteriocin [395]. In a subsequent study, Winkowski et al. [396] found that L. bavaricus MN, when coinoculated with L. monocytogenes into three model beef systems, beef cubes and beef cubes with gravy andlor 0.5% glucose, significantly inhibited Listeria growth at 4 and 10°C The inhibition was greater at 4°C than at 1O"C, and increased with addition of glucose to gravy or with use of a higher inoculum of this Lactobacillus strain. Other bacteriocin-producing strains of L. bavaricus were reported [222,223]. Some of the many other bacteria that produce antilisterial bacteriocins are Lactobacillus salivarius M7 [46], Lactobacillus curvatus FS47 and LTH 1174 [148,374,379], L. sake [3 191, L. sake Lb674 and LTH673 [ 172,3741, Lactobacillus plantarum MCS [65], Leuconostoc carnosum LA54 [ 1981, Leuconostoc mesenteroides [771, Propionibacterium thoenii P 127 [ 2421, Carnobacterium piscicola [55,56,252], and Enterococcus spp. [2 1,1971.

MODIFIED ATMOSPHERE Buchanan and Klawitter [54] investigated aerobic versus anaerobic incubation in relation to growth and survival of Listeria in TPB at pH 4.5. Under aerobic conditions, L. monocy-

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togenes Scott A was undetectable after -50 h at 37"C, survived without change in numbers at 10 and 5"C, and grew to 107and 10' CFU/mL at 28 and 19°C in 100 and -500 h, respectively. When the experiments were repeated under anaerobic conditions, a similar trend in Listeria growth and survival as related to incubation temperature was observed. However, anaerobic incubation was more conducive to Listeria growth or survival than aerobic incubation. Anaerobic incubation at 19°C decreased the length of the lag phase from 80.6 (aerobic) to 27.3 h and the generation time from 19.1 (aerobic) to 6.8 h. Although anaerobic incubation at 37°C initially decreased the count by -2 logs, the population gradually increased to a level close to that of the initial inoculum. The authors suggested that anaerobiosis improved recovery of acid-injured cells at 37°C and led to subsequent long-term survival of repaired cells. Using similar experimental conditions, George and Lund [ 1521 examined the effect of anaerobiosis on growth of two L. monocytogenes strains when incubated at 20°C in TPB and Tryptone Soya Broth which was supplemented with yeast extract (3 g/L) and glucose (10 g/L) (TSYGB) and adjusted to pH 4.5 with HCI. Contrary to findings of Buchanan and Klawitter [54], the authors noted that the anaerobic condition (generated through flushing with nitrogen), when compared with aerobic incubation, inhibited growth in both media. When the investigators changed incubation conditions for TPB from aerobic to anaerobic, the generation time of Listeria increased from 4.22-4.98 to 6.12-6.7 1 h, increased the lag-phase duration from 45.8-47.5 to 73.8-80.1 h, and decreased the maximal population from 9.55-10.09 to 8.74-8.86 logs. Although these results are conflicting, it is generally believed that Listeria grows well under both aerobic and anaerobic conditions [54,58]. The capacity for anaerobic growth at refrigeration temperatures makes L. monocytogenes a potential threat to the safety of foods packaged under vacuum or modified atmosphere. Sous vide-processed foods also fall into this category. Such foods are vacuum packaged, then cooked, chilled, and finally stored refrigerated. Two gases, N2 and CO2, are commonly used in modified atmosphere packaging. For packaging of fresh meats, vegetables, and fruits, limited levels of O2 or air may be incorporated to maintain food quality. Aerobic microflora are greatly inhibited by modified atmosphere packaging; however, some psychrotrophic or anaerobic pathogens, such as L. monocytogenes, A. hydrophila, Y. enterocolitica, and Clostridium botulinum are potentially capable of growing under these conditions. L. monocytogenes can grow in food which has been packaged under vacuum or N2 gas. However, incorporation of CO2 improves the antilisterial activity of the packaging atmosphere [23,130,140,178,2151. Growth of L. innocua was not seen in cottage cheese packaged under 100% CO2during 28 days of storage at 5°C; however, the organism grew after 7 days in containers packaged under air and 100% N2 [140]. Fang and Lin [130] reported that growth of L. monocytogenes was inhibited in raw pork tenderloin packaged under 100% CO2 when stored for 10 days at 20°C or 20 days at 4°C. Avery et al. [23] also found that when L. monocytogenes was inoculated into packaged fresh beef striploin steaks (pH 5.3-5.5) counts of the pathogen decreased slightly under saturated CO2 atmosphere during storage at 5 to 10°C but increased by 3 logs in vacuum-packaged steaks [23]. Kraemer and Baumgart [215] investigated growth of L. monocytogenes at 4, 7, or 10°C in sliced frankfurter-type sausage that was packaged under 0, 20, 30, 50, or 80% CO2,with the remainder being under N2packaging Growth of L. monocytogenes decreased as levels of CO2 increased, and complete inhibition occurred under 80% CO2. Packaging under 50% CO2 resulted in only partial inhibition. Concurrent with this work, Farber and Daley [132] observed that L. monocytogenes was inhibited by 270% CO2 in modified

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atmosphere--packaged turkey roll slices stored at 4 and 8°C; however, the pathogen grew in packages containing 30 and 50% CO2. Because of the threat from some foodborne pathogens, especially C. botulinum, modified atmosphere-packaged foods rely heavily on refrigerated storage to prevent outgrowth of C. botulinum and toxin production. However, refrigeration alone can not guarantee food safety [317]. Growth of L. monocytogenes, A. hydrophila, and Y. enterocolitica was observed in vacuum-packaged sliced roast beef stored at - 1.5"C [ 1781. Adding additional microbiological hurdles should increase the safety of modified atmosphere-packaged foods. Wederquist et al. [390] found that inhibition of L. rnonocytogenes increased when 0.5% oodium acetate, 2% sodium lactate, or 0.26% potassium sorbate were added to vacuum-packaged bologna stored at 4°C. Safety of raw pork tenderloin packaged under a modified atmosphere was improved by incorporation of nisin [ 129,1301. Furthermore, Degnan et al. [ 851 observed that inoculation of pediocin-producing P. acidilactici into vacuum-packaged beef wieners decreased numbers of the coinoculated L. monocytogenes during storage at abusive temperatures.

NONTHERMAL PROCESSING TECHNOLOGIES Processing of food by nonthermal means is not new. For centuries, relatively shelf-stable foods were produced by adding salt and curing agents. Food fermentation, which has been known and practiced for a long time, also may be considered a nonthermal process to extend the shelf life of perishable foods like milk, meat, and fruit juice. Increased consumer demand for foods with a fresh-like taste and texture have led to development of several novel-technologies. Some newly developed physical treatments which inactivate microorganisms without significantly increasing the temperature of the food can yield products with fresh-like qualities. Such nonthermal physical treatments with potential applications in nonthermal processing of food include irradiation, high hydrostatic pressure, high-intensity pulsed light, high-intensity pulsed electric fields, and oscillating magnetic fields. These modern nonthermal technologies are gradually gaining acceptance from regulatory agencies and consumers, with irradiation now approved in the United States for use on selected foods such as strawberries. The ability of these nonthermal processes to control L. monocytogenes will be discussed in this section. Although these technologies are perceived as nonthermal, some heat may be generated during their application. However, provisions are always made to dissipate this heat, and thus control of pathogens is accomplished mainly by nonthermal means.

Irradiation The entire electromagnetic spectrum consists of at least six distinct forms of radiation that differ in wavelength, frequency, and penetrating power, of these forms, microwaves and ultraviolet and gamma radiation are of primary interest to food manufacturers. In food processing, microwave radiation is mainly used for its heating properties; thus discussion of effects of this form of radiant energy on L. monocytogenes are not covered in this section. Ultraviolet (UV) radiation, which is nonionizing, ranges in wavelength from 136 to 4000 and has some application in food processing. The poor penetrating power of UV radiation restricts its use to a few specialized beverage applications, eradication of

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airborne contaminants and treatment of food contact and ?on-food contact surfaces. Gamma radiation, which has a shorter wavelength (0.1- 1.4 A) than UV, is better suited for external and internal decontamination of foods. Information concerning the ability of gamma and ultraviolet radiation to inactivate L. monocytogenes in laboratory media will be reviewed now. Findings from similar food-related studies are discussed elsewhere in this book.

Gamma Irradiation Literature on sensitivity of L. monocytogenes to gamma irradiation is less controversial than that addressing thermal inactivation. Results from gamma irradiation studies conducted in the United States [72,123,181] and Hungary [371] are strikingly similar, with reported D-values ranging from 0.28 to 0.6 1 kGy for 12 different L. monocytogenes strains. In addition, exposure to a gamma radiation dose of 1.7-4.0 kGy was generally sufficient to reduce numbers of L. monocytogenes, L. ivanovii, and L. seeligeri by six to seven orders of magnitude. Overall, these findings suggest that Listeria spp. are likely to be at least equally, if not slightly more, resistant to gamma radiation in culture media than are other commonly encountered non-spore-forming foodborne pathogens such as S. typhimurium (D-value = 0.28 kGy) [377], S. aureus (D-value = 0.24 kGy) [377], and Y. enterocolitica (D-value = 0.1 1 kGy) [ 1241. Although differences between L. monocytogenes strains likely account for most of the observed variation in D-values, radiation sensitivity of L. monocytogenes is also affected by age of the culture, irradiation menstruum, and the type of medium used to enumerate the pathogen after irradiation. According to Huhtanen et al. [ 1811, 1.5- and 2.5-h-old cultures of L. monocytogenes were somewhat more resistant to gamma radiation than those incubated 5 and 18 h before exposure. Furthermore, surviving cells previously exposed to high radiation doses were no more resistant than the parent culture. Consequently, observed differences between sensitivity of young and old cultures probably resulted from innate differences between strains rather than from development of radiation-resistant mutants. These authors also reported that 12-h-old centrifuged cultures of L. monocytogenes were most resistant to 1.0 kGy gamma radiation when resuspended in fresh culture media or the original culture supernatant liquid followed in order by phosphate buffer and distilled water. Inability of distilled water effectively to scavenge cell-damaging free radicals produced during irradiation is likely responsible for decreased resistance of the pathogen in water than in culture media that contain high concentrations of free radical-quenching organic compounds. It is not surprising that L. monocytogenes is more resistant to gamma radiation when present in foods than in culture media. Two independent investigations [ 123,2921 have shown that D-values for radiation resistance are markedly affected by the type of plating media used to enumerate the pathogen after irradiation. In both studies, a significantly higher ( P < .OS) D-value resulted from increased recovery of the pathogen with nonselective or semiselective rather than highly selective plating media. These findings indicate that substantial numbers of listeriae were sublethally injured during exposure to gamma irradiation. Since repair and subsequent growth of injured cells is frequently inhibited by some of the selective agents used in highly selective media, D-values for organisms exposed to irradiation or any other potentially injurious treatment always should be determined using a plating medium with low selectivity. Andrews et al. [9] found that sensitivity of L. monocytogenes in TSB to gamma radiation was affected by broth temperature (-80, 4, or 20°C) during treatment and the initial count (103,106,and 109CFU/mL). Under this wide range of conditions, the organ-

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ism exhibited D-values of 0.4 1-0.62 kGy. L. monocytogenes was significantly more resistant to irradiation at room temperature (20°C) than at refrigeration (4°C) or freezing (-80°C) temperatures. D-values obtained with an initial count of 109CFU/mL were significantly lower than those with 106CFU/mL. Andrews and Grodner [8] later reported that gamma radiation was more effective in inactivating L. monocytogenes when split into two equal doses than when the same dose was applied as a single treatment. At 2OoC, the split dose of gamma radiation with 1-2 h between treatment decreased D-values from 0.50-0.58 kGy (single irradiation) to 0.41-0.42 kGy. However, a similar trend was not observed at refrigeration (4°C) or subfreezing (- 80°C) temperatures.

Ultraviolet Radiation In 1971, Collins [73] determined the susceptibility of L. monocytogenes to UV radiation emitted from a 14-W cold cathode mercury vapor lamp. Tryptone Soy Agar plates containing 109 L. monocytogenes were exposed to a radiation output of 40 W/cm2 at 40 cm from the source for 30, 60, 90, and 120 s and then incubated for 3 days at 37°C. Populations of L. monocytogenes decreased 10-fold during the first 60 s of irradiation (Dvalue of 60 s) after which the rate of inactivation increased sharply with a D-value of 15 s. L. monocytogenes was much more resistant to radiation than E. coli or Serratia marcescens, which are commonly used to test the effectiveness of UV lamps. Yousef and Marth [402] also reported that L. monocytogenes was inactivated by exposing the bacterium to UV energy. Following 4 min of exposure to short-wave (254 nm) ultraviolet energy (100 pW/cm2), numbers of L. monocytogenes (strain Scott A) decreased approximately 7 logs on Tryptose Agar plates that were previously spread with a 24- or 48-h-old culture of the test organism. In contrast, L. monocytogenes numbers remained constant after 10 min of exposure to long-wave (364 nm) UV energy. Increasing the intensity of short-wave UV radiation to 550 pW/cm2 nearly doubled the rate at which L. monocytogenes was inactivated. These investigators also found that dry rather than moist Listeria cells were more resistant to radiation. Exposing a dried film of L. monocytogenes cells in a Petri plate to short-wave UV energy (100 pW/cm2) decreased the population by 2 rather than 7 logs for moist cells on Tryptose Agar. Fortunately, when present in food processing environments, numbers of listeriae appear to be relatively low. Hence, results from the aforementioned study suggest that UV energy may be of some practical importance in reducing airborne contaminants, including listeriae, in food production and storage areas.

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High-Intensity Pulsed Light Pulsed light, which has wavelengths ranging from -200 nm (UV) to 1 mm (near-infrared) with peak emissions at 400-500 nm, inactivates microorganisms with flashes of intense sunlight-like radiation. Currently employed pulsed light has intensities about 20,000 times that of sunlight. Literature currently available on the effect of pulsed light on L. monocytogenes is limited to one 1995 report by Dunn et al. [98]. They found that L. monocytogenes present on surfaces was inactivated to a greater extent by pulsed light than by UV energy. Treatment with a single flash of pulsed light at 0.5-1.0 J/cm2 inactivated 10s CFU/cm2 of various microorganisms, including L. monocytogenes, on an agar surface, and several such flashes inactivated up to 10' CFU/cm2. Although pulsed light is much more effective in inactivating organisms than UV light, both types of radiation have limited penetrating power and thus are only useful for inactivating microorganisms on surfaces of foods

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and packaging materials or in transparent food ingredients, such as water and some beverages. When these authors used pulsed light to treat wieners that were previously surface inoculated with L. innocua, populations decreased about 100-fold, with similar pulsed light treatments being shown to be effective in extending the shelf life of baked foods, seafood, and meat.

High Hydrostatic Pressure Although most microorganisms except certain ocean-dwelling species grow best under normal atmospheric pressure, exposure to hydrostatic pressure >600 atm often induces cellular changes that can be lethal to non-spore-forming organisms. Hence, exposure to high hydrostatic pressures has been suggested as another means of inactivating certain spoilage and pathogenic organisms in raw and pasteurized milk as well as in meat, poultry, seafood, fruits, and vegetables. Recognizing the proven ability of high hydrostatic pressure to inactivate Salmonella spp. in laboratory media, Styles et al. [368] examined the behavior of L. monocytogenes strains Scott A and CA in phosphate-buffered saline solution during exposure to pressures of 35,000-50,000 psi (-240-345 MPa) at ambient temperatures. Strains Scott A and CA were fairly barotolerant, exhibiting D-values of 56.4 and 31.6 min, respectively, in the presence of 35,000 psi. At 50,000 psi, the D-values for Scott A and CA were 2.9 and 6.7 min, respectively. When these experiments were repeated using raw milk and UHT processed milk, 60 and 80 min at 50,000 psi were required, respectively, to inactivate an L. monocytogenes population of 1 X 106 CFU/mL. Thus both Listeria strains were more resistant to high hydrostatic pressure when suspended in milk than in buffer. Working with a higher range of pressures, Patterson et al. [293] found that exposure to 375 MPa (54,400 psi) for 15 rnin at ambient temperatures was sufficient to inactivate > 105L. monocytogenes CFU/mL in a phosphate buffer, whereas similar reductions in Y. enterocolitica, S. typhimurium, S. enteritidis, E. coli 0 157:H7, and S. aureus required 275, 350, 450, 700, and 700 MPa, respectively. Sensitivity to high pressure varies among Listeria strains, with the type of media also influencing the degree of protection against inactivation. L. monocytogenes was more resistant to inactivation by pressure when present in UHT milk than in buffer or poultry meat. Bacterial inactivation during high-pressure treatment of foods is also temperature dependent. Resistance of L. innocua to inactivation by high hydrostatic pressures at different temperatures was studied by Gervilla et al. 11541 in ewe's milk. Applying 200 MPa (29,000 psi) of pressure at different temperatures (2-50°C) for up to 15 rnin resulted in 51-log decrease in population, whereas treatment at 500 MPa (72,500 psi) for 5 min decreased the count of L. innocua from 107-108CFU/mL to < I CFU/mL regardless of temperature. High-pressure treatment was least effective at 20-30°C; this temperature dependence was most obvious at 350 MPa. Results of kinetic studies yielded D-values of 3.12 and 4 rnin at 2 and 25"C, respectively, when L. innocua was treated in ewe's milk at 400 MPa. Effectiveness of high hydrostatic pressure also depends on the growth history and physiological state of treated bacteria. Lanciotti et al. [220] grew L. monocytogenes in BHI broth at different temperatures (3-37"C), pH (5.0-6.5), and a, (0.94-0.99) before treatment with high pressure. Cultures of L. monocytogenes grown or preconditioned at lower temperatures (3-2OoC), pH 6 or high a, value (20.96) were most tolerant of high hydrostatic pressures.

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Lanciotti et al. [22 1] investigated the effectiveness of continuous homogenization at pressures of 15 to 200 MPa (2 175-29,000 psi) on microbial inactivation in milk and two biphasic (oil and aqueous) model food systems. The authors found that homogenization markedly reduced the initial load of microorganisms, including L. monocytogenes, and changed the microstructure of treated foods in a way to minimize growth of survivors. L. monocytogerzes populations decreased linearly at a rate of 0.0025 log CFU/g per bar as the pressure increased. Homogenization in both model systems at 40-90 MPa resulted in a 100-fold decrease in numbers of L. monocytogenes, with the remaining population decreasing an additional 10-fold after 10 days of storage at 3-4°C. Treatment at 190 MPa reduced the initial population of 107CFU/g to < 1 CFU/g. The authors suggested that decreasing space availability, as evidenced by the small water droplet sizes, may account for the stability of processed foods.

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COMBINED TREATMENTS Since total reliance on any single preservation method (e.g., heat, acidity, salt) usually causes quality deterioration, many food processors use several treatments in combination to process and preserve food. The well-known ‘‘hurdle concept” emphasizes the combined use of antimicrobial factors to inhibit growth or eliminate microorganisms from food. When preservation factors (hurdles) are combined, an additive antimicrobial effect often is observed. However, combined hurdles sometimes act synergistically to enhance microbial inhibition and inactivation beyond the additive effect. In other circumstances, however, one hurdle may negate the antimicrobial effect of another hurdle. Further complications may arise when hurdles are applied in sequence, with time gaps, rather than simultaneously. When used intermittently, a mild hurdle may stress an organism and elicit an adaptive response which will in turn protect the microorganism against subsequent exposure to more severe hurdles. This phenomenon of adaptation and protection is receiving great attention in relation to efficacy of preservation by multiple hurdles and microbial safety of the resulting food. In this section, examples illustrating the interaction between hurdles will be presented in relation to control of L. monocytogenes in food. It should be cautioned, however, that the outcome of interaction between hurdles depends heavily on the conditions under which these hurdles are applied. A two-hurdle interaction was demonstrated by Johansen et al. [ 1871, who found that antilisterial activity of lysozyme was synergistically enhanced by low pH values. Another example of a two-hurdle interaction was presented by Mainsnier-Patin et al. 12491, who found that adding nisin to skim milk dramatically reduced the heating time required to inactivate L. monocytogenes. Results of a study by Conner et al. [75]illustrates the negative interaction between two hurdles, refrigeration and high acidity. The investigators observed that at maximum growth-limiting pH values, L. rnonocytogenes populations decreased from -104 to < 10 CFU/mL in 1-3 weeks at 35°C; whereas at 10°C, listeriae survived for 6- 12 weeks. Interaction between multiple hurdles was presented by Bal’a and Marshal1 [25] who investigated the combined effect of NaCl (2.5-7.8%), pH (5.4-7.8), temperature (5, 15, 25, and 35”C), and sublethal levels of monolaurin (2-8 pg/L) against L. monocytogenes grown on double (salt-pH) gradient plates. Addition of monolaurin to the gradient plates reduced salt and pH tolerance of the pathogen. Complicated interactions between preservation factors (hurdles) were evident in a recent study by Lou [2371, who noted that antilisterial activity of nisin was affected by pH and the presence of NaCl. Addition of NaCl

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(3.5-7.5%), to Trypticase Soy Broth decreased the bactericidal action of nisin against L. monocytogenes. However, the presence of 3.5-5.5% NaCl interacted synergistically with nisin to inhibit outgrowth of the pathogen on Trypticase Soy Agar plates. Inhibition of L. monocytogenes by multiple hurdles was studied by Buchanan and coworkers [58]. A factorial design was used to determine the combined effect of incubation temperature (5-37”C), initial pH (6.0-7.5), sodium chloride (0.5 vs 4.5%), sodium nitrite (0-1000 ppm), and atmosphere (aerobic vs anaerobic) on growth of L. monocytogenes in TPB. Although lag periods, generation times, and maximum populations were all affected by these five interacting variables, sodium nitrite was most listeriostatic when used in conjunction with low pH, increased sodium chloride, refrigeration temperatures, and anaerobic conditions that simulated vacuum packaging. Research using predictive microbiological modeling is likely to be valuable in assessing the safety of foods preserved by multiple hurdles. Additive, nullifying, or synergistic antimicrobial effects of multiple hurdles can be estimated by predictive models. Consistent with these objectives, Buchanan et al. [5 1,52,57] attempted to predict behavior of L. monocytogenes in response to an array of extrinsic factors. Buchanan et al. [57] used a factoriallsupplemental central composite design to assess quantitatively the effects of temperature (5, 10, 19,28, 37”C), pH (4.50, .5.25, 6.00, 6.75, 7.50), sodium chloride (0.5, 1.5, 2.5, 3.5, 4.5%), sodium nitrite (0, 50, 100, 150, 200, 1000 ppm), and atmosphere (aerobic vs anaerobic) on the growth kinetics of L. monocytogenes strain Scott A in TPB. After growth curves were constructed from each experiment using regression analysis to obtain ‘‘best fit” Gompertz equation curves, results were analyzed by response surface analysis to generate a polynomial model that could mathematically predict lag periods, exponential growth rates, generation times, and maximum populations for L. monocytogenes in association with any of the five variables examined. Overall, changes in response of the organism to the five environmental factors were most evident as altered specific growth rates and lag periods. L. monocytogenes also achieved similar maximum populations in all instances except those that involved growth of the pathogen under environmental extremes in the presence of high concentrations of sodium nitrite. As a result of these and other studies, Buchanan’s group developed useful mathematical models to quantify behavior of L, monocytogenes in response to multiple environmental factors or hurdles [53]. These models were incorporated into a computer program called the Pathogen Modeling Program. As of 1997, the program is available as version 5.0 for Microsoft Windows and can be downloaded from a USDA site on the internet or requested from the developers. The L. monocytogenes module of this program can be used to predict lag time, growth rate, maximum population, and time required to attain a given count of Listeria under a wide range of environmental conditions. Interaction between hurdles becomes even more complicated when the history of Listeria cells to be inactivated by the multiple hurdles is considered. Adaptation of L. monocytogenes during sublethal exposure to various preservation techniques (or stress) may protect the pathogen against subsequent exposure to the the same, different, or any combination of stresses at normally lethal levels. Kroll and Patchett [216] reported that adaptation to pH 5 greatly increased survival of L. monocytogenes at pH 3 as compared with the unadapted cultures. According to Lou and Yousef [238,239], adaptation of L. monocytogenes to sublethal levels of acid, ethanol, and hydrogen peroxide and starvation increased resistance of L. monocytogenes to lethal levels of these factors and heat. This stress adaptation, or ‘‘hardening,’’ complements the hurdle concept, since such hurdles in foods can be applied simultaneously or sequentially. When applied sequentially, hurdles

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may not deliver the desired effect. Stress adaptation to the first encountered hurdle, which ' 'hardens' ' pathogens and increases their resistance to subsequent preservation factors, may counteract hurdle build-up.

SURVIVAL, ATTACHMENT, AND BlOFlLM FORMATION ON SURFACES L. monocytogenes is a very hardy organism, being able to survive up to 2 1 years in refrigerated laboratory media [SO] as well as 10 days in tap water incubated at 22°C and 6, 3, and 1 day in distilled water stored at 22, 30, and 40"C, respectively [87]. Moreover, this pathogen is also relatively resistant to drying. These observations have led to questions concerning the ability of L. monocytogenes to survive on various types of materials common to food processing facilities. In an early study, Durst and Sawinsky [ 1001 moistened various inert materials with a 24-h-old NB culture containing 1O9 L. monocytogenes CFU/mL and stored the materials in sterile Petri plates at ambient temperature. L. monocytogenes survived <24 h on glass, iron, and aluminum, <48 h on paper and plastic, 7 but not 42 days on porcelain, 6 but not 12 months on wood, and at least 1 year on gauze. L. monocytogenes also survived more than 1 day on stainless steel [324], 165 days on contaminated wool stored at 8-22°C [26], 105 days on dried threads stored at room temperature [376], 20-30 days on tiles [369], and at least 20 days on 250-pm diameter glass beads [391]. Of particular interest to the dairy industry is a 1987 study by Stanfield et al. [363] which examined survival of three L. monocytogenes strains on exterior surfaces of waxed cardboard and plastic milk containers. Both container types were contaminated by swabbing their surfaces with a heavy suspension of an 18- to 24-h-old unstressed or stressed (heated at 56°C for 30 min) L. monocytogenes culture, and then containers were stored at -0.8-6.6"C for 14 days. Unstressed cells of L. monocytogenes were recovered after 14 days of storage from at least one site on the surface of plastic and waxed cardboard containers. L. monocytogenes, like several other foodborne pathogens, can attach to surfaces and form biofilms. When biofilms are formed, they are hard to remove by normal cleaning. Biofilms also protect microorganisms, including L. monocytogenes, from antimicrobial or sanitizing agents and often serve as a source of recontamination for processed foods. Herald and Zottola [ 1681 examined the ability of a culture of L. monocytogenes grown at 10, 21, and 35°C in Trypticase Soy Broth at pH 5, 7, and 8 to attach to stainless steel. A small stainless steel chip was placed inside a culture vial containing L. monocytogenes and then incubated at 21 or 35°C for 18-24 h or at 10°C for 36-48 h. Following incubation, analysis of the chips using scanning electron microscopy (SEM) revealed that the pathogen adhered to stainless steel at all pH values and temperatures studied; however, cells with fibrils were observed only at 2 1 and 10°C. Amounts of exopolymeric attachment material were greater when the organism was incubated at 10 rather than 35°C and increased with the length of incubation. The ability of L. monocytogenes to attach to various surfaces on food processing facilities was subsequently reported by other researchers. Mafu et al. 12471found that L. monocytogenes attached to stainless steel, glass, polypropylene, and rubber, which are common materials in food-contact surfaces, after short contact times (20-60 min) at both ambient and refrigeration temperatures. Formation of extracellular material around the attached cells was revealed by SEM after 60 min of incubation

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at both temperatures. According to Krysinski et al. [217], L. monocytogenes adhered to stainless steel and polyester or polyester-polyurethane conveyor belts at levels of -2 X 104CFU/cm2 after incubating these materials in a culture medium at 35°C for 24 h. Blackman and Frank [38] observed variable adherence of L. monocytogenes to surfaces of stainless steel, Teflon, nylon, and polyester floor sealant after 7 days of incubation in TSB at 21°C with markedly less attachment occurring at 10°C. Kim and Frank [204] found that L. monocytogenes grown in a minimal medium exhibited an attachment rate 50-fold higher than cells grown in TSB. L. rnonocytogenes attachment to Teflon and buna-n rubber and secretion of biofilm matrix materials were also reported by Mosteller and Bishop [269]. Since floor drains frequently harbor L. monocytogenes, Spurlock and Zottola [361] investigated growth of the pathogen in a model floor drain and its attachment to freestanding cast iron drains at ambient temperature. L. monocytogenes grew and remained on these drain surfaces at 106-108 CFU/cm2 regardless of drastic changes in pH from alkaline (pH 9.0) to acidic (pH 4.5). Attachment of L. monocytogenes to cast iron was clearly seen in SEM images taken after several hours of contact, with attachment material also being observed on drains after 9 and 29 h of incubation in TSBYE and 0. I % reconstituted nonfat dried milk, respectively. Environmental flora in food processing facilities may interfere with or enhance attachment of L. monocytogenes to surfaces or its growth in biofilms. Sasahara and Zottola [334] found that in a flowing system, Pseudomonas fragi, a bacterium which strongly attaches to and produces exopolysaccharide materials on surfaces, enhanced surface attachment of L. monocytogenes, but P. fragi itself failed to attach to these surfaces in this flowing system. In contrast, Jeong and Frank [I861 reported that when environmental isolates from meat or dairy plants were statically incubated in 0.2 and I .O% TSB at 10°C, these bacteria either inhibited or minimized attachment of L. rnonocytogenes to stainless steel, although considerable L. monocytogenes attachment and biofilm growth occurred in all instances. In pure culture biofilms, L. monocytogenes populations began to increase after 9 and 13 days and reached 106CFU/cm2 after 17 and 25 days, respectively [ 1861. L. monocytogenes on surfaces can be protected by the presence of food components, and this protection increases as the layer of food thickens, possibly from the slower loss of moisture from the thick food layers. When compared with phosphate-buffered saline (PBS) solution, various milk residues (e.g., raw milk and pasteurized whole milk) enhanced survival of L. monocytogenes on stainless steel and buna-n rubber, and sometimes promoted growth of Listeria [ 1671. However, cottage cheese whey, as a residue, did not increase survival of L. monocytogenes. With PBS as a residue, L. monocytogenes populations (initially, -104 CFU/cm2) decreased -1 log at 6°C and 75.5% relative humidity (RH) after 10 days or became undetectable at 25°C and 32.5% RH after 3-5 days. However, numbers of L. monocytogenes inside a single layer of pasteurized whole milk increased >2 logs at 25"C/75.5% RH after 3 days and remained unchanged at 6OU32.5 or 75.5% RH and 25"C/32.5% RH. Of particular interest to dairy processors are the findings of Al-Makhlafi et al. [7], that attachment of L. rnonocytogenes to silica surfaces preabsorbed with milk proteins was highest with P-lactoglobulin and lowest with bovine serum albumin. Preabsorbed a-lactoglobulin and p-casein had an intermediate effect on attachment of Listeria. Although L. monocytogenes attaches to all types of surfaces, the ability of this bacterium to form a biofilm on buna-n rubber, a gasket material, is relatively low. Buna-n rubber is bacteriostatic to L. rnonocytogenes under certain conditions, with this activity remaining after 20 cycles of a simulated clean-in-place process [ 167,3201. According to

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Ronner and Wong [320], buna-n rubber was strongly bacteriostatic to L. monocytogenes growing in Peptone Glucose Phosphate (PGP) broth (a low nutrient medium) and slightly bacteriostatic in TSB. Four of seven strains of L. monocytogenes growing in PGP formed less dense biofilm populations on buna-n rubber than on stainless steel. Temperature and moisture also affect survival of L. monocytogenes on surfaces. Palumbo and Williams (284a) suspended a mixture of seven L. monocytogenes strains in seven menstrua (distilled water, tryptone broth, nonfat dry milk, canned milk, glycerol, light Karo syrup, and beef extract), and then dried these cell suspensions on glass plates which were stored at 5 or 25°C and I-75% RH. Enhanced survival was observed at 5°C rather than at 25°C and at lower rather than higher RH. In a subsequent study, Helke and Wong [ 1671 investigated survival of L. monocytogenes on surfaces under 32.5 and 75.5% RH and temperatures of 6 and 25°C. The authors found that survival of Listeria was higher at 6°C than at 25°C and, in contrast to the previous study, survival was greater under humid (75.5% RH) rather than dry (32.5% RH) conditions. Microorganisms embedded in biofilms are more resistant to heat, sanitizers, and other antimicrobial agents than are freely suspended (planktonic) cells. Frank and Koffi [ 1461 prepared L. monocytogenes in three states: (a) planktonic cells, obtained by growing the bacterium in TSB for 38 h at 21"C, (b) adherent single cells, prepared by immersing glass slides in a planktonic cell culture for 4 h at 2 I "C; the attached Listeria was mostly single cells, and (c) adherent microcolony cells, made by incubating the glass slides with attached L. monocytogenes cells for 14 days at 2loC, during which the slides were periodically washed with a saline solution and incubated in fresh media; the attached cells in this instance were mostly in microcolonies. The investigators found that Listeria was more sensitive to banzalkonium chloride (n-alkyl dimethyl dichlorobenzyl ammonium chloride, a quaternary ammonia sanitizer) and dodecyl benzene sulfonic acid, an anionic acid sanitizer (DBSA) when present in the planktonic rather than in the adherent single cell or microcolony state. Contact with either sanitizer ( 100-800 ppm) at ambient temperature immediately reduced populations of the planktonic cells from 1Oh CFU/ml to undetectable levels. In contrast, adherent single cells (initially 105- 106CFU/cm2) and adherent microcolony cells (initially 106- 107CFU/cm2) decreased 3-5 and 2-3 logs, respectively. The few remaining adherent single cells became undetectable after 16 min of sanitizer exposure, with adherent microcolonies surviving a maximum of 20 min. Survival of these adherent cells was not caused by depletion of sanitizers, since the remaining sanitizers produced similar inactivation when new microcolony slides were treated. When planktonic cells were heat-treated ( 5 min at 55 or 70°C) in the presence of 400 ppm benzalkonium chloride or 200 ppm DBSA, the combined treatment, regardless of temperature, decreased populations -5 logs from an initial - 106CFU/mL, whereas similar treatments decreased counts of microcolonies only -2 and >5 logs, respectively. In a subsequent study, Lee and Frank [226] investigated the sensitivity of stainless steel-adhering single and microcolony cells (initially 1O5 L. monocytogenes CFU/cm2) on stainless steel to hypochlorite and heat. They found that adherent single cells on stainless steel were more sensitive to hypochlorite and heat inactivation than adherent microcolony cells. Exposure to a hypochlorite solution, which contained 200 ppm residual chlorine, for 30 s decreased the population of adherent single and microcolony cells by 4.8 and 2.6 log units, respectively, with microcolony cells surviving up to 5 min of exposure to this agent. Although heating at 65°C for 30 s resulted in a 3.8-log reduction of both types of adherent cells, only microcolony cells were detectable after 3 min of heating. Adherent single and microcolony cells became undetectable after 30 and 60 s of heating at 72"C,

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respectively. Combined exposure to 65°C for 30 s and 200 ppm chlorine decreased the number of microcolony cells to undetectable levels. Krysinski et al. [217] obtained adherent L. monocytogenes cells by growing the organism in a culture medium for 24 h at 25°C that contained pieces of stainless steel, polyester belt, or polyester-polyurethane conveyer belt and then tested the effectiveness of 10 sanitizers and 6 cleaners in inactivating or removing adhering L. monocytogenes cells after 10 min of exposure. Although L. monocytogenes attached to these surfaces at similar levels (-2 X 104CFU/cm2), protection provided by these surfaces against sanitizers or cleaning agents varied with the substrate, with polyester belt being most protective followed by polyester-polyurethane belt and stainless steel. Although most of the sanitizers and cleaners that were tested inactivated Listeria attached to stainless steel, none of these agents could effectively eliminate cells attached to polyester-polyurethane belt. However, detergent cleaning followed by sanitizing, a practice commonly followed in industry, was more effective in controlling adherent L. monocytogenes cells than when either cleaning or sanitizing was used alone. Besides sanitizers, effectiveness of listeriaphages in inactivating surface-adherent L. monocytogenes was also investigated [326]. Adherent cells were obtained by immersing chips of stainless steel or polypropylene in a L. monocytogenes culture for 1 h at 26°C. Treatment of adherent L. monocytogenes cells with a phage suspension (3.5 X 108PFU/ mL) reduced populations of the pathogen by -3.4 logs, with mixtures of three different phages proving to be most effective. Although use of QUATAL (containing 10.5% Nalkyldimethyl-benzylammonium HCl and 5.5% glutaraldehyde as active ingredients, Ecochimie LtLe, Quebec, Canada) at 50 pprn destroyed the adherent Listeria flora, a combination of 108PFU/mL phage and 30 pprn QUATAL resulted in similar destruction.

SANITIZERS Sanitizers have been widely used in the food industry to decrease populations of pathogenic and spoilage organisms in food production and processing facilities. Most of these sanitizing agents belong to one of four categories: (a) chlorine-containing compounds, (b) iodophors, (c) quaternary ammonium compounds frequently called “quats,” or (d) acid sanitizers. Additionally, ozone has been used for decades in some European counties, and application of this sanitizer in the US food industry is likely to increase. When in aqueous solutions, chlorine-containing compounds release hypochlorous acid which accounts for their bactericidal action. Iodophors, water-soluble complexes of elemental iodine, and nonionic surface-active agents owe their bactericidal activity to release of free elemental iodine and hypoiodous acid, which is enhanced under acidic conditions. In contrast, quaternary ammonium compounds are best classified as noncorrosive germicidal cationic detergents that remain active at relatively high pH values. Finally, acid sanitizers such as phosphoric and citric acid-containing compounds are frequently used in conjunction with rinsing agents in automated cleaning systems better known as clean-in-place (CIP) systems. Unlike iodophors, acid sanitizers are nonvolatile and retain their bactericidal activity at temperatures below 100°C. Sanitizing agents must reduce populations of a given test organism at least 5 logs during 30 s of exposure at ambient temperatures before the particular agent is deemed to be effective.

Chlorine Compounds In the absence of organic debris, chlorine rapidly inactivates most non-spore-forming bacteria even when used at the very low concentrations found in chlorinated drinking

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water. Although the actual mechanism of disinfection is not fully understood, germicidal activity of chlorine has generally been attributed to hypochlorous acid (HOCI), which is generated in aqueous solutions of sodium hypochlorite and other chlorine-containing compounds. Although HOCl can in turn dissociate to form the hypochlorite ion (OC1-) and hydrogen ion (H'), depending on the pH of the solution, the neutral electric charge of the former suggests that HOCl can more easily penetrate the bacterial cell membrane than OCI-. Thus it is not surprising that the germicidal activity of HOCl is 80 times that of OC1-. After diffusing into the cell, HOCl is thought to inactivate the organism by inducing formation of toxic oxygen species or combining with proteins, which may in turn inhibit key enzymatic reactions and alter cell membrane permeability. Numerous studies have dealt with the lethal effects of various chlorinated sanitizing agents on L. monocytogenes. Beginning in 1969, Baranenkov [26] found that hypochlorite could effectively control L. monocytogenes on the surface of hen's eggs. Chloramine also was later shown to be listericidal when used under acidic conditions at concentrations of 0.1-0.2% [ 2581. Subsequently, Lopes [236] reported that two solutions of chlorine-based sanitizers (one containing 8.5% sodium hypochlorite with 8%)active chlorine and the other containing 25.8% sodium dichloro-s-triazinetrione) containing I00 pprn active chlorine both reduced L. monocytogenes populations by more than 5 logs after 30 s of exposure. These findings were subsequently confirmed by Rossmoore and Drenzek [324]. Further tests by Lopes [236] revealed that the organic chlorine-based sanitizer was slightly more effective against L. monocytogenes than the sodium hypochlorite-based sanitizer; the former had a lower pH which would in turn lead to higher concentrations of HOCI, the most bactericidal form of chlorine. A chlorine dioxide-based sanitizing agent also has been approved by the FDA for use in the food industry. According to its manufacturer [ 121, the unusual effectiveness of this formula against L. monocytogenes and other microorganisms results from a special activator which converts large quantities of stabilized chlorine dioxide to the free form. Following the published report by Lopes [236], Brackett [41] determined the germicidal effect of reagent-grade sodium hypochlorite and household bleach on two L. monocytogenes strains (Scott A and LCDC 8 1-861 ) previously associated with outbreaks of foodborne listeriosis. After 20 s of exposure to 2 5 0 ppm available chlorine, both compounds led to substantial reductions in numbers of viable L. monocytogenes in phosphate buffer. However, Listeria populations remained relatively stable for an additional 4.6 min, and in several instances listeriae survived 1 5 min with free residual chlorine levels that approached 40 ppm. Since 10 ppm available chlorine was ineffective, results of this study indicate that the minimum chlorine concentration needed to kill L. monocytogenes lies between 10 and 40 ppm. Effectiveness of chlorine against L. monocytogenes also was examined in depth and later reviewed by El-Kest and Marth [103-1051. Cells of L. monocytogenes strain Scott A were harvested from 24- and 48-h-old slants or broth cultures, washed by centrifugation in 20 mM phosphate buffer solution or 0.3 12 mM phosphate buffer dilution water, and then exposed at 25°C to sodium hypochlorite solutions at pH 7 (25°C) that contained 0.510.0 ppm available chlorine. Using a solution containing 5 ppm available chlorine, numbers of survivors decreased -6 logs after only 30 s, with the organism no longer being detectable by direct plating on TA after 1 h. (Results from Rosales et al. [32 I ] also showed that populations of L. monocytogenes, L. ivanovii, and L. seeligeri decreased >5 logs following 30 s of exposure to distilled water (pH 7) containing 2 2 5 pprn hypochlorite [i.e., 223.8 ppm available chlorine]). Exposing L. monocytogenes to 0.5, 1.0, 2.0, 5.0, and 10.0 ppm available chlorine resulted in corresponding D-values of 61.7, I 1.3, 6.7,

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4.9, and 4.7 s. Although disinfecting activity clearly increased with increasing concentrations of available chlorine, the effectiveness of sodium hypochlorite also was affected by several additional factors. Increased resistance of L. monocytogenes to chlorine was observed using (a) 24- rather than 48-h-old cultures, (b) cells harvested from broth rather than agar slants, and (c) cultures exposed to solutions containing 20 mM rather than 0.3 12 mM phosphate. Five and 10 ppm of available chlorine was partially neutralized in the presence of 0.05 and 0.1% peptone (nitrogenous compound) [103]. Given the findings indicating that hypochlorite concentrations of up to 400 ppm were of little use against L. monocytogenes, L. ivanovii, or L. seeligeri when these organisms were suspended in reconstituted NFDM (10% solids) [321], it is clear that antimicrobial activity of chlorine can only be maintained if organic material is effectively removed before exposure. In addition to the factors just discussed, Lee and Frank [225] found that resistance of L. monocytogenes cells in late exponential phase to hypochlorite solution (1-5 ppm available chlorine) was greater when the organism was grown at 35°C than at 6 or 21°C. Exposure to 1 ppm available chlorine for 5 min at ambient temperature decreased populations of the organism previously grown at 6, 21, and 35°C by 3.4, 3.1, and 2.1 logs, respectively. Furthermore, L. monocytogenes grown in a nutrient-poor medium ( 15-fold diluted TSB) was 10-times more resistant to chlorine than when grown in regular TSB [225]. Additional work by El-Kest and Marth [105] demonstrated that populations of L. monocytogenes decreased most rapidly in sodium hypochlorite solutions at 5°C followed by 35 and 25°C. Marked variation in chlorine sensitivity also was observed among the three L. monocytogenes strains tested. However, since dissociation of HOCl to OC1- and Hi increases with increasing pH, resistance and/or survival of L. monocytogenes in the presence of chlorine compounds ultimately depends on the pH of the suspending medium. For example, exposing the pathogen to 1 ppm available chlorine for 30 s led to population decreases of -4.0, 3.0, and 0.7 logs at pH 5, 7, and 9, respectively. Hence, for chlorine to be effective against listeriae and other microorganisms, it is imperative that such solutions have pH values <7. Although the work of El-Kest and Marth [103-1051 clearly indicates that the minimum listericidal concentration of free chlorine lies between 1 and 5 ppm (similar to that observed for many other non-spore-forming bacteria) depending on pH, temperature, the presence of organic material, and bacterial strain, earlier studies [41,10I] conducted under less controlled conditions showed minimum listericidal concentrations of free chlorine that were markedly higher. Similar problems also were probably encountered by Mustapha and Liewen [273], who found that a minimum of -100 ppm sodium hypochlorite was required to reduce L. monocytogenes populations >4 logs in sterile distilled water during 2-5 min of exposure. Chlorine is used extensively in fresh vegetable processing. Therefore, Zhang and Farber [408] investigated the efficacy of several chlorine-based compounds against a cocktail of five L. monocytogenes strains on the surface of freshly cut lettuce and cabbage at refrigeration and ambient temperatures. Sanitizers tested by these investigators included chlorine from a hypochlorite-containing bleach, chlorine dioxide and a sodium chloritebased oxy-halogen compound. Immersing Listeria-contaminated vegetables in solutions containing 200 ppm chlorine, 5 ppm chlorine dioxide, or 200 ppm Salmide for 10 min resulted in maximum reductions of 1.3- 1.7,0.8-1.1, and 0.6 logs, respectively, for lettuce, and 0.9-1.2, 0.4-0.8, and 1.8 logs for cabbage. The presence of surfactants reduced the effect of chlorine. The authors also tested trisodium phosphate and lactic acid on lettuce

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and cabbage. Trisodium phosphate (0.1 and 0.2%) failed to inactivate listeriae, whereas 0.1% lactic or acetic acid reduced populations by only 0.5 and 0.2 log, respectively. Many researchers have investigated the ability of commonly used sanitizers to inactivate L. rnonoc.ytogenes on various types of food contact surfaces. Mustapha and Liewen [273] found that destruction of L. rnonocytogenes was greater on smooth rather than pitted stainless steel surfaces. However, cells incubated on either surface for 1 h were more resistant to the lethal action of sodium hypochlorite than those remaining on such surfaces 24 h before exposure. Lower moisture levels on stainless steel surfaces incubated 24 rather than 1 h may have enhanced the listericidal effect of sodium hypochlorite. In contrast to these findings, Rossmoore and Drenzek [324] reported that L. rnonocytogenes populations decreased 5 logs on relatively moist surfaces of glazed and unglazed ceramic tile as well as stainless steel chips following exposure to 100 ppm sodium hypochlorite as directed by the manufacturer. Furthermore, in no instance was L. monocytogenes more resistant than single cultures of Pseudornonas or Serratia. However, when the same three surfaces were treated with 1 and 10% solutions of milk and blood, Listeria populations decreased 1-4 logs in the presence of 100 ppm sodium hypochlorite. As mentioned earlier, commonly used sanitizers are generally less effective against L. rnonocytogenes in biofilms than when the cells are freely suspended. Lee and Frank [226] reported that microcolonies of L. rnonocytogenes adhering to stainless steel (-105CFU/cm2) decreased 2.6 logs after 30 s of exposure to 200 ppm chlorine (from a hypochlorite solution), with some cells surviving a 5-min treatment. Mosteller and Bishop [269] found that 200 ppm chlorine was sufficient to inactivate more than 5 logs of freely suspended L. rnonocytogenes cells. However, a similar treatment failed to inactivate 3 logs of L. rnonocytogenes when a milk biofilm (initially 1O4-1O5CFU/cm2)was formed on surfaces of Teflon and buna-n rubber. Resistance to sanitizers, including chlorine, increased when L. rnonocytogenes biofilms were prepared on surfaces of polyester or polyester-polyurethane instead of stainless steel [2 171. Therefore, although freely suspended L. rnonocytogenes can be controlled by 100 ppm chlorine, a higher level of chlorine is required to eliminate L. rnonocytogenes from biofilms.

Ozone Ozone, a powerful sanitizing gas, is a better alternative to chlorine in many food processing applications. Although used in European countries for decades, ozone is only approved in the United States for treatment of bottled drinking water. Recently, a panel of experts representing academia, food processors, and utility companies self-affirmed the GRAS status of ozone, thus permitting its use in food processing applications [ 1591. Ozone can be applied as a sanitizer in its gaseous form or as ozonated water. Ozonated water is bactericidal to various microorganisms, with vegetative cells being more sensitive to ozone than molds or bacterial spores. Use of ozone, in the form of ozonated water, in food preservation and for decreasing microbial loads of meat and poultry and of food plant effluents has been investigated [ 127,192,3441. Several factors affect the bactericidal activity of ozonated water; organic matter such as food components quickly react with ozone and reduce its effectivity. Restaino et al. [316] investigated the lethality of ozonated water with and without 20 ppm organic matter, soluble starch (SS), or bovine serum albumin (BSA), against four gram-positive (including L. rnonocytogenes) and four gram-negative (E. coli, S. typhirnuriurn, Y. enterocolitica, and P. aeruginosa) bacteria, two yeasts (Candida albicans and

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Zygosaccharomyces bailii), and mold spores (Aspergillus niger). Initial ozone concentrations of 0.15-0.20 ppm produced by the ozone generator were higher in deionized water with or without 20 ppm SS than in BSA-containing deionized water. Biphasic inactivation curves were observed for bacteria and yeasts. Vegetative cells were inactivated instantly (decreased >4 logs) after contact with ozone, with a much slower decrease in microbial counts occurring during extended incubation. Gram-positive bacteria were generally more resistant to ozone than gram-negative organisms, with the four gram-negative species having similar sensitivity to ozone; however, L. monocytogenes was an exception. Contact with ozone instantly inactivated >5 logs of L. monocytogenes but only -3 logs of the other gram-positive species. The presence of organic matter during ozonation decreased the lethality of ozone; however, the type of organic matter was more important than the concentration. Incorporating 20 ppm SS had little effect on lethality of ozone toward Listeria, whereas 20 ppm BSA significantly decreased the inactivation rate.

Antiseptic Soaps Cross contamination of foods by food handlers or raw products in food service facilities is a potential threat to public safety. Kerr et al. [ 1991 found that 12 and 7% of food workers carried Listeria spp. and L. monocytogenes on their hands, respectively. Therefore, eliminating L. monocytogenes from hands should decrease the incidence of Listeria in many foods and enhance overall food safety. According to one report [32 I], full-strength solutions of three commercially available antiseptic soaps, namely Mikro-x, Isoderm, and Zerobac were strongly listericidal, with populations of L. monocytogenes, L. ivanovii, and L. seeligeri decreasing 7 logs following 30 s of exposure. Isoderm (a chlorine/quaternary ammonium compound-based soap) remained almost equally effective when diluted 1 :4, whereas Zerobac (an iodophor-based soap) retained strong listericidal activity at a dilution of 1:8. In another study [2 101, fingers of human volunteers were inoculated to contain 105 or 109L. monocytogenes CFU/finger to test the effectiveness of moist soap and a commercially produced finger wipe containing isopropyl alcohol and citric acid as active ingredients. Overall, numbers of listeriae on fingers were generally reduced no more than 2-4 logs after 5 s of rubbing in phosphate buffer and moist soap, respectively. Therefore, a population decrease of approximately 2 logs can be attributed to physical removal of the pathogen during rubbing. In contrast, L. monocytogenes populations consistently decreased 2 4 logs after rubbing fingers with finger wipes for 5 s. Thus, strong listericidal activity of these particular finger wipes and the ease with which they can be used should make such products beneficial for food handlers in the food service industry. In 1996, McCarthy 12531 checked inactivation of L. monocytogenes on latex gloves by five commercially available hand-washing sanitizers. The latex gloves were artificially contaminated by dipping them for 30 s into a PBS solution or crab cooking water that contained - 1 O5 Listeria CFU/mL and then were treated with various hand-washing sanitizers. Dipping contaminated gloves into PBS containing a commercial chlorine bleach solution (50 and 100 pprn chlorine), Zepamine A ( I95 ppm active quaternaries) or UltraKleen (a peroxide-based powder at 56 g/3.8 L) decreased counts of L. monocytogenes on surfaces of gloves to undetectable levels, whereas treatment with Zep-i-dine (25 ppm titratable iodine) and Zep Instant Hand Sanitizer that contains 60% ethanol reduced populations only 2 logs. Using nutrient-rich crab cooking water instead of PBS dramatically

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decreased the effectiveness of both 50 ppm chlorine and Zep-i-dine and slightly decreased the effectiveness of 100 ppm chlorine and Zepamine A. According to this study, only Ultra-Kleen maintained the same effectiveness in cooking water.

Other Sanitizing Agents Interest in the listericidal activity of non-chlorine-based sanitizers dates back to at least 1969 when Raranenkov [26] reported that iodine monochloride could effectively eliminate L. monocytogenes from the surface of hen’s eggs. Shortly thereafter, creosote [59], phenol [258,303], formaldehyde [303], and sodium hydroxide [258] were added to the list of listericidal agents along with mercuric [258] and quaternary ammonium compounds (e.g., cetylpyridinium bromide) [258]. Sodium hydroxide at concentrations of 1 -2% solubilizes the cytoplasmic membrane of Listeria [304], whereas bactericidal concentrations of ethyl alcohol, phenol, and formaldehyde inhibit certain key enzymes, including succinic and xanthine dehydrogenase [ 3031. During the 1970s, several sanitizing agents were evaluated for treating soil samples inoculated with L. mnnocytogenes. According to Vranchen et al. [380], 5 days of exposure to 5 L of 3% formaldehyde solution was required before L. monocytogenes was eliminated from 1 m2of soil. In a later study [ 101, 1 m2 of soil was Listeria-free 3 h after treatment with 0.5 L of an aqueous solution containing 3% quaternary ammonium compound. Although sanitization of soil has little direct bearing on the food industry, such practices may be useful in decreasing Listeria populations on farms that have experienced cases of listeriosis in domestic livestock. Following reports of foodborne listeriosis in the mid 1980s, a series of studies were done to determine the listericidal activity of non-chlorine-based sanitizers that are routinely used by the food industry. According to experimental evidence presented in 1986 by Lopes [236), acid anionic, iodophor, and quaternary ammonium compounds are effective against L. mnnncytogenes when used at concentrations recommended by manufacturers. Numbers of L. monocytogenes were reduced more than 5 logs after 30 s of exposure to two different acid anionic sanitizers that contained 200 ppni of active ingredients (5% dodecyl benzene sulfonic acid and 30% orthophosphoric acid or 2.6% sulfonated oleic acid and 15% orthophosphoric acid). Similar reductions in Listeria populations were obtained with a quaternary ammonium compound diluted to contain 200 pprn of the active ingredient n-alkyl dimethyl benzyl ammonium chloride ( 12- 16 carbon atoms in the alkyl group). An iodophor sanitizer diluted to contain 12.5 ppm titratable iodine was equally effective against this pathogen. Thus all sanitizers tested showed effective antilisterial activity when used at concentrations recommended by the manufacturer. Two years later, Rosales et al. [321] found that populations of L. monocytogenes, L. ivanovii, and L. seeligeri decreased >5 logs following exposure to aqueous solutions containing 12.5- 100 ppm iodophor (pH 2.7-5.0), 12.5- 100 ppm quaternary ammonium compound (pH 4.9-6.8), 100-400 ppm acid sanitizer (pH 2.4-3.l), 400 ppm phenolic compound (pH 7.9), and 50- 100 ppm of a combined quaternary ammonium compound/ acid sanitizer preparation (pH 2.8-3.0). When these experiments were repeated using 10% reconstituted nonfat dry milk (10% solids) rather than aqueous solutions of sanitizers, reductions in Listeria populations of >5 logs only were observed for two of three iodophors, one of three quaternary ammonium compounds, and one quaternary ammonium compound/acid sanitizer preparation at concentrations of 200-400, 400, and 400 ppm,

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respectively. Although all aqueous solutions proved to be listericidal at concentrations recommended by the manufacturer, only one iodophor, quaternary ammonium compound, and phenolic sanitizer were listericidal at recommended concentrations when the test organism was suspended in milk. Information from two additional investigations dealing with listericidal effects of various non-chlorine-based sanitizers also became available in 1989. In the first study, Mustapha and Liewen [273] reported that aqueous solutions containing 100-800 ppm of one quaternary ammonium compound (i.e., n-alkyl dimethyl dichlorobenzyl ammonium chloride) exhibited greater listericidal activity than similar solutions of sodium hypochlorite when L. monocytogenes was exposed to these sanitizing agents in vitro. Moreover, a 50-ppm aqueous solution of the quaternary ammonium compound was equally effective when the pathogen was present on smooth as well as pitted surfaces of stainless steel chips, with populations decreasing >4 logs following short-term exposure. In the second of these two investigations, Rossmoore and Drenzek [324] examined the ability of four quaternary ammonium compounds as well as peroxyacetic acid, glutaraldehyde, MCI (S-chloro 2-methyl 4-isothiazolin 3-one and 2-methyl 4-isothiazolin 3-one), dodecyl benzene sulfonic acid/orthophosphoric acid, and sulfonated oleic acid/orthophosphoric acid to inactivate L. monocytogenes, Pseudomonas, and Serratia on glazed/ unglazed ceramic tile and stainless steel. When used as recommended by the manufacturer, peroxyacetic acid, glutaraldehyde, MCI, and one of four quaternary ammonium compounds reduced numbers of listeriae >5 logs regardless of the type of surface tested, and population decreases of 3-5 logs were noted for the three remaining quaternary ammonium compounds. Since L. monocytogenes was consistently more sensitive to these sanitizers than the other two organisms tested, destruction of Pseudomonas spp (i.e., a frequently used group of indicator organisms for general sanitation) also should guarantee elimination of listeriae from properly treated surfaces. To better simulate conditions that are likely to exist in dairy and meat processing facilities, these researchers repeated the study just described [324] using surfaces that were precoated with 1 and 10%solutions of milk or blood before exposure to the same sanitizing agents. Not surprisingly, destruction of listeriae by most sanitizers was only 1-4 logs in the presence of increasing concentrations of milk and blood, with the latter being most detrimental to germicidal activity. However, since peroxyacetic acid and glutaraldehyde maintained peak listericidal activity in the presence of up to 10%milk and blood, these two sanitizers appear to be best suited for controlling listeriae within milk and meat processing facilities. Since water-based chain conveyor lubricants also may serve as a potential source for spoilage and pathogenic microorganisms, including L. monocytogenes, incorporation of sanitizing agents into lubricants has been suggested as one means of minimizing the spread of microbial contaminants in food processing facilities. Although L. monocytogenes populations in inoculated samples of sanitizer-free lubricant (pH 9.5) decreased only 2 logs during 14 days of storage at ambient temperatures [324], numbers of listeriae decreased more than 5 logs following 30 min of exposure to lubricant containing as little as 25 ppm glutaraldehyde [323]. Furthermore, data collected during a field investigation [324] showed that addition of 85 ppm glutaraldehyde to a conveyor chain lubricant reduced general bacterial contamination along a dairy floor conveyor belt by an average of 4.4 logs/60 cm2. Although L. monocytogenes was not directly used in the latter study, this pathogen still appears to be equally, if not more, sensitive to glutaraldehyde than Pseudom-

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onus and other microbial contaminants. Hence, if L. rnonocytogenes was present initially, this bacterium was likely to be eliminated during exposure to glutaraldehyde. As is true for steel and tile surfaces, conveyor lubricants also come in contact with various organic materials in food processing facilities. Hence, Rossmoore and Drenzek [324] examined behavior of L. rnonocytogenes in lubricants containing 25-50 ppm glutaraldehyde, 5- 10 ppm MCI, and 500- 1000 ppm parachlorometaxylenol in combination with 1% added milk and blood. In the presence of 1% milk, 50 ppm glutaraldehyde was most effective, with numbers of listeriae decreasing >5 logs following 3 h of exposure. However, in samples containing 1% blood rather than 1% milk, only 1000 ppm parachlorometaxylenol retained sufficient bactericidal activity to reduce Listeriu populations >5 logs within 24 h. Although parachlorometaxylenol exhibited similar activity in the presence of milk, addition of 5-10 ppm MCI was of little value in decreasing numbers of listeriae in lubricant containing 1% milk or blood. In addition to lubricants, several water-based cooling system fluids used in the dairy and meat industry also are subject to sporadic contamination with pathogenic microorganisms, including L. rnonocytogenes. Consequently, Rossmoore and Drenzek [324] also examined the potential benefit of adding low concentrations of glutaraldehyde, parachlorometaxylenol, and MCI to sweet water (i.e., potable refrigerated water containing a corrosion inhibitor) and an aqueous solution of 35% propylene glycol, both of which are commonly used in the cooling section of pasteurizers and other types of heat exchangers. According to this report, L. rnonocytogenes populations in inoculated samples of sweet water (pH 9.3) and 35% propylene glycol (pH 8.8) decreased only 1 and 3 logs, respectively following 14 days of storage at 3.5"C. In sharp contrast, addition of 25 ppm glutaraldehyde to sweet water and propylene glycol containing 1% milk completely inactivated L. rnonocytogenes populations of 105 CFU/mL in less than 1 h, as did addition of 100 ppm parachlorometaxylenol to propylene glycol. Inclusion of 100 ppm parachlorometaxylenol in sweet water and 10 ppm MCI in both coolants was at best only marginally effective, with the pathogen surviving at least 48 h in several instances. Thus, although a low concentration of glutaraldehyde will inactivate L. rnonocytogenes in sweet water and propylene glycol, use of parachlorometaxylenol for such a purpose should be limited to solutions of propylene glycol.

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Conventional Methods to Detect and Isolate Listeria monocytogenes CATHERINE W. DONNELLV University of Vermont, Burlington, Vermont

Die Methode ist alles German proverb (Raiovich [ 1221)

INTRODUCTION Listeria monocytogenes is a nonfastidious organism that can be subcultured on most common bacteriological media (i.e., Tryptose Agar, Nutrient Agar, and Blood Agar); however, attempted isolation or reisolation of Listeria from inoculated or naturally contaminated food and clinical specimens by use of nonselective media is often unsuccessful. Difficulties encountered in isolating L. monocytogenes date back to initial characterization of this pathogen in 1926 when Murray and his coworkers [ 1061 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. Direct plating, cold enrichment, selective enrichment, and several rapid methods all 225

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can be used in various combinations to detect L. monocytogenes in food, clinical and environmental samples. Early attempts to isolate small numbers of Listeria from samples containing large populations of indigenous microflora relied on direct plating and often ended in failure. In 1948, Gray et al. [64] introduced the cold enrichment procedure as an alternative method to isolate L. monocytogenes from highly contaminated samples. Although this method has contributed much to our 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 the development of various enrichment broths, as well as plating media and methods, used to isolate Listeria spp., including L. monocytogenes, from clinical, environmental, and food samples. Numerous enrichment broth and plating media formulations have been used during the past 50 years for selective cultivation of Listeria, the most important of which are detailed in Appendix I. Detection and isolation of Listeria remains complicated by the inability of researchers to identify a single procedure that is sufficiently sensitive to detect I;. monocytogenes in all types of foods within a reasonable time. Furthermore, many selective enrichment broths and plating media fail to allow repair and/or growth of sublethally injured Listeria frequently present in processed foods [26] or food processing environments. Despite these inherent shortcomings, research efforts in response to foodborne listeriosis outbreaks have led to development of numerous regulatory procedures, including the U.S. Food and Drug Administration (FDA) and the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) procedures [74,76] which 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 both healthy and sublethally injured Listeria in the wide range of foods currently being examined, these methods and others that are less widely accepted will undoubtedly undergo further modifications as selective enrichment broths and plating media used in these procedures continue to be improved.

COLD ENRICHMENT Difficulties in isolating L. monocytogenes typically arise when small numbers of Listeria are present in environmental and clinical food samples containing large numbers of indigenous microorganisms. Hence, numbers of Listeria must be increased, 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. [106], Biester and Schwarte 1131 observed that Listerella (Listeriu) 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, these authors 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. [64] reported that in three of five bovine listeriosis cases, L. monocytogenes was only isolated

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after brain tissue diluted in Tryptose Broth, was stored for 5-13 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 showed the ability of L. monocytogerzesto multiply to detectable levels in the presence of other microbial contaminants during extended storage at 4°C. Gray’\ cold enrichment method, in which samples hornogenized 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 1621, 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 from a variety of specimens, including food. In 13 studies summarized by Bojsen-Mgller 1171, Listeria was identified in 995 tissue and organ specimens from naturally and experimentally infected domestic animals. Using both direct plating and cold enrichment procedures, Lipteria 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. [ I3 I ] stressed the importance of colcl enrichment for recovery of L. monocytoqenes from cottage cheese manufactured from milk inoculated with this pathogen. Using direct plating, L. monocytogenes was recovered from 43 of 1 12 (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 Listeriu in 59 of 1 12 (52.7%) samples. Thus, cold enrichment was necessary to detect this pathogen in 16 of 1 12 ( 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 [ 1321, Camembert [ 1331, and brick cheese [ 1351 manufactured from pasteurized milk inoculated with the bacterium. 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 nonpsyc hrotrophic organisms, Gray and Killinger [62] indicated that, at times, growth of Listerig was too rapid to attribute enhanced growth of this pathogen to mere multiplication. When this procedure was first described in 1948, Gray et al. [64] suggested possible involvement of an inhibitory factor in bovine brain tissue that suppressed growth of competing organisms. However, this theory has been dispelled by subsequent studies which demonstrated enhanced growth of Listeria during cold enrichment of such diverse samples as mouse liver [ 1441, oat silage [61], feces [ 1171, sewage [46], cabbage [66], raw milk 11441, and cheese 1131-1351. A more plausible explanation is that in many clinical specimens, Listeria may exist within monocytes, rnacrophages, or other phagocytic cells, with colcl storage facilitating release of the intracellular organism. More recent research on the role of cold-shock proteins, cold-acclimating proteins, and other mechanisms which enable psychrotrophic growth of L. monocytogenes may help further explain the preferential growth of Listeria during cold enrichment [K,8I]. For instance, anteiso-C15 fatty acid reportedly plays a critical role in adaptation of L. monocytogenes to cold temperatures [4], with mutants deficient in this fatty acid being shown to be cold sensitive.

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As previously reviewed by Ryser and Marth [ 1361, over 20 media formulations have been successfully used to cold enrich a diverse group of samples that were either naturally or artificially contaminated with L. monocytogenes. Since incubation at 4°C is in itself partially selective for growth of L. monocytogenes, nonselective broths such as Tryptose Broth and Oxoid Nutrient Broth No. 2 (ONB2) rapidly emerged as media of choice, with Tryptose Broth generally recognized as being 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 2-12 months [139]. Following incubation, plates were examined under oblique lighting for typical bluish green, Listerialike colonies. Although growth of L. rnonocytogenes is favored at 4OC, other organisms, including Proteus, Hafiia, Pseudornonas, enterococci, and certain lactic acid bacteria, also can 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 inhibitory agents to various nonselective cold enrichment broths. In 1972, Bojsen-MQller [ 171 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. rnonocytogenes 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 [68]. Recovery of L. monocytogenes also is enhanced when cold enrichment is used as a secondary enrichment preceded by a selective primary enrichment at 30-37°C. Bannerman and Bille [7] subjected numerous cheese and cheese factory environmental samples to secondary cold enrichment in FDA Enrichment Broth (Listeria Enrichment Broth [LEB]) that were previously incubated at 30°C for 48 h (primary warm enrichment). 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 cold enrichment offers no advantages over selective enrichment [70]. In addition, the lengthy incubation period necessary for cold enrichment makes this procedure impractical for routine regulatory analysis of foods.

SELECTIVE ENRICHMENT AND PLATING AT 30-37°C The principle of enrichment at elevated temperatures (30-37°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. [63] isolated L. monocytogenes from contaminated material that was inoculated into Nutrient Broth containing 0.05% potassium tellurite and incubated at 37°C for 6-8 h before being plated on Tryptose Agar with or without 0.05% potassium tellurite. Even though subsequent studies showed both potassium telluritecontaining media to be partially inhibitory to Listeria [80,89,111,122], Gray and his colleagues can still be credited with introducing both the first cold-enrichment procedure and

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the first warm enrichment media for selective isolation of L. rnonocytogenes. 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 30-37°C. Mavrothalassitis [99] reported an optimum incubation temperature of 30°C for enrichment of L. rnonocytogenes from heavily contaminated samples. Results from at least two additional studies [33,109] also showed that laboratory cultures of L. rnonocytogenes, L. seeligeri, and/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 [ 1361 previously reviewed the wide range of media formulations that have been developed for selective enrichment of L. rnonocytogenes from environmental and clinical food specimens.

Selective Agents Modest, nonspecific nutritional requirements of L. rnonocytogenes have led to difficulties in formulating media that enhance growth of this pathogen. Consequently, efforts have primarily focused on inhibition of the indigenous bacterial fIora by taking advantage of the resistance of L. monocytogenes to various selective agents and antibiotics. The major advances that have contributed to our present-day ability to isolate Listeria from heavily contaminated environments are shown in Table 1. Although tnany inhibitory agents have proven to be at least somewhat useful for selective isolation of L. rnonocytogenes from

TABLE 1 Recognition of Selective Agents Useful in Isolation of Listeria Year

Compound

Role in selective media

References

1950

Potassium tellurite

20,63,80,83,89,100,111,122,145

1960

Lithium chloride/ phen ylethanol

1966

Nalidixic acid

1971

Acriflavin(e)/ trypaflavin(e)

Selective/differential for Listeria, which reduces tellurite to tellurium, producing black colonies Amplification of Listeria in the presence of gram-negative bacteria Inhibitory to gram-negative bacteria through interference with DNA gyrase Inhibitory to gram-positive cocci

1971

Polyniyxin B

1986

Moxalactam

1988

Ceftazidime

Prevents growth of gramnegative rods and streptococci Broad spectrum; inhibitory to many gram-positive and gram-negative contaminants, including Staphy Lococcus, Proteus, and Pseudomonas Broad-spectrum cephalosporin antibiotic

38,49,65,66,68,87,92,100,132, 133,144 1,16,42,45,60,77,80,112,113, 124,141

3,15,36,41,42,47,65,75,78,79, 1 12,113,122,123,125,126, 127 17,35,38,92,112,127,142 74,87,103,112

7,03,96,109

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naturally and artificially contaminated biological specimens, others have demonstrated very little value when added to basal media, as previously reviewed by Ryser and Marth [ 1361. Throughout the following discussion of selective agents, one must keep in mind that formulating media selective for L. monocytogenes is not a straightforward process, as many selective agents can partially inhibit growth of this pathogen, particularly when the organism is sublethally injured.

Potassium Tel Iu rite Many selective media, including the early formulation by Gray et al. [63], contain inhibitory substances that are now of questionable value. As previously described, in 1950, Gray et al. [63] 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. [ 1 1 I] observed that potassium tellurite prevented growth of numerous L. monocytogenes strains. Other investigators [80,83, 89,100,1221 have substantiated these findings and have discouraged the use of potassium tellurite as a selective agent. The advantage of adding potassium tellurite to selective media is that the resulting L. monocytogenes 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 [ 1391. A modification of Vogel Johnson agar (MVJA) was evaluated by Buchanan et al. [20] 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. [23] 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 both 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 [ 1451 reported that potassium tellurite prevented repair of heatinjured L. monocytogenes.

Lit hiu m C hIo ride/P he ny Iet ha no I Using the combination of phenylethanol and lithium chloride, McBride and Girard [ 1001 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 confirmed by other investigators, resulting in the earlier widespread use and acceptance of McBride Listeria Agar (MLA) as a plating medium for L. monocytogenes [38,49,65,66,68,87,92,100,132,133,144].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 [92,93]. Ryser and Marth [ 134,1351 and Yousef and Marth [ 1521 reported that increasing the lithium chloride concentration to 0.5% (0.05% lithium chloride in the original formulation [loo]) increased selectivity of the medium without appreciably decreasing recovery of healthy Listeria [ 134,135,1521.

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Nalidixic Acid Beerens and Tahon-Caste1 [ 111 were first to report the usefulness of nalidixic acid in isolating L. rnonocytogenes from heavily contaminated pathological specimens. Increased isolation of Listeria using media containing nalidixic acid primarily resulted from inhibition of indigenous gram-negative bacteria [60]. The benefits of adding nalidixic acid to otherwise noninhibitory media were soon confirmed in many laboratories [ 16,42,77,80, 1 12,113,1411. After discovering the benefits of adding nalidixic acid to enrichment broth [ 1 11, Ralovich et al. [ 1241 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. [45] 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 [60].

Trypaf lavine/Acriflavi ne Despite successful use of nalidixic acid, Ralovich et al. [125,126] 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 inhi bitor of gram-positive cocci, in media containing nalidixic acid. This medium soon became known as Trypaflavine Nalidixic Acid Serum ,4gar (TNSA). The end result was the selective inhibition of virtually all other bacteria, whereas growth of L. monocytogenes was only slightly decreased [ 15,1111. Following successful use of this medium in many European studies [ 15,78,112,113,125], Ralovich et al. [ 1221 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. rnonocytogenes with the naked eye. In 1972, Seeliger [140] reported that the combined use of acriflavine and nalidixic acid greatly suppressed gram-negative organisms and fecal streptococci without apparently affecting recovery of L. rnonocytogenes. These findings were subsequently confirmed by Bockemuhl et al. [ 161, 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 [3,42,47,65,79] led to widespread use of trypaflavinehalidixic acid as Listeriaselective agents. In 1974, Hofer [75] 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 [123]. According to Gregario et al. [65], use of nalidixic acid together with either 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 [36,41,78,123], a Serum Agaror Blood Agar-based medium containing trypaflavine, acriflavine, and nalidixic acid ap-

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peared to be satisfactory for selective isolation of L. monocytogenes from samples containing a mixed microbial flora. In 1984, Rodriguez et al. [ 1271 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. [ 124,1261. 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 [ 18,671, which is commercially available in Europe.

Potassium Th iocyanate In 1961, Fuzi and Pillis [55] proposed a medium containing 0.35% potassium thiocyanate for selective enrichment of L. monocytogenes. Although reported useful by some researchers [42,88,141], others found that potassium thiocyanate inhibited L. monocytogenes [83,89,125]. 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 [66] and milk [68,144] and other dairy products [85]. In 1972, Ralovich et al. [125] endorsed Levinthal’s Broth and Holman’s Medium, both of which contain nalidixic acid and trypaflavine, for selective enrichment of Listeria. Results obtained by Slade and Collins-Thompson [ 1441 demonstrated that growth of L. monocytogenes in ONB2 containing both nalidixic acid and potassium thiocyanate can be improved by adding acriflavine.

Thallous Acetate During the early 195Os, thallous acetate was employed as a selective agent for lactic acid bacteria; however, it was not until 1969 that Kramer and Jones [83] recommended the combined use of thallous acetate and nalidixic acid in Listeriu-selective media. Three years later, Khan et al. [80] 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 [89] demonstrated that the combined use of thallous acetate and nalidixic acid completely suppressed growth of E. coli strains that were previously resistant to nalidixic acid. Greater inhibition of grampositive bacteria also occurred when both selective agents were used together rather than separately. Although Leighton [891 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 the rough form. In view of this experience, most of the currently used formulations of Listeria-selective media omit thallous acetate.

Polymyxin B In 1971, Despierres [35] reported that the combination of polymyxin B and nalidixic acid was useful for recovering L. monocytogenes from feces, with these antibiotics preventing growth of many background organisms, including Enterococcus faecalis. That same year, Ortel [ 1 121 proposed another medium containing polymyxin B and bacitracin to isolate L. monocytogenes from stool samples. According to Bojsen-MQller [ 171, gram-negative rods and enterococci failed to grow in Tryptose Phosphate Broth containing polymyxin B, whereas growth of L. monocytogenes was relatively unaffected. After examining six different enrichment and isolation media, Rodriguez et al. [ 1271 concluded that little if

Methods to Detect and Isolate L. monocytogenes

233

any benefit was gained by adding polymyxin B to media already containing nalidixic acid and acriflavine. Doyle and Schoeni [38] 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 I1 developed by Rodriguez et al. [ 1271. Although the selective enrichment broth developed by Doyle and Schoeni gained some attention [92], the necessity for polymyxin B in this medium remains somewhat questionable. Siragusa and Johnson [ 1421 successfully isolated L. monocytogenes from yogurt using a medium containing polymyxin B, nalidkic acid, and acriflavine. Their medium reportedly prevented growth of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and thus making it particularly suitable for isolating L. monocytogenes from certain fermented dairy products.

M o x a lacta m Results from antibiotic susceptibility tests [ 1 121 led Lee and McClain [87] to add moxalactam (a broad-spectrum antibiotic which is inhibitory to many gram-positive and gramnegative bacteria, including Stuphylococcus, Proteus, and Pseudomonas) to MLA containing 0.25% phenylethanol and O S % lithium chloride. The result was a highly selective medium for recovery of L. monocytogenes from raw beef and many other foods. This medium, Lithium chloride-Phenylethanol-Moxalactam(LPM) Agar, is recommended by the USDA-FSIS for isolating L. monocytogenes from raw meat and poultry [ 1031 and also has been incorporated into the current FDA procedure as a second selective plating medium [74].

Ceftazidi m e Bannerman and Bille [7] 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 found to be superior to FDA-Modified McBride Listeria Agar (MMLA) [93,96], recovering approximately 50% more L. monocytogenes isolates frorn soft cheese and cheese manufacturing environments, than did FDA-MMLA. Except for ii few enterococci, the combination of acriflavine and ceftazidime inhibited all other non-Listeria organisms, including yeasts and molds. However, van Netten et al. [109] 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 both 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 ISOLATION AND ENRICHMENT OF LISTERIA Isolation Media

McBride Listeria Agar MLA was the first widely used plating medium for selective isolation of L. monocytogenes. This medium, introduced by McBride and Girard [ 1001 in 1960, is prepared from Pheny-

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lethanol 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 [9] dates back to 1959, nearly I year before the original formulation appeared in the literature [ 1001. 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, with the anhydride form reportedly being less inhibitory to L. rnonocytogenes than glycine [87]. In most instances, MLA2 was more Listeriaselective than Nalidixic Acid Agar [49,112], Acriflavine Nalidixic Acid Agar [ 1441, or Acridine Nalidixic Acid Agar [49]. The selectivity of MLA2 can be further improved, without affecting recovery of Listeria, by increasing the lithium chloride content to 0.5%. With the addition of sheep blood, this medium became partially differential and inhibitory to background microflora, and hence it was better suited than MLA2 for recovering L. monocytogenes from brick [ 1351, feta [ 1 151, and blue cheese [ 1 161, as well as cold-pack cheese food [ 1341. An earlier report in which glycine was found partially to inhibit L. monocytogenes [87] prompted many individuals to prepare the aforementioned forms of MLA with glycine anhydride, which is far less inhibitory to Listeria. Nevertheless, two widely used formulations of the original MLA containing glycine have been commercially available since 1985 from Difco Laboratories, Detroit, MI and Bethesda Biological Laboratories (BBL) Cockeysville, MD. Although addition of blood provides one means of identifying possible L. monocytogenes colonies (virtually all are at least somewhat P-hemolytic) and enhances growth of the pathogen in certain B vitamin- and/or amino acid-deficient media, many individuals prefer 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. [96] 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 FDAMMLA [93,94,96] and MLA2 [58,66,68,96,126,144], which contain glycine anhydride, were the MLA formulations of choice for isolating Listeria spp. from foods, particularly dairy, vegetable, and seafood products, with the FDA formulation serving for many years as one of two plating media (the other being LPM agar) in the widely used FDA procedure [95].

LPM Agar In 1986, Lee and McClain [87] added 4.5 g of lithium chloride and 20 mg of moxalactam to MLA2 and named their new medium Lithium chloride-Phenylethanol-MoxalactamAgar. Although this selective medium (commercially available in the United States from BBL and Difco Laboratories) 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 [27], which produces black L. monocytogenes colonies, each with a black halo following 24 h of incubation.

Oxford/MOX Agar In 1989, Curtis et al. [34] developed an agar medium that eliminated the need for oblique illumination. Their medium, Oxford Agar, was prepared from Columbia Agar base to which a number of selective agents, including colistin sulfate (20 mg/L), fosfomycin (10

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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, with this new medium being designated Modified Oxford Agar (MOX) [27]. In May of 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 FDA-MMLA with Oxford Agar (OXA). In the present version of the FDA method [74], two selective media must now be used, either both PALCAM and OXA or OXA and LPM (or LPM plus esculin and Fe3+).These changes have decreased reliance on the sometimes tedious Henry illumination technique and have brought U.S. regulatory procedures into closer compliance with international regulatory protocols.

PALCAM Agar In 1988, van Netten et al. [ 1081 reported that RAPAMY Agar, a modification of TNSA developed by Ralovich et al. [ 1261 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 surface-plated on RAPAMY and nonselective agar, with growth of all non- Listeria organisms tested, except Enterococcus fizecalis and Enterococcus faecium, being completely inhibited on the selective medium. Like OXA [34], RAPAMY Agar also produced distinctive black Listeria colonies that were surrounded by a dense black halo from esculin hydrolysis. Although such characteristic colonies were present 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 having total aerobic plate counts of 5 1O6CFU/gand enterococcus counts of 5 1 02CFU/g. However, as might be expected, high populations of enterococci severely hampered detectioin of Listeria spp. in chicken, minced meat, and mold-ripened cheese. Further attempts by van Netten et al. [107] 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 Staph~ZOCOCCUS 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 [ 1091, which contains polymyxin B and lithium chloride along with half or less the concentration of acriflavine and ceftazidime found in AC A.gar. It is recommended that PALCAM Agar plates be incubated for 48 h at 30°C under microaerobic conditions (5%

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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 isolation.

Other Selective Plating Media Interest in foodborne listeriosis during the 1980s led to development of many additional Listeria-selective media for examining milk and dairy products. In 1984, Martin et al. [98] developed Gum Base Nalidixic Acid Medium (GBNA)-a synthetic agar-free solid medium superior to the MMLA of Bearns and Girard [9] for isolating L. monocytogenes from raw milk [68]. Bailey et al. [5] 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 [66] based on the enrichment broth of Doyle and Schoeni [38], from which acriflavine was omitted and Fe'' was added, compared favorably with the original formulation of MLA [ 1001. Supplementation of selective [66] and nonselective [30] 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. As indicated previously, attempts to isolate L. monocytogenes from food products have focused on enhancing the selectivity of currently available blood-free plating media which are normally viewed under oblique illumination, as well as development of alternative media that incorporate differential agents other than blood to aid microbiologists in identifying Listeria colonies in mixed cultures. In 1987, Buchanan et al. [20] 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 (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 [9] 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. In a subsequent study, Buchanan et al. [22] found that MVJ was slightly superior to ARSMMLA 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 new media functioned well. In a follow-up study, Buchanan et al. [21] 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 LEB I in a three-tube/24-h most probable number (MPN) method, comparable isolation rates were obtained for both MVJ and LPM Agar. The differential capability of MVJ was again extremely useful in selecting presumptive Listeria colonies.

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 colo-

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n

Mirror

FIGURE1 Oblique illumination technique developed by Henry [73].Angles of reflected light (p) and transillumination (a)equal 45" and 135", respectively. nies are observed under oblique illumination with a binocular scanning microscope. Using the Henry technique [73] in which plates are examined under obliquely transmitted white light at an angle of 45" (Fig. I), Listeria colonies are small, round, finely textured, bluish green to bluish gray with an entire margin. In 1984, Martin et al. [98] compared the appearance of L. rnonocytogenes 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 [73]. Noting that the angle of transmission in the Henry method is 135O, Lachica [84] 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 X magnification hand lens while colonies were directly illuminated with a high-intensity beam of light that traveled perpendicular to the bench surface (Fig. 2). This View

W!Jso 5x Hand Lens

FIGURE2

Modified Henry technique developed by Lachica [841. Angle of transillumination (a)equals 135".

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latter method has eliminated many of the problems (i.e., reproducibility and convenience) associated with the classical technique developed by Henry [73] nearly 60 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 are not readily adaptable for routine use in large testing laboratories.

p-Hemo Iysis Addition of blood to solid media also can be used to differentiate Listeria, including L. rnonocytogenes, from other microorganisms. When grown on media containing blood, such as MLA, L. rnonocytogenes colonies are typically surrounded by a narrow zone of P-hemolysis. In some instances, P-hemolytic activity is so weak that the clearing zone cannot be observed until the colony is gently removed from the agar surface. In 1989, Blanco [ 141 proposed overlaying previously inoculated plates of blood-free Listeria selective agar with a thin layer of blood agar so that the P-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.

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. Golden et al. [57], Hao et al. [66], and Cassiday et al. [28] collectively compared 20 selective plating media for their ability to recover uninjured cells of L. rnonocytogenes 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 106L. monocytogenes CFU/g or mL. 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. [57] 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 14 plating media used in this study failed to recover L. rnonocytogenes 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 102and 104 L. rnonocytogenes 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 111 (MRIM III), MLA2, FDA-MMLA, and MDA were judged to be satisfactory when Brie cheese contained 1 1 0 6Listeria CFU/g. However, subsequent results from the same laboratory [29] indicate that LPM Agar was superior to these four media for isolating Listeria rnonocytogenes 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

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inoculum levels, L. rnonocytogenes was readily quantitated by direct plating on MDA, GBNTSM, and MLA2. However, this same investigative team [29] later obtained even better results using LPM Agar. One year earlier, Hao et al. [66] successfully recovered L. rnonocytogenes 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 citra.te, 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 [56,72,85,90] in which LPM Agar outperformed other popular plating media, including FDA-MMLA, RIM 111, and/or MVJ for recovery of L. rnonocytogenes from raw milk, ice cream, yogurt, soft cheese, and/or vegetables inoculated with the pathogen. In addition, Rodriguez et al. [ 1281 found that. Rodriguez Isolation Medium (RIM) 111 containing 6 rather than 12 g of acriflavine hydrochloride was superior to the original formulation of MLA for isolating L. rnonocytogenes 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 [7 1,74,76,94]. 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. [28] evaluated 10 selective plating media for their ability to enumerate L. rnonocytogenes in artificially contaminated dryand country-cured ham as well as raw oysters. According to their results, MDA, FDAMMLA, and LPM Agar recovered approximately equal numbers of uninjured Listeria from dry-cured ham. However, ease in differentiating L. rnonocytogenes 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 111, MVJ, and University of Vermont Agar (UVM)] that were deemed acceptable for isolating Listeria from country-cured ham. Unlike both types of ham, high populations of indigenous microflora in rilw oysters greatly complicated detection of Listeria on virtually all 10 plating media. Although MRIM I11 and MVJ supported less growth of Listeria than other marginally acceptable plating media, including MLA2, FIIA-MMLA, and GBNTSM, MRIM I11 and MVJ were somewhat more reliable for differentiating L. rnonocytogenes from background contaminants. Therefore, these authors hesitantly recommended MRIM I11 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. [90], recognition of L. rnonocytogenes in inoculated samples of raw ground beef and scallops was only possible using LPM Agar. Among the three other plating media tested, RIM I11 and the original formulation of MLA proved to be insufficiently selective, whereas MVJ was inhibitory to the L. rnonocytogenes strain tested. Unlike these findings, Garayzabel and Genigeorgis [56] indicated that LPM Agar and RIM 111 were acceptable for detecting Listeria in raw meat with both media superior to FDA-

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MMLA. Bailey et al. [5] found that LPM Agar and GBNA fortified with lithium chloride and moxalactam were both 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 30-37°C. Plates containing popular selective media such as LPM Agar or MOX Agar normally are incubated for 48 h, whereas plates containing pure or near-pure cultures of Listeria on nonselective media can generally be examined after 24 h. Since growth of L. monocytogenes is reportedly enhanced under conditions of reduced oxygen [ 1391, inoculated plates [38,107,108,131-133,1351 as well as selective enrichment broths [38] have been incubated under microaerobic conditions (5% 02:10% CO2:85% N2). These latter conditions are recommended when using PALCAM Agar.

Enrichment Media Several food-related listeriosis outbreaks during the 1980s emphasized the need for more sensitive Listeria detection methods. 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 one or two selective enrichments. An outbreak of listeriosis which was epidemiologically linked to consumption of pasteurized milk [53] led Hayes et al. [68] to develop a two-stage enrichment procedure for isolating L. rnonocytogenes 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 either Stuart Transport Medium or selective enrichment broth containing 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 most useful for identifying positive raw milk samples. Slade and Collins-Thompson [ 1441 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 ( 1 6 ) during cold enrichment as a result of fewer lactic acid bacteria and less lactose being present, which 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, since 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 7-14 days of cold enrichment in Tryptose Broth, L. monocytogenes was

Methods to Detect and Isolate L. monocytogenes

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most frequently isolated after plating samples enriched in Thiocyanate Nalidixic Acid Broth on either MLA+blood or Tryptose Agar. A “shortened” enrichment procedure and a two-stage cold/selective enrichment procedure were developed in Canada by Farber et al. [44] 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+blood after 1 day of incubation ai: 30°C and surface plating the 30-day-old cold enriched FDA Enrichment Broth culture (initially incubated 7 days at 30°C) on MLA2+blood. Collectively, these steps detected Listeria spp. in 31 of 51 (60.8%) positive raw milk samples. Although I I isolation:; were made after 1 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+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. [44], also detected Listeria spp. in raw milk samples. Using this procedure, Listeria spp. were isolated from 12 samples that were negative using the shortened enrichment procedure. Similarly, 10 samples that were 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 [38], who also observed that Listeria spp. could be more readily isolated from raw milk and soft, surface-ripened cheese [39] during the first 2 weeks of cold enrichment. Food-associated outbreaks of listeriosis along with the discovery of L. monocytogenes in many European varieties of soft- and smear-ripened cheese prompted two Swiss investigators, Bannerman and Bille [7], 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 procedure just described [44] 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 1099 (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 surface-plating 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. [68] and Slade and Collins-Thompson [ 1441; however, the need for cold enrichment in these procedures increased the length of analysis to 30-40 days. Hence, although cold enrichment will likely remain an important research tool, the time

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constraints of this method negate its use in any isolation procedure that is to be adopted by the food industry as a “standard” method. Rodriguez et al. [129] developed a complicated scheme to isolate Listeria from raw milk which more importantly paved the way for subsequent development of several widely used enrichment media, including UVM Enrichment Broth [27,37]. Their protocol included three noninhibitory collection (primary enrichment) media, three selective (secondary) enrichment media, and one selective plating medium, RIM 111, all of which were previously described by Rodriguez et al. [ 1271. The three selective enrichment media used in this protocol contained nalidixic acid and trypan blue with or without polymyxin B, whereas 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 I11 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 11. A portion of Collection Medium A also was diluted in Collection Medium C, which was streaked on to RIM I11 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, since 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 colony 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. Since transfer of the culture after 24 h of cold enrichment provides little opportunity for appreciable growth of L. monocytogenes, 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 [93,96] and USDA [102,103] for enrichment of food samples containing L. monocytogenes were modifications of media proposed by Ralovich et al. [126] and Rodriguez et al. [127] as modified by Donnelly and Baigent (University of Vermont Medium) [37], respectively. Donnelly and Baigent 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 [127]. This medium, designated LEB, by Donnelly and Baigent [37], 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 phosphate-2-hydrate ( 12.0 g/L), potassium phosphate monobasic (1.35 g/L), esculin (1.O g/L), nalidixic acid (40 mg/L), and acriflavine HCl (12 mg/L). McClain and Lee [ 1021 modified this formula to contain 20 mg/L nalidixic acid, and this

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formulation was known as USDA LEB I. These authors further modified LEB I to contain 25 mg/L acriflavine and used this medium, LEB 11, for secondary enrichment of meat and poultry samples. USDA-FSIS currently recommends use of UVM Broth (LEB I) for primary enrichment of meat and poultry samples [27,76].

Fraser Broth Fraser Broth [54] is a modification of USDA LEB I1 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 samples. Since Listeria will turn Fraser Broth black from esculin hydrolysis within 48 h of incubation [ 191, this broth has now replaced USDA LE,B I1 in the USDA protocol as the preferred secondary enrichment medium for meat and poultry samples [76]. In 1986, Doyle and Schoeni [38] used the microaeraphilic 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% 0,:10% CO,: 85% N,, 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 using DSSEB rather than cold enrichment and direct plating, respectively. Another enrichment procedure, which is partially based on microaerobic incubation, was developed by Skovgaard and Morgen [143] 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% CO,) of the sample in USIIA LEB I is followed by aerobic secondary selective enrichment in USDA LEB 11, 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. [66] 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 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, both with and without an

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additional 5 mg of Fe3+/L,led to the recommendation of Modified Doyle/Schoeni Selective Agar I1 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. Although these two plating media have gained widespread acceptance, their efficacy in isolating L. monocytogenes from cabbage and other vegetables should 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 [74], UVM Broth [76], and Fraser Broth [76], all of which are commercially available from BBL or Difco Laboratories. Truscott and McNab [ 1471 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, L-PALCAMY Broth, which was developed by van Netten et al. [ 1091, has shown superior results to USDA LEBs I and I1 as well as the Tryptose Broth-based antibiotic medium of Beckers et al. [ 101 for detecting L. monocytogenes in naturally contaminated cheese, minced meat, fermented sausage, raw chicken, and mushrooms. However, given wide variations in both the type and number of naturally occurring microbial contaminants in our food supply, development of a single enrichment broth for truly optimal recovery of Listeria from all types of food appears unlikely.

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 and poultry products, respectively. 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 injured cells. In response to these concerns, the USDA-FSIS and FDA protocols have been modified to enhance recovery of injured Listeria. Working in cooperation with the International Dairy Federation (IDF), other official European agencies have developed somewhat similar protocols which are 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.

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FDA Method The FDA method, originally developed by Lovett et al. “33,961, is the most frequently used procedure in the United States for detecting L. monocytogenes in milk, milk products (particularly ice cream and cheese), seafood, vegetables, and food processing environments. The original protocol [93] has been modified as shown in Figure 3 [74]. The enrichment medium (LEB M52 [74]) consists of TSBYE supplemented with monopotassium phosphate (anhydrous) 1.35 g/L; disodium phosphate (anhydrous) 9.6 g/L; and pyruvic acid (sodium salt 10% w/v aqueous solution) 11.1 ml/L. A 25-mL liquid or 25-g solid sample is added to 225 mL of LEB without selective agents, mixed, and incubated at 30°C for 4 h. Following addition of selective agents (acriflavine HCl 10 mg/L; nalidixic acid sodium salt 40 mg/L; cycloheximide 50 mg/L), the sample is incubated an additional 44 h at 30°C for a total incubation period of 48 h. LEB was modified by increasing its buffering capacity, thereby positioning this medium to be used in conjunction with DNA probe and other rapid methods which are less sensitive than conventional cultural methods. In a further modification for nondairy foods, the acriflavine concentration was reduced from 15 to 10 mg/L so as to conform to that used for milk; and dairy products. After 24 and 48 h, LEB cultures are streaked onto OXA [34] and LPM [87] Agar prepared either with or without esculin/Fe3+.PALCAM [ 1091 agar may be used in place of LPM agar. This I

1

Add 25g or 25 ml sample to 225 ml LEB

I

1

Stomach or blend 6 Incubate 4h at 30°C

Add selective agents acriflavine, nalidixic acid and cycloheximide A

II 20h

U 44h

30°C

Streak to F

a

n

d

30°C

Streak to

I

LPM

I

35°C

1

24,48h

U 30°C 24,4811

I

II 35°C

24,48h

L Examine for Listeria-like colonies

FIGURE3 FDA procedure for isolating

I_.

monocytogenesfrom foods. (From Ref. 74.)

246

Donnelly

substitution brings the FDA method in closer alliance with other protocols used outside the United States and decreases reliance on Henry’s oblique illumination technique. OXA and PALCAM plates are incubated (with optional use of a C02-air atmosphere) at 35°C for 24-48 h, with LPM plates being incubated at 30°C for 24-48 h. LPM plates can be viewed using Henry illumination, or alternatively esculin and ferric iron salt may be added to LPM to obtain Listeria colonies with black halos as also appear on OXA and PALCAM. It is recommended that five or more typical colonies be picked from OXA and PALCAM or LPM and transferred to TSAYE for confirmation. The selection of five colonies increases the likelihood that multiple species of Listeria, if present, will be identified. TSAYE plates are incubated at 30°C for 24-48 h or 35°C if colonies are not being used for wet mount motility confirmation. Purified isolates are subjected to a series of standard biochemical tests, with a total of 10-1 1 days being required to isolate and confirm the presence of Listeria in food samples via the FDA procedure. Present versions of the FDA procedure have greatly shortened and simplified the isolation of Listeria spp. from many foods as compared with earlier methods that were developed to detect the pathogen in clinical specimens, with revised procedures affording many improvements over the original FDA protocol. In 1987, Doyle and Schoeni [39] compared the original FDA classic cold enrichment and shortened enrichment procedures for their ability to recover L. rnonocytogenes from 90 samples of commercially produced, soft, surface-ripened cheese that was previously identified as likely to contain L. rnonocytogenes. Although L. rnonocytogenes 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. rnonocytogenes from 5 of 21 (23.8%) and 8 of 21 (38.1%) samples that were positive following cold enrichment. Furthermore, since 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. [40] when these same three enrichment procedures were used to isolate L. rnonocytogenes from raw milk samples after HTST pasteurization. Researchers in Canada [45] and England [ 1181 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 [39,40] found cold enrichment superior to the FDA method for analysis of soft, surface-ripened cheese, where 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 the 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. rnonocytogenes.

International Dairy Federation Method Using the original FDA method as a starting point, the IDF initiated development of a “reference” method in 1988 [146] to recover L. rnonocytogenes from dairy products.

Methods to Detect and lsolate L. monocytogenes

24 7

Development of the IDF method essentially followed that of the FDA protocol as previously reviewed by Ryser and Marth [ 1361 with the eventual elimination of both preenrichment for detecting sublethally injured Lister-ier and the KOH treatment of the enrichment broths before plating on Listc.r-icr-selective media. The present IDF method [4a] received AOAC approval in I993 based on results from an AOAC collaborative study [ 1474 which assessed the ability of this method to recover L. nzorzoc‘~toSeizL’.sfrom inoculated samples of raw milk. ice cream, Camembert cheese. Limburger cheese, and skim milk powder. The AOAC-approved IDF method (Fig. 4) closely resembles the FDA protocol (see Fig. 3) with the sample enriched i n IDF enrichment broth which contains the same concentrations of selective agents found in LEB. 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 either LPM without esculin/Fe” or PALCAM. This method which requires a minimum of 4 days to obtain presumptive results continues to be popular among Europeans for detecting Lister-icr in dairy products.

USDA-FSIS Method The USDA-FSIS devised a method for detecting L. i,zonoc~itoserie.sin meat and poultry products (Fig. 5 ) 1761. The original USDA protocol developed in 1986 by Lee and McClain [87,103] differs from both the original and revised FDA procedures in that both primary and secondary enrichment steps are included for detecting Listrr-ici. The original USDA procedure enabled Lister-iir detection within 3 days compared with 9- 1 1 or 5-6 days using the original and revised FDA methods, respectively. The original USDA procedure was revised in May of 1989 [27] and differs from the original method in that (a) LEB I1 has been replaced by Fraser Broth [ 541 as the secondary enrichment medium; (b) LPM Agar has been replaced by MOX; and ( c ) the regulatory sample size has been increased to 25 g. Fraser Broth and Modified Oxford Agar will both blacken during incubation, because Lister-io spp. and other contaminants can hydrolyze esculin, with colonies of Listerici exhibiting black halos on Modified Oxford Agar following 24-48 h of incubation. However.

FIGURE4

IDF procedure for isolating L. rnonocytogenes from milk and dairy products. (From Ref. 4a.)

Donnelly

248 Add 25g Meat sample to 225 ml UVM Broth stomach 2 min.

U

Incubate at 30°C for 20, 2411

1

0 1 ml + I0 ml Fraser Broth

U 26 f 2h 35°C

I

Streak to MOX

U 35°C

U 48h

24, 4811

35°C

Examine for black colonies

-+

ifnegative

+

Streak to MOX

U 35°C 24, 48h Examine for black colonies

FIGURE5

USDA procedure for isolating L. monocytogenes from meat and poultry products. (From Ref. 76.)

MOX is more selective than LPM or Oxford Agar [34], with staphylococci and streptococci both generally unable to grow on MOX. Reported inadequacies in the prior [27] USDA procedure were related to the 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 [6,82]. Kornacki et al. 1821 compared recovery of L. monocytogenes from Fraser broth incubated for 26 versus 48 h. L. monocytogenes was isolated from 60 of 1088 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 hours, 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 therefore recommended several modifications. All Fraser broth enrichment cultures should be streaked following 24-26 h of incubation regardless of color. Once cultures have been streaked to MOX, Fraser broth cultures should be reincubated at 35°C for an additional 24 h. MOX plates streaked from 24- to 26-h Fraser broth enrichment cultures should be examined for the presence of Listeria-like colonies. If present, isolation should proceed. If absent, a second MOX plate should be streaked from the 48-hr Fraser broth enrichment culture. Ferron and Michard [48] compared the FDA and

Methods to Detect and Isolate L. monocytogenes

249

USDA enrichment procedures using 300 pastry samples supplied by 100 different suppliers in western France. The USDA procedure was deemed superior, detecting 69% of all positive samples compared with the FDA procedure which detected only 34%.

Netherlands Government Food Inspection Service Using the Netherlands Government Food Inspection Service (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 plated 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) [ 1091, after which presumptive Listeria colonies are black and surrounded by a dense black hole from txulin hydrolysis. Lund et al. [97] examined 300 raw milk samples for the presence of Listeria using three primary enrichment media. A total of 84 positive sarnples 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 Listeriapositive 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. [ 1 101 evaluated the impact of more than one test procedure on recovery of Listeria species from naturally contaminated seafood and seafood products. A total of 21 1 samples were evaluated using five different protocols. The FDA procedure [95] 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 which were subsequently found to harbor Lzsteria via other procedures. The overall incidence of Listeria increased I 1.7% using more than one testing procedure. Hayes et al. [69] assessed the USDA-FSIS and cold enrichment procedures for recovery of 1,. 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. [70] during an examination of foods obtained from the refrigerators of patients with active clinical cases of listeriosis. A total of 2229 food samples were examined in this study, of which 11% were positive for L. monocytogenes. Overall, the USDA-FSIS [27], FDA [95], and NGFIS [ 1091 methods were not statistically different in their ability to isolate Listeria from 899 samples included in the comparative evaluation. The FDA procedure [95] 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 protoco'ls)to 87-9 1% for combined protocols.

CONSIDERATIONS FOR RECOVERY OF INJURED L/ST€R/A Most conventional and rapid detection procedures for Listeria use highly selective enrichment media to facilitate growth over competitive background flora. However, these highly

250

Donnelly

selective enrichment 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 of food processing facilities. Sublethal injury of Listeria as a result of heating, freezing, drying, irradiation, or exposure to chemicals (i.e., sanitizers, preservatives, acids) is well documented [ 1,12,24,25,26,28,31,33,56,58,59,101,130,132,138].Under ideal conditions, such injury is reversible with Listeria being capable of repairing sublethal damage in foods. Repair of heat-injured L. monocytogenes has been reported in whole and 2% milk stored at 4°C [105]. 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. All current detection procedures, with the exception of cold enrichment, involve selective enrichment and/or selective plating. Cold enrichment is not feasible for routine testing, since several months of incubation may be necessary to obtain positive results. By failing to consider recovery of injured Listeria, current methodologies 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 both thermally stressed and nonstressed Listeria [31,86,145,148,149,151].Furthermore, when examined for ability to recover quantitatively thermally stressed Listeria, LEB agar, modified McBride's Agar (MMA), LPM Agar [87], and FDA enrichment broth agar showed significantly impaired recovery [221. Warburton et al. [ 1501 examined the ability of the modified FDA and USDA methods to recover stressed cells and low levels of L. monocytogenes in food and environmental samples. Although the modified FDA and USDA 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 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 [26] developed an enrichment medium capable of resuscitating heat-injured Listeria. This medium, Listeria Repair Broth (LRB), permits complete repair of injured Listeria within 5 h at 37°C after which various selective agents can be added to inhibit the growth of competing microflora upon 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 [95],phosphate-buffered Listeria Enrichment Broth (PEB; Gene-Trak Systems, Framingham, MA), or UVM Enrichment Broth [ 1031. Final Listeria populations in selective enrichment media after 24 h of incubation at 30°C were 1.7 X 108to 9.1 X 10' CFU/ mL compared with populations in LRB which consistently averaged 2.5 X loll to 8.2 X 10'' CFU/mL [26]. Studies with LRB were extended to examine the potential for repair of freeze-injured and sanitizer-injured L. monocytogenes [50,138]. Although variation in susceptibility of L. monocytogenes to freeze injury was recorded, in general, L. monocytogenes is not severely injured by freezing [43,58,114]. Percentage of injury ranged from only 40 to 60% after Listeria populations were frozen at -9" to - 11"C for 24 h [50].As storage time increased, an increase in percentage of injury increased to a maximum of only 70-80%. To examine reversibility of freeze injury, low-level populations of freeze-injured L. monocytogenes

Methods to Detect and lsolafe L. monocytogenes

251

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, with the pathogen again attaining high populations in LRB. Sallam and Donnelly [ 1381 examined the ability of four commonly used dairy plant sanitizers tc) 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. [52] 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 which 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, NC) assay. Of 80 sites 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 97.5-98.8% [52]. Flanders et al. [51] also evaluate 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 to 75.3% to 82.2-94.4%. The combination of LRBC and UVM detected 94.4% of positive samples, whereas LRBS and LRBC identified 91.1% of positive samples. Pritchard et al. [121] 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 medium, 56 were from LRB, and 57 came from LRBC. A total of 26 samples (32.5% of positive samples) were identified by either LRB or LRBC but not by UVM media. Combining UVM with either LRB or LRBC again substantially increased the number of positive samples identified. When results from UVM and LRB are combined, 65 to 80 (8 1.3%) positive samples were identified. Using both UVM and LRBC, 74 of 80 (92.5%) positive samples were identified. Despite the improved recoveries obtained by combining medja, these results illustrate the severe limitations associated with the current regulatory procedures used to assure absence of Listeria in foods and food processing environments. Ryser et al. [ 1371 evaluated the ability of UVM and LRB to recover different strainspecific ribotypes of L. monocytugenes 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 the above 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

Donnelly

252

(including 4 known clinical ribotypes), 12 L. innocua, and 8 L. welshirneri ribotypes were identified from selected positive samples (15 samples of each product type, 2 UVM and 2 LRB isolates/sample). Twenty-six of 36 (13 L. monocytogenes) ribotypes were detected using both UVM and LRB; 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 (1 1 L. monocytogenes) different Listeria ribotypes, respectively, with some Listeria ribotypes being confined to a particular product. More importantly, striking differences in both 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 (both clinical and nonclinical ribotypes) in two and four samples, respectively (Table 2). These findings stress the complex microbial ecology of Listeria in foods and the limitations of existing detection procedures fully to characterize the total Listeria population. Furthermore, two of the L. monocytogenes ribotypes missed using UVM were known clinical ribotypes which were linked to sporadic and epidemic cases of human listeriosis in England and Scotland [ 1041. Continuing work [ 1201 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 < .OS) percentage of Listeria-positive samples than when either LRB or UVM are used

TABLE 2 Ribotypes of Listeria spp. Recovered from 10 Samples of Raw Chicken Following Primary Enrichment in UVM or LRB and Secondary Enrichment in Fraser Broth

No. of isolates Listeria spp.

Ribotype 1-909-3 5-418-3 5-415-4 5-4 13-2 2-864-3 1-916-la 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

L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L.

innocua monocytogenes innocua monocytogenes welshimeri monocytogenes rnonocytogenes innocua innocua innocua monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogeries

UVM

LRB

0 2 4 2 2 3

1 0 0 0 0 3 0

2 5

0 0 0 0 0 0 0 0

Recognized clinical ribotypes associated with known epidemic or sporadic cases of human listeriosis. Source: Adapted from Ref. 137.

5

1 1 3 2 1 1 1

I

Methods to Detect and lsolate L. monocytogenes

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alone. These findings, combined with reports of L. innocm being able to outgrow L. monocytogenes in UVM (and Fraser Broth) [32,119] suggest that different ribotypes of L. monocytogenes may vary somewhat in nutritional requirements or their ability to compete with other ribotypes of L. monocytogenes and/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. Roth and Donnelly [ 1301 assessed survival of acid-injured L. rnonocytogenes in four different acidic foods and also examined the efficacy of LRB and UVM to recover acidinjured Lisreria from such foods. L. monocytogenes was injured in lactic (pH 3.0) and acetic (pH 3.5) acids. Two levels of injury were produced anld monitored; one population with 99.999b 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 non-fat 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, sainples were enriched in both LRB and UVM followed by standard isolation/ identification procedures with survival of healthy L. monncytogenes 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 in that 4°C storage was bacteriostatic and 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 Listeriu compared with UVM (3/54). Hence, use of LRB is recommended when examining acidic foods for L. monocytogenes.

REFERENCES 1. Ahamad, N., and E.H. Marth. 1990. Acid injury in Listeria monocytogenes. J. Food Prot. 53126-29. 2. Albritlon, W.L., G.L. Williams, W.E. DeWitt, and J.C. Feeley. 1980. Listeria monocytogenes. In: E.H. Lennette, A. Balows, W.J. Hausler, Jr., and J.1’. Truant, eds. Manual of Clinical Microbiology. 3d ed. American Society for Microbiology, Washington, DC, pp. 139142. 3. Al-Ghazali, M.R., and S.K. Al-Azawi. 1988. Effects of sewage treatment on the removal of Listeriu 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-C,, fatty acid in the growth of Listeria monocytogenes at low temperatures. Appl. Environ. Microbiol. 63:3887-3894. 4a. Association of Official Analytical Chemists. 1996. 17.10.0 1 .AOAC official method 993.12. Listeriu monocytogenes in milk and dairy products. In: Official Methods of Analysis of the Association of Official Analytical Chemists, AOAC International, Gaithersburg, MD. 5. Bailey. J.S., D.L. Fletcher, and N.A. Cox. 1989. Recovery and serotype distribution of Listeriu monocytogenes from broiler chickens in the southeastern lJnited States. J. Food Prot. 52: 148- 150. 6. Bailey. J.S., and N.A. Cox. 1992. Universal preenrichment broth for the simultaneous detection of Salmonellu and Listeria in foods. J. Food Prot. 55:256-259. 7. Bannerman, E.S., and J. Bille. 1988. A selective medium for isolating Listeria spp. from heavily contaminated material. Appl. Environ. Microbiol. 54: 165- 167. 8. Bayles, D.O., B.A. Annous, and B.J. Wilkinson. 1996. Cold stress proteins induced in

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134. Ryser, E.T., and E.H. Marth. 1988. Survival of Listeria monocytogenes in cold-pack cheese food during refrigerated storage. J. Food Prot. 5 1 :6 15-62 1,625. 135. 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. 136. Ryser, E.T., and E.H. Marth. 1991. Listeria, Listeriosis and Food Safety. New York: Marcel Dekker. 137. 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. 138. Sallam, S. and C.W. Donnelly. 1992. Destruction, injury and repair of Listeria species exposed to sanitizing compounds. J. Food Prot. 55:77 1-776. 139. Seeliger, H.P.R. 1961. Listeriosis. New York: Hafner. 140. Seeliger, H.P.R. 1972. Reviews-A new outlook on the epidemiology and epizoology of listeriosis. Acta Microbiol. Hung. 19:273-286. 141. Seeliger, H.P.R., F. Sander, and J. Bockemuhl. 1970. Zum kulturellen Nachweis von Listeria monocytogenes. Z. Med. Mikrobiol. Immunol. 155:352-368. 142. 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: 147160. 143. Skovgaard, N., and C.-A. Morgen. 1988. Detection of Listeria spp. in faeces from animals, in feeds, and in raw foods of animal origin. Int. J. Food Microbiol. 6:229-242. 144. 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. 145. Smith, J.L. and D.L. Archer. 1988. Heat-induced injury in L. monocytogenes. J. Indust. Microbiol. 3:lOS-110. 146. Terplan, G. 1988. Provisional IDF-Recommended Method: Milk and Milk Products-Detection of Listeria monocytogenes. Brussels. International Dairy Federation. 147. 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. 5 1 :626-628,638. 147a. 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. 148. 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:lOS-118. 149. 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 monoc-ytogenes. J. Food Prot. 54:669-676. 150. 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. 151. Werner, B.S., and D.V. Lim. 1990. Growth of Listeria monocytogenes in different media. Abstr. Ann. Mtg, Amer. Soc. Microbiol., Anaheim, CA. May 13-19, Abstr. P-41. 152. Yousef, A.E., and E.H. Marth. 1988. Behavior of Listeria monocytogenes during manufacture and storage of Colby cheese. J. Food Prot. 5 1 :12- 15.

Rapid Methods for Detection of Listeria CARLA.BATT Cornell University, Ithaca, N e w York

INTRODUCTION Presence of Listeria monocytogenes in food products is a safety problem that warrants attention and improvements in detection and tracking. Although normal, healthy adults are primarily unaffected by this pathogen, infants and immunocompromised persons are at far greater risk [38]. The traditional techniques developed :sincethe 1980s for detecting and enumerating L. monocytogenes are not sufficiently rapid to assure the safety of perishable food products before consumption. Regulations limiting contamination of ready-toeat foods to a “zero-tolerance” have been the driving force behind development of rapid tests, prompting an intense effort in both commercial and academic laboratories. These techniques are only useful as a survey tool and as a method to track an alleged foodborne outbreak. The need to develop quicker and more precise methods for detecting Listeria is also a function of the similarity between L. monocytogenes and other members of the Listeria genus. Distribution of a ready-to-eat food containing L. monocytogenes typically leads to a class I recall. This chapter is an attempt to review objectively most of the literature on the subject published to date with emphasis on experiences from my laboratory. My group has explored many different formats to detect L. monocytogenes, and over the years, several different rapid methods have been assessed. We have focused on L. monocytogenes because of its significance to humans (1 400 cases occurred per year during the late 1980s [38]) and its usefulness in models for development of rapid methods.

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Microbiology-Based Methods Classic microbiology-based methods for detecting and enumerating Listeria involve enrichment in selective media, which may include incubation at refrigeration temperatures [31,49]. Selective enrichment media which allow only Listeria to grow have also been developed. The wide range of Listeria-selective plating media currently available is daunting. Even though several comparative studies have been reported, no single detection scheme appears to be so vastly superior as to be adopted universally [ 141. Recovery of injured Listeria cells has emerged as another important issue. Sublethal thermal processing in addition to other intrinsic and/or extrinsic factors can injure Listeria. Although injury is not a new phenomenon, the potential significance of injured Listeria in foods deserves greater consideration in the formulation of enrichment/recovery media.

RAPID METHODS FOR DETECTION OF L. MONOCYTOGENES Antibody- (monoclonal or polyclonal) and nucleic acid probe-based systems, the latter alone or in conjunction with amplification, have been developed to detect both L. monocytogenes and Listeria spp. Determining whether to use nucleic acid probes or antibodies to detect pathogenic microorganisms is partly a matter of personal preference, with factors such as simplicity, cost, speed, and sensitivity also being of importance. Amplificationbased methods (most notably polymerase chain reaction, PCR) have superior sensitivity as compared with standard nucleic acid probes or immunoassays. However, PCR is somewhat more complicated in terms of setup and operation (Fig. 1). Only recently have reagent additions in PCR been simplified and the process made more amenable to routine use as seen by the introduction of the BAX system by Qualicon (Wilmington, DE). When coupled to more direct measures of PCR product accumulation [3], these advances will likely result in a system suitable for routine testing.

Nucleic Acid-Based Probes Since 1987, nucleic acid probes have become a viable tool to detect viruses, bacteria, and other microorganisms in food, clinical, and environmental samples. Target sequences that can be used include (a) ribosomal RNA, (b) mitochondrial DNA, (c) plasmid DNA, and (d) chromosomal DNA. The key criterion for selection of any target nucleic acid is that its presence defines the organism in question with little or no probability of existing in another microorganism that might be found in the same ecological niche. There is no way of ensuring that a targeted nucleic acid sequence will be found only in the microorganism for which the detection system is being developed. This is especially true where the sequence is cryptic and chosen simply because of its uniqueness within a selected test population. In cases where a specific toxin gene sequence is selected, there is an assumption that it will not be widely distributed in nature. The use of 16s rRNA as a distinct signature for a bacterium has become a universal method when no other obvious nucleic acid sequence uniquely defines the desired target [80]. Databases of 16s rRNA sequences covering a wide diversity of microorganisms can be searched to identify regions that are characteristic of the targeted microorganism. A DNA probe based on the sequence for 16s rRNA which can detect all Listeria spp. [49,50]

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FIGURE1 Polymerase chain reaction. has been developed by Gene Trak Inc. (Framingham, MA). Although the exact sequence is proprietary, it is clearly derived from one of the variable regions of the 16s rRNA. A novel solution hybridization assay has been formatted where final quantification is accomplished using an enzymatic marker [48]. Briefly, 16s rRNA is released by alkaline lysis from cells grown in an enrichment broth. Then a capture tag consisting of the complementary sequence to a unique region of 16s rRNA and a poly-A (polyadenylic acid) tail is allowed to hybridize to the target 16s rRNA. This hybrid is then removed from solution through the poly A tail using a poly-T (polythymidylic acid) s,equencethat has been immobilized on a polystyrene solid support (Fig. 2). Detection is accomplished using an antibody coupled to horseradish peroxidase and directed against a fluorescein marker covalently linked to the detector probe. The detector probe recognizes sequences in 16s rRNA as spatially distinct from the region recognized by the capturleprobe. Therefore, oxidation of a substrate (tetramethyl benzidine) in the presence of hydrogen-peroxide by horseradish peroxidase indicates the presence of Listeria. A more recent refinement of this approach uses a 16s rRNA probe that is specific for L. monocytogems [58a]. Unique 16s rRNA sequences that define L. monocytogenes have been reported [2 11, but achieving specificity in the assay requires precise temperature control. Virulence genes are frequent targets for nucleic acid--based probe methods, since these genes are essential for pathogenicity and are typically conserved among a given species. A probe derived from a putative delayed-type hypersensitivity (DTH) factor isolated from L. monocytogenes 1/2a hybridized to all L. monocytogenes serogroups and L. ivanovii but not to any other Listeria spp. tested [64]. The exact nature of the DTH gene

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FIGURE2 Sandwich hybridization capture assay. has not yet been reported, and therefore its role in L. rnonocytogenes pathogenicity cannot be determined. It does, however, appear to be an effective tool for detecting Listeria, although its species specificity is not absolute for L. rnonocytogenes. For example, the DTH gene appears to be absent from L. rnonocytogenes serogroup 4a yet present in L. ivanovii. Thus far, it has only been used as a nucleic acid probe in colony hybridization assays and the entire 1.1-kb DTH, which contains a fragment labeled with 32P,served as the probe. A sequence from what was first believed to be a putative L. rnonocytogenes a-hemolysin gene [33] was reportedly specific for L. rnonocytogenes [22,24]. However, subsequent analysis showed that this gene encoded for a major secreted protein (msp) rather than a hemolysin [34]. Despite its nebulous quality, this sequence has proven to be useful in developing nucleic acid-based detection systems for Listeria. Initially, a colony hybridization protocol was used where suspect colonies were transferred to nitrocellulose filters and probed with this 32P-labeledfragment. Good specificity was shown toward Listeria spp. which were P-hemolytic (CAMP-positive). Subsequent refinements of this approach have included the evaluation of four synthetic 20-bp oligonucleotide probes in lieu of the entire 500-bp fragment. Two probes which were tested against a range of Listeria spp. hybridized to all L. monocytogenes isolates and one weakly hemolytic isolate of L. seeligeri [24]. The origin of this probe has been clarified by the reported cloning and sequencing of an invasion-associated protein (iap) [52]. Pathogenicity of L. rnonocytogenes depends on a number of factors, including the production of one or more hemolysins. Transposon mutagenesis (Tn916) disrupts the coding sequence for listeriolysin 0 and renders L. monocytogenes avirulent to mice. The gene coding for listeriolysin 0 has been cloned [23,54,61] and sequenced [61]. Interestingly,

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when the listeriolysin 0 gene is introduced into Bacillus subtilis, the organism gains the ability to grow in macrophage-like cells in culture [8]. The listeriolysin 0 gene is presumably unique to L. monocytogenes and therefore is an obvious target for developing a detection system. It does, however, share some amino acid homology with other hemolysins, including streptolysin 0 and pneumolysin. The listeriolysin 0 gene has been used as a probe in Southern hybridization analysis of DNA purified from several Listeria spp. [ 191. A 610-bp fragment internal to the region coding for listeriolysin 0 appears to hybridize only with hemolytic strains of L. monocytogenes. However, under nonstringent conditions, a probe derived from sequences on the 3’ of the listeriolysin 0 gene hybridized with hemolytic strains of L,. ivanovii and L. seeligeri. Although some nucleotide sequence conservation between the hemolysin genes in Listeria apparently exists, a detailed sequence analysis will be required to determine the exact extent of homology. Datta et al. [23] used two synthetic oligonucleotide listeriolysin 0 probes in a colony hybridization assay to detect L. monocytogenes and obtained good specificity [24]. Such listeriolysin probes can likely be adopted to several assay formats for analyzing food samples.

Nucleic Acid Amplification-Based Methods The sensitivity of a nucleic acid-based detection system is a function of several parameters, including the number of copies of the target within a single cell. The use of 16s rRNA has the obvious advantage in that each cell contains over 100 copies which in turn makes such an assay far more sensitive than an assay based on a single copy target. As an alternative to using high-copy number target sequences, nucleic acid-based amplification methods employing the ligase chain reaction (LCR) [78,79], PCR [4,9,15, 32,35,36,39,63,66,72], and most recently nucleic acid sequence-based amplification (NASBA) [ 12,741 have been reported.

Ligase Chain Reaction LCR is an amplification method that uses target DNA as a template for ligation of oligonucleotides designed to abut one another. By using a pair of diametrically opposed complementary oligonucleotides, each ligated pair can serve as a template for subsequent rounds of amplification (Fig. 3). LCR can be used to discriminate between two target sequences that differ only in a single nucleotide because of the extreme sensitivity of DNA ligases to mismatches on the 5’ end of the substrate. The strength of LCR therefore lies in its specificity as compared with PCR, which is more sensitive. Temperature cycling allows products from one round to dissociate from their target and then anneal and serve as a template in a subsequent round. Key to the process is the use of a thermostable DNA ligase which retains activity after being exposed to temperatures sufficient to dissociate the products [2]. The 16s rRNA which is sufficiently diverse for phylogenetic determinations also can serve as a target for LCR-based assays. In studies documenting the utility of LCR as a means to detect L. monocytogenes, 32P-labeledoligonucleotides were used and the ligated products were detected by autoradiography after electrophoretic separation of the substrates and products [79]. Subsequent improvements included use of nonradioactive labels and a capture step which obviated the need for electrophoresis [78]. Enhanced sensitivity was achieved by introducing a preliminary PCR amplification which utilized a common set of 16s-rRNA primers. This approach is a generic model for developing a PCR-LCR assay for virtually any microorganism.

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FIGURE3

Ligase chain reaction.

Polymerase Chain Reaction PCR involves the enzymatic amplification of a targeted nucleic acid sequence using a thermostable DNA polymerase and flanking oligonucleotide primers that uniquely define the target. The most commonly used DNA polymerase is from Thermus aquaticus and is termed Taq polymerase. Since amplification is exponential, the target can be amplified over one million times with respect to other sequences within the cell through cycles of denaturing, annealing, and extending. The power of PCR prompted its obvious application for detection of L. monocytogenes. A wide variety of PCR-based assays have been developed for L. monocytogenes which target several different genetic sequences. These sequences are largely derived from virulence genes which are unique to L. monocytogenes and essential for the organisms pathogenicity. These virulence factors, all of which were previously described in Chap. 5 , include (a) listeriolysin (&A), a gene encoding a thiol-dependent hemolysin which is involved in escape from intracellular vacuoles [ 1,3,13,15,36,46], (b) invasion-associated protein (icy)[ 16,44,56], (c) phospholipase B @lcB) [20], and (d) DTH [76]. In addition to virulence genes, any other nucleic acid sequences which are unique to L. monocytogenes can serve as targets for PCR-based assays. Several genes sequences sufficiently divergent to differentiate species including the ribosomal RNA operon and its

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intergenic regions are likely to be highly conserved among all L. monocytogenes but PCRbased assays have been employing 16s rRNA 14 I ,44,75,77], the intergenic spacer region that lies between the I6 and 23 rRNA. (In some instances, the assay was diagnostic only in the size and restriction pattern of the PCR products providing not definitive identification [ 26,401.) Finally, cryptic sequences have been discovered which are unique to L. morzocytogenes and use repetitive element sequence-based PCR (rep-PCR) [4S] and subtractive hybridization [ S I ] . Distribution and conservation of these cryptic sequences within all L. rnono~yt0geize.sstrains cannot, however, be intuitively deduced and must be proven by large-scale screening studies. Formats for PCR-based assays are varied and differ in their complexity as well as utility. The most comtnon “read-out” for PCR-based assays is gel electrophoresis accompanied by ethidium bromide staining, with the presence of a particular PCR product being diagnostic. The disadvantages of gel electrophoresis are the lack of quantification and the difficulty in automating post-PCR processing. Alternative means of detecting PCR products posthybridization include (a) reverse dot-blots. (A labeled PCR product is captured by an oligonucleotide primer immobilized on a membrane [ IS].), (b) microtiter plate capture (the labeled PCR product is captured specifically or nonspecifically in the well of a microtiter plate [ 1 S]), (c) macroporous hydrophobic cloth [ 1 I 1, (d) immunodetection of RNA:DNA hybrid [ 101, (e) fiberoptic biosensors [73]. A 5’ nuclease PCR detection assay was first developed and perfected using L. monocytogenes a\ the target organism (Fig. 4). As a nucleic acid target, listerolysin 0 (hlyA) was chosen iis the nucleic acid target, because this sequence is unique to L. nzorzocytogenes. We have previously used this gene as a target for a reverse dot-blot PCR assay [IS]. Although this assay was extremely sensitive, the post-PCR handling steps, including product capture and secondary enzyme-conjugate addition, introduced potential problems in assay throughput and contamination. The latter is of particular concern, since PCR product contamination through aerosols frequently leads to false-positive results. Initial work in my laboratory has reliably demonstrated the ability of the 5’ nuclease PCR detection assay to quantify L. monocytogenes in pure culture [3]. The specificity of the PCR primers and reactions and the parameters that were used in this assay have been documented and were supported by our data [ IS). Among all Listerici spp., significant ARQs and amplification products, the latter observed on ethidium bromide-stained agarose gels, were only obtained for L. monocytogerzes. Furthermore, addition of competing organisms did not affect the assay until the ratio of competing to target organisms exceeded 10”.

The 5’ nuclease PCR detection assay using the hlyA fluorogenic probe was linear over a range of 5 X 10” to S X 10’ L. monocyfogerzes CFU with SO CFU [3] easily detected. The “yes” or “no” assignment is an accurate scoring method which can be used for positive and negatijre samples. Non-L. monocytogrnes strains can give a weak positive signal only when >S X 105copies of the template are present. Even then, the signal generated is >30 times weaker than the signal obtained from an equivalent number of L. r?zorzoc:~togene.s templates. This assay is now being used as a format to develop methods for detecting L. r,zonocytogsnL.s in dairy, feed, and clinical samples.

Nucleic Acid Sequence-Based Amplification Nucleic acid sequence-based amplification (NASBA or 3SR [29]) is a system where nucleic acid targets are amplified using a series of enzymes, including a RNA polymerase

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and a reverse transcriptase (Fig. 5). A target RNA molecule is first reverse transcribed to cDNA using reverse transcriptase. RNAase H is added to digest the template RNA which occurs only after hybridization with cDNA. The newly formed cDNA is then used as a template for a second round of synthesis again using reverse transcriptase. The primer for this second round carries a T7 promoter as a tail on its 5’ end and therefore introduces this promoter sequence into the second round of synthesized cDNA. At this stage, the T7 promoter containing cDNA is a substrate for RNA synthesis by T7 RNA polymerase. Copious amounts of T7 RNA polymerase-synthesized RNA are then produced. This increase in RNA can be detected easily by gel electrophoresis or sandwich hybridization, since amplification is typically on the order of 106fold, Since NASBA uses RNA templates, it is amenable to detection of L. monocytogenes with 16s rRNA. Probes specific for L. monocytogenes have been developed [74]. NASBA assays have used hZyA mRNA as the target with sensitivities as low as 10 CFU/g being reported [12]. In this latter study, enrichment was used to induce hZyA, a problem in mRNA based methods where the initial level per cell cannot be predicted.

Problems in Amplification Methods Two major problems with PCR-based assays (and in general all methods that employ enzymatic amplification) are false negatives caused by PCR inhibition and false positives resulting from detection of nonviable cells. The former has been addressed by development of several template purification methods which range in complexity and utility.

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Target cell capture. In general, the most noted example of cell capture involves use of immunomagnetic beads to which target-specific antibodies are attached [70]. The beads are used to capture cells from solution and then the recovered cells are subjected to DNA extraction or culture enrichrnent [35]. L. rnonocytogenes also has been recovered after centrifugation and washing to remove inhibitory compounds in milk [20] and other foods [63]. Detergent or solvent extraction. Phenol, chloroform, and ether are examples of solvents that can remove compounds that inhibit PCR [43]. Sodium iodide will generally solubilize food components and make the isolation of amplifiable DNA possible [%I. Detergents including Tween 20 also can enhance the sensitivity of PCR by solubilizing inhibitory compounds [68]. A two-phase solvent extraction using polyethylene glycol and dextran is reportedly effective for soft cheeses [%I. Filtrution. For liquid foods, most notably milk, filtration is a simple means of concentrating cells [20,72]. Certain filters are amenable to solvent solubilization which aids in DNA release. DNA capture. In addition to cell capture, target DN,4 can be captured after cell lysis. DNA can be absorbed onto several matrices in a nonspecific manner; that is, silica [43]. PCR cocktail. Few of the PCR inhibitors are known in specific terms. For L. monocytogenes and its detection in milk, calcium is thought to be a PCR inhibitor. Conse-

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quently, increasing the amount magnesium in PCR is useful [7]. Addition of bovine serum albumin or proteinase inhibitors might help spare the DNA polymerase during amplification [65]. Viability has been a frequently cited but still unresolved problem. Amplification of archival DNA from various sources documents the ability of DNA to “survive” well beyond the life of the organism [57].Therefore, false positives often arise from samples whose processing history ensures that all L. monocytogenes are nonviable. Two approaches addressing viability of target cells in PCR-based assays have been proposed. The first is to have a mandatory culture enrichment period, where a positive result would require growth of the target organism. The second approach involves targeting of mRNA rather than DNA, since mRNA is less stable than DNA and should degrade in a manner that parallels cell death. Efforts to use mRNA as a template for detecting viable L. monocytogenes have been reported [42]. The utility of this approach may be limited because of strain differences in target gene expression which will alter the number of mRNA molecules per cell. Second is the difference in the history of the contaminating L. monocytogenes strain in the food before and after processing. Since mRNA destruction is a kinetic process, thermal processing and the time between processing and assay will be critical. Our efforts to pursue mRNA as a target in a single-step PCR 5’ nuclease assay have used the hlyA gene as a target [ 3 2 ] . The thermostable DNA polymerase Tth has both reverse transcriptase and DNA polymerase activity. It can be used in a single buffer reaction that contains a temperature-sensitive chelator which controls the availability of manganese. Manganese is critical for Tth switching from reverse transcriptase to DNA polymerase activity [62]. A correlation between viability (as determined by plate counts or staining with a fluorogenic esterase substrate) and the ARQ of the assay was observed. Selection of PCR primers that hybridized to the most distal portions of the hlyA gene gave a more accurate result in monitoring viability as compared with PCR primers that amplified an internal region. A second means to ensure that only viable L. monocytogenes cells will be amplified is to have a requisite enrichment period before the PCR assay. Although this might seem to be the antithesis of “rapid” methodologies, the enrichment need only be a few hours and total assay times of less than 8 h are still reasonable. We have used membrane filtration to concentrate cells from liquid foods, including raw milk [30]. Hot detergent facilitates filtration after which the collected cells, still on the membrane filter, are placed onto a nutrient-soaked absorbent pad. The cells are enriched for less than 4 h, processed using a chelating reagent, and then boiled. The total assay time is less than 8 h and sensitivities of
Antibody-Based Detection Systems Use of immunological assays for detecting bacteria is by no means new. For example, the classic methodology for Salmonella involves a series of enrichment and selective plating media followed by a fluorescent polyclonal antibody assay for final confirmation of the organism. The limitations of polyclonal antisera are obvious, with bacterial cross reactions hindering their use for primary identification of a genus. They have been used, however, for serological analysis of isolates, thus proving their effectiveness in establishing the epidemiological relationship between suspected outbreaks of foodborne illnesses. One of the earliest demonstrations of immunological detection of Listeria was reported by Eveland [28], who detected L. monocytogenes in spinal fluid from a patient with meningitis

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using rabbit polyclonal antibodies raised against heat-treated L. rnonocytogenes (serotypes 1, 2, 3, 3b, 4a, and 4b) conjugated directly to fluorescein isothiocyanate. The future for the use of immunological reagents as a rapid method for detecting microorganisms improved dramatically with the advent of hybridoma technology [5 11. Monoclonal antibodies are produced by hybridomas, which are the result of fusing an antibody-secreting splenocyte to a plasmacytoma cell. These hybridomas can then be grown in culture and the antibody subsequently harvested from the medium. Alternatively, hybridoma cells can be injected into an appropriately primed mouse and allowed to establish a tumor. This ascites tumor is a highly productive source of monoclonal antibodies which are secreted into the interstitial fluid. One of the first monoclonal antibodies against Listeria antibodies was raised by immunization with semipurified flagella extracts of Listeria spp. [30]. This antibody reacted with all Listeria spp. except L. gruyi, L. rnurrayi, and L. denitrijkans and did not react with other gram-positive bacteria, including Staphylococcus aureus and Streptococcus faecalis. An assay using these antibodies has been formatted where the bacteria are spotted onto a nitrocellulose filter and then detected with the monoclonal antibody in conjunctiori with a secondary peroxidase-coupled antibody. A monoclonal antibody has been characterized which reacts with a heat-stable antigen from Listeria [ 17,581. The antibody was raised by immunizing mice with a heattreated L. rnonocytogenes lysate and subsequently fusing the splenocytes to a mouse myeloma fusion partner. Hybridomas were screened by a direct binding assay using a heat-treated lysate from an L. rnonocytogenes culture. Although the exact nature of the antigen is unknown, it has been commercialized by Organon-Teknika (Durham, NC) and is available. It requires (as do all rapid methods developed to date) a preenrichment step, after which the culture is collected and heated to produce an extract. Detection of this Listeria antigen is accomplished using an ELISA format with two different monoclonal antibodies which first capture and then detect the trapped antigen. The two monoclonal antibodies recognize different epitopes on the antigen, thereby avoiding competition. The monoclonal antibody used for detection is directly conjugated to horseradish peroxidase, and tetramethylbenzidine is used as the chromogenic substrate. The total time involved in the actual assay is approximately 2 h. The heat stability of this antigen (which varies in size frorn 30 to 38 kD) is potentially problematic, because samples that are heavily contaminated with Listeria are then thermally processed. In this situation, a false-positive reaction is possible, although given the current need for enrichment, only viable Listeria will be detected. Some efforts to document the utility of this assay have been reported [ 5 ] . Hybridomas which produced monoclonal antibodies ag,ainst L. rnonocytogenes were isolated by immunizing mice with both live and heat-killed L. rnonocytogenes [82]. Only a limited number of Listeria strains were tested using a radioimmunoassay and shown not to cross react with some of the hybridomas isolated. McLauchlin et al. [59] characterized two monoclonal antibodies, CL 17, which recognized L. rnonocytogenes serotypes 1/ 2 , and 3; and CL2, which reacted with serotypes 4b, 4 (not 4b), and L. innocua serotype 6a. These monoclonal antibodies could be used to detect L. monocytogenes in soft cheese by direct fluorescence microscopy, although some samples which were known to contain L. rnonocytogenes (based on standard microbiological tests) were negative by this analysis [601. As mentioned previously, selective detection of L. rnonocytogenes is of great importance, and for this reason, we have isolated a monoclonal antibody which will specifically recognize I,. rnonocytogenes [ 371. We have characterized several murein monoclonals

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produced by fusing spleen cells isolated from BALB/c mice immunized with live L. monocytogenes to NS-1 plasmacytoma cells. The immunogen consisted of live L. monocytogenes Scott A cells that were injected directly into the spleen of the mice [71]. Live cells (as opposed to heat- or formalin-killed cells) and direct injection were chosen to provide the most direct presentation of an unaltered (or minimally processed) antigen to the spleen. Hybridomas were screened by direct ELISA assay, and of the 150 hybridomas tested, three reacted most strongly with L. monocytogenes Scott A [27]. Although monoclonal antibodies Mab 20-10-2, Mab 36-6-12, and Mab 59-9-16 reacted to some extent with L. innocua and L. ivanovii in the direct binding assay, greater specificity for L. monocytogenes was seen in an indirect ELISA assay (Fig. 6). These antibodies were used to trap L. monocytogenes, which was then detected by a rabbit anti-L. monocytogenes polyclonal antiserum. Siragusa and Johnson [69] also attempted to isolate a monoclonal antibody specific for L. monocytogenes. These antibodies resulted from immunizations with heat-treated L. monocytogenes as previously reported [ 171. Both immuno-dot-blot of heat-treated whole cells and Western analysis of sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separated cell extracts were used to demonstrate reactivity. Unfortunately, their monoclonal reacted not only with L. monocytogenes but also with L. welshimeri, L. innocua, and possibly others. Later efforts by this group targeted another antigen that produced monoclonal antibodies which preferentially reacted with L. monocytogenes [6]. Further specificity in terms of serotype-specific monoclonal antibodies which detect L. monocytogenes 4b has also been reported [47].

1 0.9

0.8

0.7 0.6 U)

g

0

0

0.5

0.4

0.3 0.2

0.I 0 L rnonocytogenes

L. ivanovii

L innouca

L seeligeri

S. faecalis

Organism

FIGURE6 Indirect ELISA using MAB20-10-2 to trap antigen and rabbit polyclonal anti-L. rnonocytogenes serum for detection. Bound antibodies were detected using goat antirabbit alkaline phosphatase conjugate and p-nitrophenol phosphate. 2 x 106cells/mL; 5 x 105cells/mL. (From Ref. 37.)

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Immu noassay Formats Unlike nucleic acid probe-based assays, most immunoassays utilize a relatively standard ELISA format. Cells are either directly absorbed or immunocaptured onto wells of a microtiter plate. The captured cells are then detected using an antibody which carries either a hapten (i.e., biotin) or an enzyme reporter, with the latter being chromogenic, fluorescent, or chemiluminescent either directly or through the use of substrates. Beyond ELISA formats, other assays employ flow cytometry-which can detect irnmunocaptured cells in solution rather than having to immobilize them on a solid support [25].Antibodies conjugated to magnetic beads also have been used not directly for detection but for capture, followed by standard microbiological plating [70].

Commercial Test Systems Many companies have entered the market with rapid methods to detect either Listeria or more specifically L. monocytogenes. Most of these assays are extensions of formats used to detect other microorganisms (or their toxins), with their components altered specifically to detect Listeria. Assays based on nucleic acid hybridization or antibody-antigen interactions are available as well as one that employs nucleic acid amplification. All of these assays require some prior enrichment to increase selectively the target population to detectable levels. At best, these assays can be completed within 24 h, although this is dependent on the food source and intended level of sensitivity. Barring approval of the test method by an appropriate regulatory agency, positive samples must be confirmed using standard microbiological culture methods. Therefore, these rapid methods are most often used as quick screening tools to examine large numbers of samples. One ELISA for Listeria has been developed by Organon-Teknika. This ELISA is formatted for a 96-well microtiter plate and the readout is colormetric. To obviate the liquid handling normally associated with microtiter plates, Tecra has developed and incorporated an antibody-coated dipstick into its unique system. After initial cell capture, an interim culture replication step helps increase cell numbers. The readout is colormetric. BioMerieux (St. Louis, MO) also has developed a immuno test strip for the Vidas instrument that employs a solid phase receptacle and a fluorescent readout. In general, most immunological assays for Listeria have not been successfully altered to specifically detect L. monocytogenes. Despite apparent successes in developing antibodies specific for L. monocytogenes (this author’s work. inclusive), incorporation of these antibodies into commercial assay formats has not yet followed. One exception is the development of an immunomagnetic bead-colony immunoassay by Vicam (Watertown, MA) which is rather complex and is claimed by the manufacturer to be specific for L. monocytogenes. Cells from the sample are collected on immunomagnetic beads which are subsequently cultured on an agar plate. Suspect colonies are than screened using a filter immunoassay. A nucleic acid hybridization assay which uses a sandwich capture format is available from Gene Trak. Two assays have been developed, one which detects all species of the genus Listeria and the other which is specific for L. monocytogenes. Both assays target 16s rRNA and have a colorimetric readout. In each assay, a capture probe binds to a region of the 16s rRNA while a second probe binds to a spatially separated region of the 16s-rRNA molecule. The complex is then removed from solution using a poly-A tail which hybridizes to a poly-T-coated solid support. Finally, a PCR-based amplification method for detecting L. monocytogenes has been

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developed by Qualicon (Wilmington, DE; a subsidiary of DuPont). Although the nature of the amplicon is proprietary, it is reportedly specific for L. monocytogenes. All assay components are contained in a single tablet which is added to the sample after processing. This assay requires gel electrophoresis to confirm the presence of the appropriate amplification product.

FUTURE DEVELOPMENTS The long-term goals in development of any rapid method dictate that the test be fast, simple, sensitive, accurate, and, for commercial purposes, inexpensive. However, at least some of these desired performance attributes are mutually exclusive: for example, as an assay is made more sensitive, the accuracy, as it is defined by the number of false positives, increases. Most attention concerning monoclonal antibodies or nucleic acid probes for Listeria identification (or in fact other microorganisms) is focused on the reporter molecules and associated detection instrumentation. Advances in chemiluminescent-based reporters which have sensitivities in excess of 100-fold greater than existing enzymaticbased systems will be applicable to Listeria detection. All of the rapid assays developed to date (July 1997) require prior enrichment, with this step taking up to 48 h. Therefore, any claims that an assay can be completed within, for example, 4 h, are not entirely truthful. Continued efforts to further improve media formulations for recovery of Listeria from foods should prove beneficial as a prelude to any rapid detection method. Another area of concern is the significance of injured Listeria cells in a given food product and their potential for recovery either during enrichment or in the food during long-term storage. As mentioned previously, antibodies or nucleic acid probes can, in theory, detect both injured and dead cells. If future rapid assays are developed to detect microorganisms in food without any prior enrichment, the significance of injured populations will need to be addressed. At issue is whether detection systems specific for L. monocytogenes are advantageous over genus detection of all Listeria. The most obvious argument for an L. monocytogenes-specific test is based on the fact that virtually all cases of human listeriosis are caused by L. monocytogenes. In an ideal world, the goal would be to create a rapid test which detects Listeria spp., which are pathogenic in humans. Until we have elucidated the factors mediating pathogenicity of L. monocytogenes, such a goal is not feasible.

ACKNOWLEDGMENTS The support of the Northeast Dairy Foods Research Center is greatly appreciated. The author thanks Mary Lou Tortorello and Jerrie Gavalchin for their assistance. The author also thanks Liz Borod for her help in the preparation of this manuscript.

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Subtyping Listeria monocytogenes LEWISM. GRAVES, BALASWAMINATHAN, AND SUSANB. HUNTER Centers for Disease Control and Prevention, Atlanta, Georgia

Most bacterial species have sufficient phenotypic and genotypic diversity to allow for identification of different subtypes. Therefore, phenotyping and genotyping systems used singly or in combination often provide useful subtyping schemes for pathogenic bacteria. The various subtyping systems reviewed in this chapter provide different degrees of discrimination among Listeria monocytogenes isolates. By using these systems in epidemiological studies to distinguish individual strains or groups of strains, it has been possible to obtain information on relationships between isolates, identify disease outbreaks, identify the source of infections in outbreaks and sporadic disease settings, and determine modes of transmission for the organism. We present a broad overview of the typing methods that have been applied to L. monocytogenes and, where appropriate, discuss briefly the strengths and weaknesses of each. In this review of the usefulness of the most commonly used subtyping methods for L. monocytogenes, we have separated them into two major categories: conventional methods (i.e., serotyping, phage typing) and molecular methods (i.e., multilocus enzyme elec-

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.

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trophoresis, DNA restriction analysis). At present, each of these methods has some utility; however, with the extraordinary developments in nucleic acid technology and the wide availability of these technologies, some conventional methods may cease to be used in the future. L. monocytogenes is among the first pathogenic bacteria for which a concerted and internationally coordinated attempt has been made to evaluate critically various available subtyping methods and to standardize the more useful methods. In 1996, Bille and Rocourt organized the World Health Organization (WHO) Multicentre Listeria monocytogenes Subtyping Study. The result from Phase I of this study were published in a special issue of the International Journal of Food Microbiology [25] and will be referenced throughout this chapter.

CONVENTIONAL METHODS Serotyping Serotyping has been a classic tool for epidemiological and sporadic case studies of L. monocytogenes [27,38]. Strains of L. monocytogenes differ in the 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 serological typing (serotyping). Strains of Listeria species are divided into serotypes based on somatic (0)and flagellar (H) antigens [71]. Flagellar antigens as well as 0 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 0 antisera (one with antibodies to factor I and the other with antibodies to both factors I and 11) and three H antisera (one with antibodies to factors A and B, one reacting with C, and one reacting with D). With antisera for 0 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 [80]. Serotype 4bX is a variant of serotype 4b and was implicated in an outbreak in the United Kingdom that was traced to contaminated piit6 [47]. 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 epidemiological investigations. In the WHO Multicentre L. monocytogenes Subtyping Study, Schonberg et al. [69] found that all 80 strains tested by serotyping were typeable. However, for only 49 (61.3%) strains was there complete agreement between the six participating laboratories on the serotype (21 of serotype 1/2a and 28 of serotype 4b). Intralaboratory reproducibility, assessed on 11 duplicate strains, ranged from 82 to loo%, 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. Schonberg et al. [69] concluded that a critical need exists for good-quality antisera prepared from standardized strains. Also, they emphasized the need to absorb these antisera completely and efficiently to produce good-quality factor sera. Serotyping has poor discriminating power when compared with other subtyping methods. Isolates from foods and the environment are frequently nontypeable with standard typing antisera. Nevertheless, serotyping provides valuable information for rapid

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screening of groups of strains isolated during suspected outbreaks. Serotype information allows elimination of isolates that are not part of an outbreak and facilitates efficient application of other more sensitive but time-consuming subtyping methods.

Bacteriophage Typing (Phage Typing) Numerous lytic bacteriophages have been identified for Listeria spp. [42], L. monocytogenes isolates can be characterized by their patterns of resistance or susceptibility to a standard set of phages, as demonstrated by Rocourt et al. [63]. Until the recent advent of molecular subtyping, phage typing was often used in conjunction with serotyping for epidemiological investigations because of its high discriminating power [4,45,63,68]. The WHO Multicentre L. monocytogenes Subtyping Study on p’hage typing was done using an international phage set in five laboratories and unique phage sets in two laboratories [44]. With the international phage set, 20-5 1% of the isolates were nontypeable. Nontypeability with the international phage set was a greater problem among strains of serogroups 1/2 and 3 (%-to 72%) than for serogroup 4 (11-22%). 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% using criteria for interpretation previously proposed for the international phage set [46]. Based on the aforementioned findings, McLauchlin et al. [44] recommended that the phages in the international set be reviewed and additions be considered to increase typeability of strains. Lemaitre et al. [41] proposed a method that facilitates detection of induced phages; this procedure may be useful for identifying additional typing phages. McLauchliri et al. [44] suggested that better interlaboratory reproducibility may be achieved by standardization of phage suspensions, propagation strains, and methodology. Use of centrally propagated phages, as is done by 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 of the need to maintain stocks of biologically active phages and control strains. Although the procedure is technically not very demanding, it suffers from considerable experimental as well as biological 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. [64] phage-typed more than 16,000 isolates in 1 year while investigating 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. rnonocytogenes in 1961 and characterized by Sword and Pickett [78] and Hamon and P6ron [35]. Monocins are resistant to trypsin, sensitive to heating at 56°C for 30 min, and stable a.t 4°C. In monocin typing, an isolate is assessed for susceptibility to a set of bactericidal peptides produced by selected strains [54,85]. Curtis and Mitchell [18] studied monocin interactions of 97 strains of L. monocytogenes using an improved production method involving standardization of the monocins against the type strain of Listeria ivanovii. Only serotype 4 strains acted as

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indicators. A typing system using 8 producer and I 1 indicator strains showed poor discrimination. Bacteriocin typing has limitations similar to those described for phage typing. Bannerman et al. [2] typed 100 strains of L. monocytogenes from sporadic cases and epidemic outbreaks by a combination of monocin typing and phage receptorheverse 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 monocinphage reversal method was simple enough to be done in a nonspecialized laboratory and was highly discriminatory and reproducible. However, they cautioned that the method and the indicator test strains must be rigorously standardized.

Antimicrobial Susceptibility Testing Antimicrobial susceptibility testing is of limited use for typing Listeria species, since L. monocytogenes susceptibility patterns have remained relatively constant for many years. However, in recent years, plasmids conferring resistance to chloramphenicol, macrolides, and tetracyclines have been found in L. monocytogenes [34,581.

MOLECULAR METHODS Multilocus Enzyme Electrophoresis Characterization of prokaryotes and eukaryotes by multilocus enzyme electrophoresis (MEE) is based on differences in electrophoretic mobility of their metabolic enzymes. These differences in electrophoretic mobility are a result of charge differences resulting from amino acid substitutions in the polypeptide sequence; these charge differences, in turn, reflect changes in the nucleotide sequence of the DNA encoding the polypeptide [72]. 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), with each ET representing a multilocus genotype. Some isolates may present null results (absence of activity for specific enzymes); this complicates analysis of MEE data. In the early 1990s, MEE was used in the United States and Europe for epidemiological investigations of listeriosis outbreaks [3,31,521 and to determine the extent to which contaminated foods are involved in sporadic listeriosis [57]. Also, MEE has been useful for taxonomic and genetic characterization studies of L. monocytogenes [7,8,56]. Boerlin et al. [8] used MEE to estimate the genetic relatedness between various Listeria species. The MEE data not only allowed 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 epidemiological investigations. Caugant et al. [ 171 coordinated evaluation of MEE for the WHO Multicentre L. monocytogenes Subtyping Study. Seven laboratories participated in the study, assaying a

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total of 24 enzymes. Reproducibility and the discriminating power of the method varied greatly between the laboratories. Null alleles were reported by five laboratories; in some instances, these could be attributed to less than optimal activity of the enzyme in the cytoplasmic extracts applied to gels, whereas in others, it was clearly related to characteristics of the strains. Caugant et al. [ 171 concluded that to asceirtain immediate epidemiological relationships of L. monocytogenes strains, one will need, in some instances, to supplement MEE with other methods providing further discrimination. Similar conclusions were reached by Norrung and Gerner-Smidt [5 11, 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 available in relatively few laboratories. For these reasons, this method presently has relatively limited application in epidemiological studies.

Chromosomal DNA Restriction Endonuclease Analysis Chromosomal DNA restriction endonuclease analysis (REA) using frequently cutting restriction endonucleases has proven useful for typing L. monocytogenes [24,29,50,83].The number and size of restriction fragments generated by digesting a given piece of DNA are influenced by the recognition sequence of the enzyme
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Restriction Fragment Length Polymorphism Analysis: Ribotyping and Other Methods Ribotyping is one form of Southern hybridization analysis in which strains are characterized for restriction fragment length polymorphisms (RFLPs) associated with ribosomal operon(s). Southern blot analyses only detect the particular restriction fragments associated with specific chromosomal loci, thereby significantly reducing the number of DNA fragments to be analyzed [75]. Ribotyping involves transferring chromosomal DNA restriction fragments that have been electrophoretically separated on a gel matrix onto a nitrocellulose or nylon membrane. After immobilization, DNA fragments are hybridized with an appropriately labeled 16+23S ribosomal RNA (rRNA) or rDNA probe [33,76]. Because the genes coding for rRNA are highly conserved, Escherichia coli rRNA [76] or a cloned ribosomal operon (rrnB) of E. coli [ 121 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 [12]. Therefore, this method may be best suited for long-term epidemiological or phylogenetic studies. Figure 1 shows a gel containing genomic DNA restriction fragments from L. monocytogenes. Figure 2 shows a typical nylon membrane containing ribotype patterns obtained after Southern blotting and probing with a cloned E. coli rrnB operon (plasmid pKK3535) labeled with digoxigenin. Ribotyping has been widely used for subtyping L. monocytogenes [ 1,13,19,31,32,39, 49,5 11, with most investigators using EcoRI as the restriction endonuclease. Baloga and Harlander [I] compared HaeIII and Hind111 to EcoRI and concluded that EcoRI was the most discriminating enzyme for subtyping L. monocytogenes. Nocera et al. [49] 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 [5 11 found ribotyping to be less discriminating than bacteriophage typing, REA, or MEE for subtyping 99 clinical, food, and slaughterhouse isolates of L. monocytogenes. Swaminathan et al. [77] 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. The overall Simpson’s index of diversity (DI) for five 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 of 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 the randomly cloned probes used by one laboratory. The investigators 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 epidemiological investigations. They recommended that ribotyping should be supplemented by other methods such as pulsed-field gel electrophoresis for molecular epidemiology of L. monocytogenes strains of serotype 4b. Probes other than rRNA and rDNA have been used to type L. monocytogenes. Saunders et al. [66] 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 between epidemiologically unrelated isolates. Ridley [62]

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FIGURE1 Genomic fingerprints of EcoRI-digested DNA o f Listeria monocytogenes strains isolated f r o m humans and food. Lanes 2 and 14, human isolates (serotype I / 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.

evaluated the same probe to type 862 isolates representing serogroups 1/2, 3, and 4. Although useful for subtyping serogroup 1/2 isolates, the method did not adequately discriminate between serogroup 4 isolates. The cloned probe evaluated by Ridley [62] and another probe derived from repeat sequences of L. monocytogenes DNA were assessed in the WHO Multicentre L. monocytogenes Subtyping Study [77]. Like ribotyping, the two cloned probes did not adequately discriminate 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 [77]. The RiboPrinter (Qualicon, Wilmington, DE) is an automated ribotyping system that generates, analyzes, and stores riboprint patterns of bacteria. The first version of the

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FIGURE2 Ribotype profiles obtained by Southern blot analysis of genomic fingerprints of EcoRI-digested DNA of Listeria monocytogenes from Figure ? .

RiboPrintcr was contigured t o generate ribotype patterns using only EcoRI and was used to generate a database of patterns for 1346 isolates of L. r i i o r i o c ~ ! ~ t o g c ’ r I c . v[ 13,371. The RiboPrintcr was used by Ryser et al. 1651 to demonstrate that different ribotypcs of L. r i i o i I o( ~j~togc~iic’.s were fiivorcd b j, d i ffe re n t selec t i ve c nri c h me n t pro tocol s. W i edma n n t:t al. I841 characterized 133 isolates of L. rilorloc:\.togc’rlc..v using the RiboPrintcr and tested for pol y morph i s nis i n vi rii 1e nce-;is soc i ii t ed ge lies. They conc 1uded that the i sol ;it e s coii 1d be separated into three distinct phylogenetic lineages: hiiman isolates were found i n lineages 1 and 2. but lineage 3 nfiis coniposeed exclusi\,ely of animal isolates. Pol y merase chain react io ti - ri bot y ping ( PC R-ri boty pi ng ) is ;i met hod that cx ploi ts iisc of oligonucleotide primers designed to be complementary t o conser\.ed regions of the 5s. 16s. and 23s regions of the rRNA genes. These primers are amplified with purified or criidc preparations of template DNA bj, PCR. The resulting PCR products may bc digested with ;i restriction cndonuclease of choice or added t o an agarose gel, elcctrophorcsed. and \,i sii;I I i zed by et h i d i ii m bromide staining . The potential of PC R- ri bo t y p i ng for discriminating between and within \.arious species of Listc~rier.;is well ;is strains of L. r i ~ o r ~ o c ~ ~ ~ thas o g ebeen i ~ ~ ~explored .~. by Sontakke and Farber 1741. who analyzed 49 strains of L. riioiioc’!’toge’iie’.s and 12 isolates of‘ other Listc~r-iuspecies. Gcnomic DNA isolated from bacteria wiis sub.jcctcd to PCR amplification using the region of DNA encoding 16s and 5s rRNA. They found that PCR-I-ibotyping distinguished beti\wn L. riiorioc:\’toSc’ric’,s serotypes 1 /2a and 1 /2b w;ith no o\-erlap i n composite ~”vfiles.The sensitii~itj~ of’ this metliod for differentiating serotypc 1 /2a and 1 /2b isolates appears to be ;is good ;is that

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of other molecular methods. However, the PCR-ribotyping method was less discriminatory for serotype 4b strains [26]. Sontakke and Farber [74] concluded that PCR-ribotyping could be considered as an alternate molecular subtyping technique. However, they recommended combining PCR-ribotyping with another highly discriminatory molecular subtyping method for confirming associations between isolates of serotype 4b.

DNA Macrorestriction Analysis by Pulsed-Field Gel Electrophoresis DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) has revolutionized precise separation of DNA fragments greater than 40 kb. Schwartz and Cantor [70] 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 being kept constant as in conventional agarose gel electrophoresis used for 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. Intact DNA is digested using one or more restriction endonucleases that cut infrequently, thus producing large fragments. Subsequent RFLP analysis allows differentiation of clonal isolates from unrelated ones. PFGE analysis has been used for epidemiological subtyping of L. tnorzocytogenes by several investigators [ 10, 1 1,14,16.36]. Figure 3 shows patterns obtained when genomic DNA from L. monocytogenes was digested with restriction endonucleases ApaI and AscI.

FIGURE3 PFGE separation of Apal (lanes 2-7) and Ascl (lanes 9-14) macrorestriction fragments of Listeria rnonocytogenes genomic DNA f r o m sporadic case isolates. Lanes 1, 8, and 15, Xbal-digest o f Escherichia col; 0 1 5 7 : H7 strain.

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Brosch et al. [ 101 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 PFGE can distinguish between closely related strains indistinguishable by other typing methods. The applicability of PFGE to subtype L. monocytogenes serotypes 1/2 and 3 was subsequently demonstrated [ 151. The applicability of PFGE for outbreak investigations was shown by Buchrieser et al. [ 141, 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 [ 198319871, California [ 19851, and Denmark [ 1985-871) 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 cow show in Illinois [20,59]. Destro et al. [21] also found that RAPD and PFGE were the most useful methods for tracing dissemination of L. monocytogenes in a shrimp processing plant. Brosch et al. [9] evaluated PFGE in the WHO Multicentre L. monocytogenes Subtyping Study. Four participating laboratories evaluated PFGE by analyzing 80 coded strains of L. monocytogenes. Two restriction endonucleases (ApaI and SmaI) were used by all laboratories; one laboratory used an additional restriction endonuclease (AscI).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, with most of the epidemiologically related strains being correctly identified. This study validated 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 subtyped satisfactorily by most other subtyping methods [9]. The major disadvantages of PFGE are the time required to complete the procedure (2-3 days), the requirement for large quantities of expensive restriction endonucleases, and the need for relatively expensive, specialized equipment for electrophoresis.

Random Amplification of Polymorphic DNA Arbitrarily primed polymerase chain reaction (AP-PCR) and random amplified polymorphic DNA (RAPD) analysis are PCR-based methods in which a single arbitrarily selected primer is allowed to anneal to nearly complementary sequences of target DNA by annealing at very low temperatures (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 was developed with longer primers [8 1,861. RAPD was first applied to subtyping of L. monocytogenes by Mazurier et al. [43]. They used a 10-mer primer (HLWL 74) to analyze 104 L. monocytogenes isolates, which 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. [43] suggested that RAPD offers an attractive alternative to phage typing. Lawrence et al. [40] used a different 10-mer primer to type 91 isolates from raw milk, food, and veterinary, environmental, and clinical sources and obtained 33 different patterns. Farber and Addison [26] 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. [48] used a 19-

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mer primer to subtype 57 L. monocytogenes isolates and reported that the method allowed the tracing of L. monocytogenes contamination in several food outlets to be traced back to a food processing plant. Boerlin et al. [6] did an extensive evaluation of RAPD by typing 100 L. monocytogenes isolates which had been characterized by serotyping, phage typing, MEE analysis, REA, and ribotyping. They found R.APD to be highly discriminating for subtyping. O’Donoghue et al. [53] found RAPD to be useful for subtyping serogroup I /2; also, they found that the method distinguished serotype 4bX strains involved in a pit&-associatedoutbreak from other serotype 4b isolates. Wernars et al. [82] evaluated RAPD in the WHO Multicentre L. monocytogenes Subtyping Study. Using three different 10-mer primers, the median reproducibility of RAPD results obtained by the six participating laboratories was 86.5% (range 0-100%). Failure in reproducibility was caused primarily by results obtained with one particular primer. The authors concluded that RAPD is a rapid and relatively simple technique for epidemiological subtyping of L. monocytogenes isolates that can produce reproducible and useful results. Despite the simplicity and high discriminating ability of RAPD, much more work is still needed before RAPD typing becomes a widely used standard technique. Its primary drawback is the inconsistent reproducibility of patterns. E%ecauseRAPD conditions are less stringent to facilitate initiation of the polymerization reaction at sites having one or more sequence mismatches, polymerization is initiated withi 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. Consequently, a well-standardized RAPD protocol must be followed and used consistently to obtain reliable results.

Repetitive Element-Based Subtyping Repetitive element-based (REB) subtyping is a PCR method incorporating use of primers based on short extragenic repetitive sequences [79] or generic rRNA intergenic spacer oligonucleotide [30]. Sequences are typically present at many sites around the bacterial chromosome such that when two sequences are sufficient1:y near 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 stain. Ericsson et al. [23] subtyped 133 strains of L. monocytogenes serotype 4b using REB. A segment of 2916 bp from L. monocytogenes ancl containing parts of the two genes inlA and inlB was amplified by the PCR. The PCR product obtained was digested with the restriction enzyme A M . The resulting fragments yielded two distinct groups, one containing 37 types and the other 96 types. These results indicate that REB may be useful for subtyping L. monocytogenes serotype 4b strains.

DNA Sequence-Based Subtyping We anticipate that within the next 5- 10 years, definitive identification of L. monocytogenes and epidemiological subtyping of strains will be based on :DNA sequencing. At this time, DNA sequence-based subtyping is a not a useful alternative, because the sequencing

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TABLE 1 Characteristics of Phenotypic and Molecular Subtyping Methods Used in the WHO Multicentre L. rnonocytogenes Subtyping Study Method

%

Typeability

Intralaboratory Interlaboratory reproducibility (96) reproducibility (%)

Discriminatory power (DI)

Reference

Recommendations For standardization, reagents should be produced and distributed by one laboratory. Serotyping is not very discriminatory, but is a useful prerequisite, especially in outbreak investigations. Centrally propagate phages and standardize phage suspensions, propagation strains and methodology. Not sufficiently discriminating to be used alone for epidemiological investigations. Develop standardized nomenclature for types. Not sufficiently discriminating to be used alone for epidemiological investigations. Highly discriminating and useful for epidemiological investigations; standardize method. Highly discriminating and useful for epidemiological investigations. One primer gave serious reproducibility problems. Standardize method and address problems with reproducibili ty.

100

82- 100

83

0.68

69

49-80a

NA

79

ND

44

MEE

100

27-9 1

ND

0.83-0.93

17

REA Ribotyping

100 I00

88-97 80- 100

98 ND

0.93-0.98 0.83-0.88

29 77

PFGE

100

ND

84

0.95-0.96

9

RAPD

I00

0.75-0.95 for three primers

82

Serotyping

Phage typing

0-100 for 3 primers

0- 100, median 86.5

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.

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methods are still too complex and appropriate targets on the genome of L. monocytogenes have not been identified. Furthermore, a database of DNA sequences of suitable targets of epidemiologically related and unrelated strains to facilitate interpretation of DNA sequencing dala is also not yet available. However, L. monocytogenes is an attractive candidate for implementing 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. rnonocytogenes. Recent characterization of numerous virulence-associated genes of L. monocytogenes also has provided critical information concerning sequence heterogeneities in these genes [ 22,60,6 1,67,73,87]. The virulence-associated genes of L. monocytogenes that have been sequenced as of November, 1997, include iup (encodes an invasion-associated protein), inlA (a family of genes involved in internalization of the organism into the host cell), hlyA (encodes a P-hemolysin), plcA (encodes a phosphatidyl inositol-specific phospholipase C which is involved in lysis of the membrane during cell-to-cell spread of L. monocytogenes), mpl (encodes a metalloprotease), actA (encodes factors involved in actin polymerization), and the lmu operon (induces delayed-type hypersensitivity reactions in L. monocytogenesimmune mice). In addition, theJEaA gene that encodes flagellin protein, thejuR gene that appears to modulate DNA topology, and 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. Others such as the inlA operon and genes encoding cell surface structures such as the cell membrane and flagella, may be more polymorphic and hence more useful for discrimination of strains. Rasmussen et al. [61] sequenced internal fragments of theJuA, iup, hly, and 23s rDNA genes from different L. monocytogenes serotypes of clinical, food, and environmental origin. A 150-bp region of the hly gene was sequenced in 75 strains, with 27 strains sequenced for the other genes. Although the DNA sequence data for hly, iupJEaA were useful for identifying three lineages, the genetic diversity within the sequencing targets was insufficient for adequate subtyping. As methods for DNA sequencing are simplified and methods for direct sequencing of 1- to 2-kb fragments are developed, DNA sequencing may become viable for subtyping L. monocytogenes.

COMPARISON OF METHODS USED TO SUBTYPE L. MONOCYTOGENES Besides the WHO Multicentre L. monocytogenes Subtyping Study, several additional studies have compared various subtyping methods using different sets of L. monocytogenes isolates. Baloga and Harlander [ 1 3 used genomic DNA fingerprinting, ribotyping, serotyping, and MEE to study 28 strains of L. monocytogenes. In theiir study, DNA fingerprinting was more discriminating than ribotyping. Norrung and Gerner-Smidt [5 I ] compared MEE, ribotyping, REA, and phage typing. Overall, phage typing was the most discriminatory, with a DT of 0.88 followed by REA, MEE, and ribotying with DIs of 0.87, 0.83, and 0.79, respectively. Differences in discrimination were observed depending on “0’’ serotype. For serotype 1, REA gave the best discrimination, whereas phage typing gave the best results for serotype 4. Nocera et al. [49] also characterized 134 strains of L. monocytogenes

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(96 of serotype 4b) from an outbreak using ribotyping and four other typing methods (serotyping, phage typing, MEE, and REA). For serotype 4b isolates, phage typing gave the highest DI, followed by REA, MEE, and ribotyping, with various combinations of these methods yielding higher DIs. Graves et al. [3 11 compared ribotyping and MEE using 305 isolates of L. monocytogenes. Although MEE was more discriminating than ribotyping for this set of isolates, neither of these methods provided adequate discrimination for serotypes 1/2b and 4b. At the beginning of this chapter, we referred to the World Health Organization (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. An overview of the study was reported by Bille and Rocourt [ 5 ] . Because several different methods were compared using a well-defined set of isolates, these studies are very useful in comparing the utility of the various subtyping methods. Table 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 11. This effort should provide a selection of standardized subtyping methods that can be employed in a variety of epidemiological investigations to make multicenter comparisons of data possible.

ACKNOWLEDGMENTS We thank Thomas Donkar and Eric Renner for their assistance with the literature review for this chapter.

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10 Foodborne Listeriosis ELLIOTT. RYSER Michigan State University, East Lansing, Michigan

HISTORICAL OVERVIEW Discovery of several listeriosis outbreaks during the 1980s that were positively linked to consumption of cheese and raw vegetables has led to inclusion of Listeria rnonocytogenes in the current “list” of bonafide foodborne pathogens. However, in retrospect, the concept of listeriosis as a foodborne illness actually can be traced back to when L. rnonocytogenes was first isolated. Nine years before Murray et al. [167] described L. rnonocytogenes in 1926, Atkinson [42] reported an outbreak of meningitis among five 2- to 9-year-old Australian children, caused by a small gram-positive, diphtheroid-type bacillus that was probably L. rnonocytogenes. Similarly, two additional listeriosis outbreaks [67,21I ] accounted for 7 of 36 listeriosis cases recorded in the literature between 19 17 and 1943 [ 1361. Hence, to explain these three small outbreaks, along with at least 35 other listeriosis outbreaks that have included 2195 documented cases since 1949 (Table I), one might reasonably postulate that food-to-human transmission of L. rnonocytogenes occurred in at least some instances. Early animal feeding studies also support the notion that listeriosis can be acquired through consumption of contaminated food. The first accurate description of L. rnonocytogenes in 1926 by Murray et al. [ 1671 included trials in which three of six 32-day-old rabbits were successfully infected via the oral route. Results from subsequent postmortem examinations agreed with the previously observed pathological findings for naturally occurring listeriosis in rabbits. Fourteen years later, Julianelle [ 1321 reported that white mice died of generalized listerial infections after consuming drinking water inoculated with 299

Ryser

300

TABLE 1 Apparent and Confirmed Common-Source Outbreaks of Listeriosis Involving 10 or More Cases Location

Year

Halle, East Germany

1949- 1957

Jena, East Germany Soviet Union Bremen, West Germany Halle, East Germany Auckland, New Zealand Anjou, France Johannesburg, South Africa Western Australia Massachusetts, USA Auckland, New Zealand Auckland, New Zealand East Cambria, England Slovakia Maritime Provinces, Canada Christchurch, New Zealand Houston, Texas, USA Saxony, West Germany Massachusetts, USA Vaud. Switzerland

1954 1956 1960- 1961 1966 1969 1975- 1976 1977- 1978 1978- 1979 I979 1979- 1980 1980 1981 1981 1981 1981-1982 I983 1983 1983 1983- 1987

Los Angeles, CA, USA Denmark Linz, Austria Los Angeles, CA, USA Los Angeles, CA, USA England England, Wales, Northern Ire 1and New York, NY, USA Denmark

I985 1985- 1987 1986 1986- 1987 1987 1987 1987- 1989

Western Australia France France Italy Illinois, Michigan, Wisconsin, USA France France Italy a

Number of cases

- 100

Possible vehicle of infection

Raw milk, sour milk, cream, cottage cheese Unknown 26 Pork, mouse 19 81 Unknown Unknown 279 Unknown 13 162 Unknown 14 Unknown 12 Raw vegetables 20 Raw vegetables, milk 10 Unknown 22 Shellfish, raw fish 11 Cream 49 Unknown 41 Coleslaw“ 18 Unknown 10 Unknown 25 Unknown 49 Pasteurized milk 122 Vacherin Mont d’Or cheese’ 142h Mexican-style cheesea Unknown 35 20 Raw milk, vegetables 33 Raw eggs 11 Butter 23 Unknown Pritt2 366

1989 1989- I990

10 26

1990 1992 1993 1993 I994

11 279 39 18 66

Shrimp Blue-mold or hard cheese Processed meats or p h i Jellied pork tonguea Pork piit6 “rilletes”” Rice salad“ Chocolate milk”

I995 1997 1997

33 14 1594

Brie de Meaux cheesea Pont I’EvCque cheese” Sweet corn

Vehicle of infection positively identified. Estimated number of cases as high as 300. Goulet. 1998. Personal communication.

Reference

44,l 13,185,186, 192,215 227 116 98 177 49 71 I25 220 122 190 144 104,156 190 210 93 69 I69 99 47.66 145 200,208 3,226 214 154 157 159,195 I94 131 139, 234 106,126,207 126 206 75 126

-c

-c

Foodborne Listeriosis

301

L. monocytogenes. From these observations, both authors concluded that ingestion of L. monocytogenes followed by penetration of the gastrointestinal tract by the bacterium is one means by which listeriosis can be acquired. As you will recall from the previous discussion of animal listeriosis, lactating cows can shed L. monocytogenes in milk for long periods as a consequence of listeric mastitis or abortion. Of greater importance are reports that clinically normal cows can shed listeriae in their milk for at least 12 months [ 123,2171. Although L. monocytogenes was not isolated from both the udder and milk of mastitic dairy cattle until 1944 [237], Burn [67] postulated as early as 1936 that milk could serve as a vehicle of infection in humans. Two years later, Schniidt and Nyfeldt [2 I I ] also suspected a causal relationship between listeriosis in humans and dairy cows during a small listeriosis outbreak in Denmark but were unable to confirm milk as the vehicle of infection. Given the preceding information, it is not surprising that consumption of raw milk was suspected as the most likely cause of the first documented foodborne outbreak of listeriosis. This outbreak, which occurred in Halle, Germany, between 1949 and 1957 (Table l), was accompanied by additional outbreaks in Jena, Germany, and Prague, Czechoslovakia, with all three outbreaks prompting numerous investigations dealing with various epidemiological aspects of the disease. In 1955, the scientific literature on Listeria that had accumulated over nearly 50 years was admirably reviewed by the late H. P. R. Seeliger in his monograph entitled Listeriosis (in German). Interest in this disease was so great that over 150 publications appeared in the literature between 1955 and 1957. Consequently, Seeliger extensively revised and updated his monograph in 1957 (in German) and again in 1961 (in English) [215] to the point where his book has been, until recently, a leading source of information on the subject. During this period of keen interest, Seeliger [2 151 and other European researchers [57,124,138,23I] emphasized the likely importance of food in disseminating listeriosis, which, in furn, resulted in some of the first studies dealing with the organism’s suggested ability to survive during pasteurization of milk, as described in Chapter 6. Although a heightened awareness of L. monocytogenes led to documentation of at least nine listeriosis outbreaks (613 cases) in the 20-year period from I960 to 1980 (Table 1 ), a lack of any clear link to food along with continued difficulties in isolating this organism from food and environmental sources are two likely reasons for a “leveling off” of interest in foodborne listeriosis during this period. Although L. monocytogenes was not yet included in the list of recognized foodborne pathogens published by the World Health Organization (WHO) in 1976 [ 5 ] ,3 years later, this pathogen was placed under the heading of “Bacteria Not Conclusively Proved to Be Foodborne” in the second edition of the well-known book Foodbornc. Infections and Intoxications, edited by Riemann and Bryan [ 1931. Ascribing a source to listerial infections has proven difficult because of a highly variable incubation period ( 1 -2 I days) before clinical symptoms appear and the unavailability of food samples for analysis at the time of onset. However, in 1981, the status of L. monocytogenes began to change when Schlech et al. [210] reported that 17 of 41 (41.5%) people died of listeriosis after consuming coleslaw from which L. monocytogenes was later isolated. This outbreak provided the first conclusive evidence that humans can contract li steriosis by consuming contaminated food and also demonstrated that foods other than dairy products can become contaminated with L. monocytogenes and thus constitute a health risk to certain segments of the population. Two years later, three additional listeriosis epidemics were documented (Table l), including one widely publicized outbreak in which consumption of pasteurized milk was epiderniologically linked to 49 cases

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of listeriosis (including 14 deaths) in Massachusetts. However, it must be emphasized that L. monocytogenes was never isolated from the incriminated milk. Any remaining doubt concerning the ability of L. monocytogenes to produce foodborne illness completely vanished in June of 1985 when consumption of Jalisco brand Mexican-style cheese was directly linked to at least 142 cases of listeriosis, including 48 deaths, in Los Angeles [ 1451. Thus the two aforementioned listeriosis outbreaks involving consumption of contaminated coleslaw and Mexican-style cheese, along with another major outbreak in Switzerland that resulted from consumption of contaminated Vacherin Mont d’Or soft-ripened cheese, have generated worldwide concern over the presence of Listeria in dairy products and many other foods, including meat, poultry, eggs, seafood, and vegetables. In response to this heightened awareness, eleven additional outbreaks (eight European, two United States, one Australian) have been identified since 1987, including two confirmed outbreaks involving pi%&(366 cases) and jellied pork tongue (279 cases) and the largest outbreak to date (1594 cases of gastroenteritis) possibly linked to Italian sweet corn (Table 1). Consequently, L. monocytogenes has now moved to the ranks of a bonafide foodborne pathogen in virtually all food microbiology textbooks [2,46,73,82, 83a, 130,162,166,180,191]. Since concern about foodborne listeriosis originally centered around dairy products, particularly soft cheeses [212], it is only fitting to begin this chapter by reviewing the known cases of listeriosis in which nonfermented and fermented dairy products were suspected and/or proven as vehicles of infection. Special attention will be given to the three cheese-related listeriosis epidemics documented in California, Switzerland, and France as well as the 1983 outbreak in Massachusetts that was supposedly linked to consumption of pasteurized milk and a more recent epidemic traced to pasteurized milk in Illinois [75]. Similar evidence for involvement/possible involvement of other foods, including red meat, poultry, eggs, seafood, fish, vegetables, and fruits in cases of human listeriosis will be presented in the remaining pages of this chapter.

RAW MILK Sporadic cases of bovine mastitis and abortion in which L. monocytogenes was intermittently shed in milk over several lactation periods have been recorded in the literature for more than 50 years. Dairy cows that appear healthy also can serve as reservoirs for L. monocytogenes and secrete the organism in milk. Once obtained from the cow, milk may be further contaminated through inadvertent contact with feces and silage, both of which often contain Listeria and are normally present in the dairy farm environment. Considering the present estimate that 3-4% of the milk supply contains detectable levels of L. monocytogenes, it is easy to understand why raw milk was suspected as one of the most likely sources of infection in several large European outbreaks of listeriosis. The first evidence for foodborne transmission of L. monocytogenes can be found in a series of anecdotal reports from Germany [ 1 13,2 151. During the reconstruction period that followed the end of World War 11, a sharp increase in the number of stillborn infants was observed at an obstetrical clinic in Halle, with approximately 100 cases recorded up to 1952. Working in an antiquated laboratory, Potel [ 1841 concluded that these stillbirths resulted from infection with Corynebacterium infantiseptica. However, in 1952, Seeliger suggested and later confirmed that this rash of stillbirths was caused by L. monocytogenes [ 1131. Unpasteurized milk as well as sour milk, cream, and cottage cheese were suspected by Seeliger [215] as possible vehicles of infection in several cases observed in Halle.

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Subsequently, Potel [ 1861 isolated identical serotypes of L. monocytogenes from a mastitic cow and from stillborn twins delivered by a woman who had consumed this milk before parturition. On this basis, Potel can be credited with the first description of foodborne listeriosis in humans. During this outbreak, raw milk for pregnant women was generally available only through the black market, which in turn suggests that consumption of raw milk was likely responsible for additional cases of listeriosis [ 1131. After this listeriosis epidemic ended in 1957, two subsequent outbreaks involving 180 and 160 cases were identified in Halle during 1960 and 1961 [98] and in 1966 [177], respectively. Fischer [98] also described a cluster of listeriosis cases that occurred in Bremen, Germany, between 1960 and 1961. Although neither the mode of transmission nor the primary reservoir for L. monocytogenes were identified in these outbreaks, involvement of milk appears unlikely, since most of these cases occurred during a time in which production and sale of milk were both rigidly controlled. Since the postwar listeriosis outbreak in Halle in which raw milk was linked to at least one stillbirth, only two additional case studies have been published in which raw bovine milk has been mentioned as a possible cause of listerial infection. In the first of these cases, reported in 1973 [61], a 28-year-old Canadian woman went into premature labor and delivered an infant who died of listeriosis 33 h after delivery. Shortly before giving birth, the mother recalled purchasing raw milk and cream; however, these products were no longer available for testing. The second case involved a 43-year-old male acquired immunodeficiency syndrome (AIDS) patient in California who contracted listerial meningitis in 1987. Following rigorous antibiotic therapy and complete recovery, the patient admitted being a regular consumer of commercially available raw milk. Although the investigating team made no attempt to confirm raw milk as the vehicle of infection, the possible role of raw milk in this case suggests that current (1997) efforts by the U.S. Food and Drug Administration (FDA) and the U.S. Centers for Disease Control and Prevention (CDC) to inform AIDS patients about the potential threat of listeriosis, salmonellosis, and other foodborne illnesses associated with consumption of certain high-risk foods (e.g., raw milk, surface-ripened cheese, undercooked poultry) should continue. Boiling has been often suggested as one means of eliminating microbial pathogens from raw milk and enhancing the safety of this product. However, given one additional report [ 1741 of a 60year-old, j mmunocompromised Indian man who developed listerial septicemia and meningitis after ingesting boiled raw milk, individuals at risk of developing listeriosis would be well advised to consume only commerciallyproduced milk that has been properly pasteurized. The threat of contracting milkborne listeriosis generally appears to be confined to susceptible individuals who routinely consume raw bovine milk, with no listerial infections yet linked to consumption of raw milk from other animals, including ewes and goats. Thus, even when given the ability of L. monocytogenes to infect humans and produce mastitis in animals, it is still surprising to learn that researchers in Yugoslavia [230] were able to link one case of neonatal listeriosis to consumption of contaminated human breast milk. According to their 1988 report, a 24-day-old infant girl contracted listeriosis after receiving breast milk from her mother. Thirteen days after onset of symptoms, L. monocytogenes serotype 4b was isolated from the infant’s cerebrospinal fluid and blood as well as the mother’s milk. The infant recovered fully 3 days after cessation of breast-feeding. Interestingly, excess breast milk from the mother was given to a newborn litter of three Doberman puppies, and all three dogs became ill with vomiting, diarrhea, and bloody stools. One of the animals died, and the same serotype of L. monocytogenes as found in breast milk was detected in a stool specimen from one of the two survivors. Although

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this is currently the only report linking listeriosis to consumption of human breast milk, the medical profession should be aware of the possibility for such transmission, particularly in apparent nosocomial cases of neonatal listeriosis.

PASTEURIZED MILK Until 1985, the only proven foodborne listeriosis outbreaks associated with dairy products involved consumption of raw milk. However, this changed when Fleming et al. 1991 epidemiologically linked consumption of a specific brand of whole and 2% pasteurized milk to 49 cases of listeriosis in Massachusetts between June and August of 1983. As seen in Table 2,42 (86%)of the cases occurred in adults and seven (14%) in mother-infant pairs. Fourteen of 49 individuals died giving a mortality rate of 29%. Two years later, Todd [225] calculated the total cost of this outbreak at $1.89 million ($1.37 million-deaths, $387,000-hospitalization, $70,00O--investigation, $6 I ,000-financial/legal costs) or $38,6 14 per case excluding legal settlements. Although all adults had underlying conditions that resulted in immunosuppression, symptoms expressed during the course of illness varied depending on the person’s age and degree of immunosuppression. Forty of 49 isolates were available for serotyping, with 32 (80%) being identified as serotype 4b, which was later defined as the epidemic strain. Two case-control studies, one matched for neighborhood of residence and the other for the patients’ underlying condition, indicated that development of listeriosis was strongly associated with drinking a specific brand of pasteurized whole or 2% milk. Further epidemiological investigations showed (a) a correlation between increased consumption of the specific brand of whole or 2% milk and contracting listeriosis, (b) a lower incidence

TABLE 2 Characteristics of Adult and Perinatal Listeriosis Cases Identified in Massachusetts Between June 30 and August 30, 1983. Number of cases (%) Case profile

Adult ~

Total cases Total fatalities Sex: M/F Clinical syndrome Meningitis Septicemia Death in utero Underlying condition Cancer Cirrhosis/alcoholism Diabetes Corticosteroid therapy Renal transplant Myelofibrosis Chronic hepatitis Intravenous drug abuse Source: Adapted from Ref. 99.

~~

42 (86) 12 (29) 271 15 13 (31) 29 (62)

Perinatal

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of disease among individuals who drank skim or 1% milk produced by the same dairy, (c) an association between several listeriosis cases in Connecticut and consumption of the same brand of whole or 2% milk, and (d) an association with a specific phage type of L. monocytogenes (phage type 2425A), which was isolated from all 19 listeriosis victims who reportedly drank the specific brand of whole or 2% milk. Although these epidemiological studies strongly suggest that this outbreak resulted from consuming whole or 2% milk, the microbiological findings were far less convincing in that L. rnonocytogenes was never isolated from the incrirninated pasteurized milk. The milk implicated in this listeriosis outbreak was processed at a single dairy factory and pasteurized at 77.2”C (1 7 1 O F ) / 18 s [ 1881, which is well in excess of the minimum requirement (7 1.7”C [ 161”F]/15 s) specified in the Pasteurized Milk Ordinance. Furthermore, no defect was identified that could have led to improper pasteurization and no source of postpasteurization contamination was ever found within the dairy factory. Shortly after the outbreak ended, a survey conducted by the CDC [99,112,119] indicated that 15 of 124 (12%) raw milk samples collected from the factory milk supply, individual farms, and a milk cooperative that supplied the factory contained L. monocytogenes. Several different serotypes were identified, including 1a, 3b, 4a/b, and 4b, the epidemic serotype. Using DNA macrorestriction analysis [ 1261, the epidemic strain was later reported to be genetically similar to isolates responsible for two subsequent outbreaks traced to p2te in the United Kingdom [ 1591and France [ 1261 (Table 10) but distinctly different from strains implicated in two North American outbreaks involving coleslaw and Mexican-style cheese [235].However, neither the phage type nor the restriction enzyme type that was epidemiologically linked to this presumed outbreaks of milkborne listeriosis was ever recovered from raw milk or the incriminated pasteurized milk. Although the epidemiological evidence gathered by Fleming et al. [99] suggests this outbreak resulted from drinking a particular brand of pasteurized whole or 2% milk, the means by which L. monocytogenes may have found its way into the milk remains unclear. Postpasteurization contamination of the milk cannot be excluded; however, it seems unlikely, since inspections failed to recover L. rnonocytogenes from the dairy factory environment. Since whole and skim milk were processed each day using the same equipment, it is also difficult to postulate a means by which only whole milk would have been subjected to postpasteurization contamination. To further support the involvement of pasteurized milk in this outbreak, the authors concluded that “intrinsic contamination of the milk and siirvival of some organisms despite adequate pasteurization is both consistent with the rr:sults of this investigation and biologically plausible”; the latter conclusion was based on the now faulty but at the time frequently quoted pasteurization study by Bearns and Girard [48]. Consequently, the apparent association of listeriosis with consumption of pasteurized milk raised immediate product safety concerns, which in turn led to numerous studies examining L. monocytogenes heat resistance (see Chap. 6). Nearly 2 years after the outbreak was reported in the New England Journal of Medicine, Donnelly et al. [81 I found that the “open-tube” method used by Bearns and Girard [48] was flawed and concluded that freely suspended L. monocytogenes cells were unlikely to survive normal high-temperature, short-time (HTST) pasteurization at 71.7”C (161°F) for 15 s. Knowing that this pathogen can exist within milk leukocytes, and that milk in the Massachusetts outbreak was not clarified but rather passed through a milk sock (coarse filter) that did not remove leukocytes, many researchers postulated that the presence of L. monocytogenes within these leukocytes enhanced the organism’s resistance to pasteurization. Subsequent studies addressing this issue have gen-

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erally shown insignificant differences in the degree of thermal resistance between freely suspended and internalized L. monocytogenes cells [84,1731. Using milk containing relatively large numbers of intracellular listeriae, several workers demonstrated that L. monocytogenes can survive minimum requirements for pasteurization; however, commingling of milk from many farms before pasteurization would result in much lower levels of listeriae (i.e., 5 10 CFU/mL) [38,146,218] with many, if not most, organisms being present only extracellularly as a result of leukocytic breakdown during the first 24-48 h of cold storage. Hence, on this basis, both the scientific community and the WHO maintain that L. monocytogenes will not survive minimal milk pasteurization at 16l0F/15 s [236]. In support of this position, L. monocytogenes has not yet been demonstrated to have survived pasteurization in a commercial dairy product that met minimum HTST pasteurization requirements. Although numerous FDA Class I recalls of pasteurized dairy products, including 2%, 1%, and skim milk as well as chocolate milk, ice milk mix, ice milk, ice cream mix, ice cream, ice cream novelties, sherbet, butter, and various cheeses, have been well publicized in the United States, in virtually all instances L. monocytogenes was present in the immediate manufacturing environment, which strongly suggests postpasteurization contamination. Given this information, the likelihood of L. rnonocytogenes having survived pasteurization at -77.2"C (17 1OF)/ 18 s in the Massachusetts outbreak appears remote at best. Since L. monocytogenes was never isolated from pasteurized milk implicated in the Massachusetts outbreak or the dairy factory environment, some investigators have questioned the role of milk [80]. In 1988, several design flaws were discovered in the case-control studies [80] that included missing questions and data on questionnaires, as well as a disproportionate number of follow-up interviews between cases and controls, any of which might have given an unfair bias. Contrary to the authors [99], cases were generally clustered around the Boston area in a manner that was not consistent with the milk distribution pattern. Furthermore, inconsistencies in data were noted between exposure to implicated milk and isolation of the L. monocytogenes strain supposedly responsible for the epidemic [go]. Discovery of these discrepancies in the various case-control studies prompted a lawsuit against the CDC; however, as one might expect, a definitive answer concerning involvement of pasteurized milk in this outbreak was never reached through the judicial system.

Chocolate Milk In July of 1994, Dalton et al. [75]reported that 54 of 60 (90%) previously healthy individuals who attended a summer picnic in Illinois developed listeriosis 9-32 h (median 20 h) after consuming one or more 8-oz cartons of pasteurized chocolate milk with four, three, and five related cases later reported in Illinois, Wisconsin [ 1891, and Michigan, respectively. Unlike most foodborne listeriosis epidemics recorded to date, gastrointestinal symptoms predominated among picnickers, with victims most commonly experiencing diarrhea (79%), fatigue (74%), fever (72%), chills (65%), headache (65%), myalgia (59%), abdominal cramps (55%), nausea (47%), and vomiting (26%) over several days. Only four individuals required short hospitalization, with one pregnant woman delivering a healthy baby 5 days after experiencing a 6-h bout of diarrhea. In support of the aforementioned findings, the clinical strain of L. monocytogenes identified as serotype 1 /2b was soon isolated from multiple unopened containers of choco-

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late milk at levels of 8.8 X 108 to 1.2 X 109 CFU/mL (Le., mean infective dose of 2.9 X 10'' CFU/person), with the product's taste and quality reportedly being poor. During a follow-up environmental survey of the implicated milk processing facility, investigators recovered the epidemic serotype of L. monocytogenes from a floor drain beneath the chocolate milk filler and from a valve connected to the chocolate: milk pasteurizer. Based on multilocus enzyme electrophoresis, ribotyping and pulsed-field gel electrophoresis, the clinical, chocolate milk, and factory environmental isolates of L. monocytogenes serotype 1/2b were indistinguishable from each other, thereby confi.rming chocolate milk as the vehicle of infection. Although properly pasteurized at >87"C/ 18 s after the addition of chocolate flavoring and immediately cooled to <8"C, the product was held unrefrigerated for 2 h in a malfunctioning and improperly sanitized tank before being pumped to the filling machine over a 7-h period. In all likelihood, this outbreak occurred as a result of postpasteurization contamination. However, given the ability for rapid growth of L. rnonocytogenes in temperature-abused chocolate milk [ 1991 (see Chap. 1 l), inadequate and/or nonexistent refrigeration during packaging (7 h), and transit (2.25 h) to the picnic are obvious contributing factors along with consumption of the product 3 days before the expiration date.

OTHER NONFERMENTED DAIRY PRODUCTS A search of the early literature has uncovered only a few inconclusive reports suggesting that listeriosis can be contracted by consuming nonfermented dairy products other than raw and pasteurized chocolate milk. After the first massive listeriosis outbreak was reported in Halle, Gerinany, between 1949 and 1957, Seeliger [215] indicated that in addition to raw milk, sour milk and cream also were considered as possible sources of infection in several cases. Although such products could certainly contain L. rnonocytogenes, the exact role of raw milk and cream in these cases was never determined. During the latter half of 1981, an apparent common-source listeriosis outbreak was recorded in East Cambria, England, in which identical phage and serotypes of L. monocytogenes were isolated from 1 I patients [ 1561. Even though epidemiological evidence indicated a possible association with consumption of pasteurized cream [45,104], the exact mode of transmission was never verified. Similarly, Ralovich [ 1901 learned of a personal account in which L. monocytogenes was isolated from a cream-based rice soup associated with illness; however, the etiological role of Listeria was never proven. Following the widely publicized 1985 listeriosis outbreak in California in which numerous deaths were directly linked to consumption of contaminated Mexican-style cheese, Los Angeles County officials instituted an active surveillance program for listeriosis and made the disease reportable [ 1521. These efforts wentually led to discovery of a cluster of 1 I perinatal listeriosis cases among Hispanics during November and December of 1987 [151]. Seven of 11 L. monocytogenes isolates obtained from the victims were of serotype 1 /2a. Although a subsequent case-control study identified butter as a possible vehicle of infection (odds ratio = 4), this first-time association between consumption of butter and listeriosis was not culturally confirmed. Absence of Listeria spp. from butter tested by the FDA during the Dairy Initiatives Program 1381 and evidence indicating that large numbers (-92-97%) of L. monocytogenes are lost in buttermilk and washings when butter is manufactured from inoculated cream [176] suggest that butter is not a major vehicle for transmission of listeriae. Nonetheless, L. rnonocytogenes can occasionally be found in hutter with at least five Class I recalls issued thus far without incident.

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Attempts to implicate frozen dairy products as vehicles of infection in listeriosis cases are of relatively recent origin. In 1986, FDA officials investigated an incident in which L. monocytogenes was isolated from amniotic fluid following the premature delivery of an infected infant who died 5 days later [ 1 I 1. After learning that the 2 1-year-old mother consumed ice cream sandwiches 3 days before delivery, all suspect product was withdrawn from store shelves; however, follow-up studies failed to incriminate ice cream as the source of infection. Since 1985, enhanced surveillance of the dairy industry by the FDA has prompted over 50 recalls of Listeria-contaminated frozen dairy products, including ice cream, ice milk, sherbet, and ice cream novelties as well as ice milk, ice cream, and milk shake mix. Consumption of such products could potentially constitute a public health risk. Hence, preliminary epidemiological results from the CDC suggesting a possible link between consumption of ice cream and a cluster of 31 listeriosis cases, including 14 deaths, in Philadelphia, Pennsylvania, appeared to have some merit [ 171. However, inability to isolate L. monocytogenes from ice cream eliminated this product as the vehicle of infection [ 18,2131. After repeated attempts to (a) identify a common epidemic strain and (b) isolate Listeria from cheese and other dairy products as well as meats and vegetables ended in failure, the CDC finally retracted their previous statement and concluded that a commonsource outbreak of foodborne listeriosis had not occurred in the Philadelphia area [ 16,2 131. Although well over 4 million gallons of frozen dairy products thought to be contaminated with Listeria have thus far been recalled from the market at a cost in excess of $88 million [19], not one case of listeriosis has been directly linked to consumption of frozen dairy products marketed in the United States. The only proven case comes from Belgium [4], in which an immunocompromised 62-year-old man developed listerial meningitis after consuming commercially prepared ice cream containing 1O4 L. monocytogenes serotype 4b CFU/g, with this pathogen coming from contaminated cream. Apparent low levels of L. monocytogenes in frozen dairy products resulting from postpasteurization contamination combined with the organism’s obvious inability to grow during frozen storage [51] are but two reasons why these products appear to constitute a very minimal risk to public health.

FERMENTED DAIRY PRODUCTS Certain species of lactic acid-producing bacteria can be used to ferment fluid milk into a wide array of dairy products, including cultured buttermilk, sour cream, and yogurt as well as hundreds of cheese varieties. Thus far, no listeriosis outbreaks have been positively linked to consumption of contaminated yogurt, sour cream, or cultured buttermilk; however, the same cannot be said for cheese. As early as the 1950s, cheese was suspected of playing a role in foodborne listeriosis. However, since June of 1985, three major listeriosis outbreaks have been directly linked to contaminated soft cheese, thus confirming its role in foodborne listeriosis. The first such outbreak involved consumption of Mexican-style cheese in California and was followed by a second major outbreak in Switzerland, which was traced to contaminated Vacherin Mont d’Or soft-ripened cheese. Most recently, Brie de Meaux and Pont 1’Eveque cheese were responsible for two smaller listeriosis outbreaks in France. These outbreaks will now be reviewed in some detail, after which several sporadic cases of cheeseborne listeriosis also will be discussed. As just mentioned, the notion that humans can contract listeriosis by consuming Listeria-contaminated cheese is not new. Along with raw milk, sour milk, and cream,

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Seeliger [2 15) suggested a possible relationship between consumption of cottage cheese and several cases of listeriosis that occurred in Halle, Germany, mentioned earlier. Nonetheless, it must be stressed that cottage cheese was never confirmed as the vehicle of infection. A search of the scientific literature revealed no additional reports suggesting involvement of cheese in cases of listeriosis during the 30 years that followed the Halle incident.

Mexican-Style Cheese: California, 1985 In June of 1985, the ability of cheese to serve as a vehicle for foodborne listeriosis became evident when consumption of Jalisco brand Mexican-style cheese was linked to a massive listeriosis outbreak in southern California. This outbreak, which later proved to be among the deadliest of all known outbreaks of foodborne disease recorded in the United States, prompted much dairy-related research worldwide, most of which is reviewed elsewhere in this book. Before dealing with the facts of this outbreak, it is appropriate to review chronologically the ‘ ‘detective work” conducted by various. governmental agencies (Fig. 1) that linked this outbreak to Mexican-style cheese and also led to a nationwide recall of the product on June 17, 1985. According to information appearing in local newspapers, the first listeriosis case associated with this outbreak was diagnosed in Los Angeles County during the first week of January 1985, with one and three additional cases being documented during the second and fourth weeks of January, respectively. Unknown to public health officials, the rate of listerial infections continued to increase, with six and nine cases being reported in Los Angeles County during February and March, respectively. Several important chance happenings led to discovery of this outbreak in the weeks that followed. The first hint of a possible problem was uncovered at the Los Angeles County-University of Southern California (USC) Medical Center-a vast medical com-

Fi Collect l o c a l l i s t e r i o s i s data

1. 2. 3.

Coordinate e n t i r e i n v e s t i g a t i o n I n i t i a t e case-control s t u d i e s Serve a s a c e n c r n l l a b o r a t o r y f o r L i S t e r i a - t e S t i n g

Los Angeles County Dept. of Health S e r v i c e s Lead I n v e s t i g a t o r

-

r--l

Orange County Health Care Agency

1. 2.

I n v e s t i g a t e c a s e s of l i s t e r i o s i s Spread news of ourbreak t o Spanish communit y

He a 1t h S e r v i c e s J a l i s c o Mexican P r o d u c t s , Inc.

4

L3ok f o r c a u s e o f contamination

FIGURE1 Primary roles o f local, state, and federal agencies in investigating the 1985 listeriosis outbreak in California.

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plex at which about 17,000 infants are delivered each year [ 128,1291.In early April, Carol Salminen, a nurse epidemiologist who monitored infection rates at this facility, uncovered five additional listeriosis cases among Hispanic motherhnfant pairs during the previous 2 weeks. Under normal circumstances, only three to five listerial infections would be observed annually at this hospital. After consulting with the medical director of the labor and delivery service, who had developed a personal interest in listeriosis and maintained a log of listerial infections over the previous 10 years, Salminen informed health officials at the Los Angeles County Department of Health Services on May 6 that nine listeriosis victims had been treated at the USC Medical Center since January of 1985. Once notified, health officials at the Los Angeles County Department of Health Services and the Orange County Health Care Agency began surveying area hospitals for additional cases. After the reported number of listerial infections increased from 16 to 67 in just a few days, Los Angeles County health officials contacted the California Department of Health Services and the CDC on May 10 for investigative assistance. At the same time, 200 area hospitals in the counties of Los Angeles and Orange were requested to report all listeriosis cases to the health department. Ten days later, health officials and epidemiologists from the CDC began the first of two case-control studies in which listeriosis victims, mostly previously healthy Hispanics, were interviewed about various environmental factors, behavioral patterns, and consumption of over 60 food items, including fresh fruits and vegetables, water, milk, and cheese. On May 29, an open package of Jalisco brand Mexican-style cheese was taken from one of the victim’s refrigerators and sent to the CDC in Atlanta, Georgia, for analysis. After CDC investigators provided preliminary confirmation of L. monocytogenes in this opened package of Mexican-style cheese on June 8, 20 packages of Mexican-style cheese of various brands, including two packages of Queso Fresco and Cotija Jalisco brand cheese, were purchased at area markets near the victim’s place of residence and sent to the CDC for analysis [145]. On June 10, results from the case-control study described earlier clearly demonstrated that individuals who had consumed Mexican-style cheeses were at increased risk of contracting listeriosis. Armed with this information, investigators immediately began a second case-control study in which individuals were questioned about the names and brands of Mexican-style cheese consumed. On June 12, statistical analysis of these data revealed a definite link between consumption of Jalisco brand Mexican-style cheese and development of listeriosis [ 1451. FDA officials were immediately advised of the impending problem with Jalisco cheese. Confirmation of L. monocytogenes serotype 4b in two unopened packages each of Jalisco brand Queso Fresco and Cotija Mexican-style cheese-the last piece of evidence needed to initiate a Class I recall of the product-was provided by the CDC on June 13. (Subsequent studies eventually identified the epidemic L. monocytogenes strain in 82% of all Jalisco brand products purchased at area supermarkets.) Armed with this information, the California Department of Food and Agriculture immediately closed the Jalisco cheese factory and announced a statewide recall for these two varieties of Mexican-style cheese, -80% of which was sold through retail outlets in Los Angeles and Orange Counties. On June 14, state officials expanded this recall to include the firm’s entire line of 44 products (predominantly cheese), consumption of which was already blamed for at least 28 deaths. Hence, in the weeks that followed, health officials were faced with the enormous task of checking -28,000 Los Angelesarea supermarkets, family-owned grocery stores, and restaurants to ascertain that all Jalisco brand products were removed from shelves. FDA officials also ordered a Class I recall of all Jalisco brand products distributed in California and in 12 other primarily western

Foodborne Listeriosis

31 1

states [6]. Three days later, this recall was expanded to include all 26 states in which these products were sold as well as the United States Protectorates of Guam, American Samoa, and the Marshal1 Islands [8]. When this recall was completed on June 22, nearly 250 tons of Jalisco brand Mexican-style cheese and other dairy products were ready for burial in a landfill site overlooking the San Gabriel Valley. Even though the number of individuals who actually contracted listeriosis after eating the tainted cheese has been a debatable issue for some time, the exact figure will never be known, since mild listerial infections in individuals who did not seek medical attention obviously went unreported. Newspaper accounts have placed the total number of listeriosis cases occurring in California between January 1 and August 15 at nearly 300, including 85 fatalities. Although about half of these cases were concentrated within the Hispanic communities of Los Angeles and Orange County, a substantial number of listeriosis victims also reportedly resided in the San Diego area, which made the collection of reliable data more difficult. In addition, at least 16 cheese-related listeriosis cases were uncovered outside California (Arizona, Colorado, Oregon, Texas, and Connecticut) with three fatalities being reported in Texas. Although the total number of listeriosis cases reported in Los Angeles County during the 12-month period immediately following the outbreak decreased to 94, the calculated annual crude incidence rate of 12 cases/million population is still approximately twice the national average [ 1521. Numbers of reported listerial infections have continued to decrease in Los Angeles County, with 1990 and 1994 rates of nonperinatal and perinatal listeriosis decreasing from 6 to 3 cased1 million population and 17 to 6 cases/100,000 live births, respectively [72,223]. The fact that these rates were previously well above the national average is not too surprising when one considers that this outbreak certainly made area physicians, hospital personnel, and public health authorities keenly aware of this disease. In all likelihood, these factors in combination with mandatory reporting of this formerly obscure illness were largely responsible for the abnormally high incidence of listeriosis in Los Angeles County. In 1988, Linnan and 14 other members of the investigative team [145] published their findings concerning 142 listeriosis cases that were linked to consumption of Jalisco brand cheese in Los Angeles County between January 1 and August 15, 1985. Although nearly 160 additional cases occurred elsewhere in California (Orange, San Diego, and Fresno Counties) and in other states, logistical concerns limited their studies to Los Angeles County. During the 7.5-month epidemic period, 93 reported listeriosis cases (65.5%) involved pregnant women or their offspring with 49 (34.5%) affecting nonpregnant adults (Fig. 2, Table 3). Forty-eight of the 142 listeriosis victims died, giving an overall mortality rate of 33.8%. Thirty deaths occurred among the 87 early fetalheonatal cases; however, no late fetal/neonatal or maternal deaths were reported. All but 1 of the 49 nonpregnant adults had a predisposing condition such as cancer (3 patients), steroid dependency (12 patients), chronic illness (23 patients), age :>65 ( 5 patients), or AIDS (3 patients), which placed these individuals at greater risk (than the normal population) of developing listerial infections [91,2121. After identifying the epidemic L. monocytogenes strain as belonging to serotype 4b, all 105 clinical isolates available for study were phage typed and compared with the strain isolated from Jalisco brand Mexican-style cheese. Results showed that 86 of 105 (82%) clinical isolates were serotype 4b, with the remaining 19 non--serotype 4b isolates originating from listeriosis victims whose illnesses were presumably not related to consumption of contaminated cheese. Of the 86 isolates identified as L. monocytogenes serotype 4b,

-

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372

n

I

N onpre g n an I

...

...

Mate

&

U 4

~~~

...

... ... ... ... ... .. ... ... .... . .... .. .. .... ... ...

0 2

.*:.

Jan

MU

May

*Pr

Jun

Jul

Week of Positive Culture

FIGURE2 Listeriosis cases classified according t o risk group in Los Angeles Country, January 1 t o August 15, 1985. Arrow designates the time of recall 11451.

TABLE 3 Clinical and Demographic Data o n 142 Listeriosis Cases Occurring in Los Angeles County, California, Between January 1 and August 15, 1985 Fetal or neonatal Variable No. of patients Mean age Race or ethnic group: number (%) Hispanic White Black Asian Fatalities (%) Epidemic phage type (%) Mean birth weight (kg) Septicemia (%) Meningitis (%) Septicemia -t meningitis (5%) Other positive culture (9%) Source: Adapted from Ref. 145.

Early

Late

Maternal

Non pregnant adults

87 32 weeks’ gestation

6 38 weeks’ gestation

93 26 yr

49 58 yr

81 (87) 10 (1 ) 0 2 (2 0 75

14 (29) 26 (53) 7 (14) 2 (4) 18 (37) 27 71 14 14 2

-

30 (34) -

2.54 88 2 6 4

-

0 3.15 17 67 17 0

-

52 0 0 48

3 13

Foodborne Listeriosis

63 (73%) sfrains were of the same phage type as strains isolated from the contaminated cheese. In several follow-up subtyping studies, clinical and cheese isolates were of the same multilocus enzyme electrophoresis type [55,110], ribotype [ 1 101, and pulsed-field gel electrophoresis type [65,163], and they also gave identical restriction enzyme [235]and random amplified polymorphic DNA patterns [74], thereby confirming the involvement of Jalisco brand cheese in this outbreak (Fig. 3). The 23 remaining clinical isolates belonging to nonepidemic phage types presumably represented non-cheese-related background cases of listeriosis that occurred throughout the year. Sporadic sale of tainted cheese in a few family-owned grocery stores and restaurants, along with a likely listeriosis incubation period of 3 days to 2 weeks, are both, at least partly responsible for those cases which occurred beyond the middle of July with secondary infections being spread by fecal shedding also a probable contributing factor [ 1531. Although these statistics are fairly typical for human listeriosis cases, the most striking feature in Table 3 is that 81 of the 93 motherhfant pairs that contracted listeriosis were of Hispanic origin. Furthermore, many of these economically disadvantaged Hispanics sought treatment at the Los Angeles County-USC Medical Center. Clustering of cases at a single medical facility was instrumental in uncovering, this outbreak of foodborne listeriosis, since this epidemic would have likely gone unnoticed if the cases had been distributed evenly among the nearly 200 major hospitals in metropolitan Los Angeles. As previously mentioned, epidemiologists and health officials used data collected from two case-control studies to trace this outbreak first to1 Mexican-style fresh cheese and then to Jalisco brand Queso Fresco and Cotija cheese. The fact that these strong-

J an

Feb

MU

APT

May

Jun

Jul

Aug

Week of Positive Culture

FIGURE3 Listeriosis cases classified according t o epidemic and nonepidemic phage types in Los Angeles County, January 1 t o August 15, 1985. Arrow designates the time of recall.

314

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flavored cheeses are a common part of the Hispanic diet and not widely consumed by other individuals was another key in determining the exact source of this epidemic. Thus recognition of this outbreak was largely possible because of the use of the Los Angeles County-USC Medical Center by many economically disadvantaged listeriosis victims and the predominance of cases among Hispanics, which in turn precipitated the involvement of Mexican-style cheeses (i.e., products which few other groups of individuals consume on a regular basis). Hence one can easily speculate that this clustering of cases would not have been observed if a nonethnic food such as Cheddar cheese, milk, and ordinary fruits or vegetables-all of which are consumed by most individuals-had been contaminated with L. monocytogenes. Under such circumstances, an epidemic would probably have gone undetected. Without the increased awareness that this outbreak brought to the scientific community, additional cases of foodborne listeriosis, including the 1987 outbreak linked to consumption of Vacherin Mont d’Or soft-ripened cheese, would likely have gone unnoticed, as the Swiss outbreak previously had for almost 10 years. Hence although Murray et al. [ 1671 can be credited with the first accurate description of L. monocytogenes, those individuals who investigated the I985 listeriosis outbreak in California (and to a lesser extent, outbreaks of listeriosis associated with coleslaw and possibly pasteurized milk in Canada and Massachusetts, respectively) can be credited with fostering the emergence of L. monocytogenes as a serious foodborne pathogen of worldwide concern. Once Jalisco brand cheese was positively identified as the vehicle of infection, local, state, and federal investigators were confronted with the task of determining how the cheese became contaminated (see Fig. 1). Additional testing of Jalisco brand cheese indicated that L. monocytogenes was present in cheese manufactured from January to mid June, thus indicating an ongoing problem at the cheese factory. Investigators then focused their attention on three areas: (a) raw milk supply, (b) adequacy of pasteurization, and (c) possible contamination of the cheese during manufacture, packaging, and/or ripening. However, before interpreting the results from these investigations, it would be prudent to deal with methods used to manufacture those varieties of Mexican-style cheese that were directly linked to cases of listeriosis and also with behavior of L. monocytogenes in the finished product. Queso Fresco, or fresh cheese (also known as Ranchero, Estilo Casero, or Quesito), is among the most popular and widely distributed Mexican-style cheeses. Unlike most cheese varieties, Queso Fresco is traditionally prepared without a lactic acid bacteria starter culture. Curd is formed by coagulating warm skim milk with rennet or a similar coagulant. The resulting curd is drained in cheesecloth, salted, packed into hoops, and pressed under weights for several days. The final product, which is consumed without additional aging, has a slightly grainy texture and can be sliced or shredded for cooking. Cotija, also known as Queso Sec0 (dry cheese) or Queso Anejo (aged cheese), is another white cheese. After cutting, the curd is pressed in large round hoops and cured at least 3 months to produce a dry, sharp-flavored, odorous cheese, which in some respects resembles Italian Parmesan. It is important to realize that these cheese-making procedures produce favorable conditions for multiplication of L. monocytogenes in the final product. The relatively high moisture content of these Mexican-style cheeses, and absence of a starter culture which leads to pH values 1 5 . 6 in the finished product, both played crucial roles in allowing L. monocytogenes to grow in the cheese during refrigerated storage [ 1211. According to Lee

Foodborne Listeriosis

3 15

[ 1421, surface and interior samples of frozen Jalisco brand cheese examined some months after the recall contained 1.4 X 104and 5.0 X 104L. rnonocytogenes CFU/g, respectively, which supports the hypothesis that the pathogen grew in cheese during refrigerated storage. Numbers of listeriae increase approximately 10-fold during cheese making as a result of entrapment within curd particles. If one assumes that the pathogen did not grow in cheese during storage, then the milk from which the cheese was prepared would have had to contain unreasonably high levels of listeriae (- 1000-5000 CFU/mL) to produce the populations observed by Lee in the finished product. It also is noteworthy that the epidemic strain of L. rnonocytogenes that was designated as strain California (CA) by Ryser and Marth [202] has since proven to be less hardy in Cheddar [202], Camembert [203], brick [204], Colby (2401, feta [ 1791, and blue cheese [ 1781 than strains Scott A (clinical isolate from the 1983 “milkborne” listeriosis outbreak in Massachusetts), V7 (raw milk isolate from Massachusetts), and/or Ohio (OH) (isolated from Liederkmnz cheese manufactured in Ohio). Comprehensive sanitation inspections of the cheese factory were conducted immediately after the recall to assess the possibility that the cheese was contaminated during manufacture, packaging, and/or storage. Although the factory received a satisfactory sanitation rating of 85 on a scale of 100, numerous problem areas were cited which included suspended filth on electrical wires near cheese vats, peeling paint above a pasteurizer vat, condensate dripping on cheese in a walk-in refrigerator, and a major ant infestation. Several L. monocytogenes isolates from environmental samples (i.e., cooler condensate, cheese curd, insects, pasteurizer) also yielded the same restriction enzyme profile as the epidemic strain [235], thereby confirming several possible modes of transmission. Although these environmental sources could have contributed to sporadic contamination of the finished product, the fact that the epidemic strain was isolated from 22 of 85 lots of cheese produced between January and mid June as well as from Cotija Fresco cheese [235] indicates that an ongoing problem existed in the factory. Hence, contact between cheese and the factory environment was likely not to be the major route of contamination in this outbreak. At the time of the recall, government officials considered faulty pasteurization as one of the most likely means by which the cheese became contaminated. Initial factory inspections uncovered various pasteurization problems related to record keeping and recording charts; however, the time and temperature at which milk was pasteurized exceeded minimum requirements (71.7”C [16l0F]/15 s). Dye testing later revealed a number of pin-sized holes in the pasteurization unit’s heat-transfer plates which separate raw and pasteurized milk. However, since further inspection demonstrated that the booster pumps of the pasteurizer had maintained a higher pressure on the pasteurized rather than raw milk side of the heat exchanger, raw milk would not have passed through the pinholes found in the pasteurizer plates. Hence pasteurization failure was no longer suspected as the source of contamination [7]. Final reports indicate that L. rnonocytogenes most likely entered the cheese during manufacture through direct addition of raw milk. Toward the end of June 1985, investigators documented that the firm received nearly 700,000 pounds (- 10%) more raw milk between April 1 and June 12, 1985, than could have been pasteurized given the capacity of their pasteurizer. Additionally, on several days only 150,000 of 200,000 pounds of milk received was pasteurized. These enormous discrepancies between raw milk received and the quantity pasteurized suggest that unpasteurized milk was deliberately mixed with pas-

316

Ryser

teurized milk for cheese making [ 1451. This conclusion also is supported by the fact that cheese supposedly prepared from pasteurized milk contained excessive levels of alkaline phosphatase-a native, heat-labile enzyme normally destroyed during proper pasteurization. However, some caution must be used in interpreting these results, since Pratt-Lowe et al. [ 1871demonstrated that California Queso Fresco cheese occasionally contains microorganisms which produce a heat-labile alkaline phosphatase similar to that found in raw milk. Under these conditions, cheese prepared from properly pasteurized milk may falsely appear as having been manufactured from raw milk. Continued preparation of Mexicanstyle cheese from a mixture of raw and pasteurized milk also is compatible with the pattern of listeriosis cases that occurred over a period of 7 months. Toward the end of July 1985, investigators visited 27 dairy farms that supplied raw milk to the cheese factory [ 145,1501. Although no Listeria spp. were detected in raw milk or milk filters from dairy farms supplying the Jalisco cheese factory, the same epidemic phage and restriction enzyme type of L. monocytogenes was isolated from jocoque (a sour cream-like product), sodium caseinate (a jocoque ingredient), and cottage cheese by-products produced by another company that shared the same raw milk source with Jalisco cheese [235]. However, no cases of listeriosis were epidemiologically linked to consumption of either of these products. According to Hird [121], the L. monocytogenes epidemic strain was uncommon in California during the 1 1-year period preceding the outbreak, with only three to five L. monocytogenes serotype 4b isolates belonging to the same epidemic phage type. Even though the epidemic strain was never isolated from raw milk, evidence described in the preceding paragraphs strongly suggests that raw milk was the probable source of L. monocytogenes. Major outbreaks of foodborne disease not only cause great human hardship but also major financial difficulties from lost product and employee wages as well as medical bills and lawsuits. Jalisco Mexican Products, Inc., was forced to close its doors and declare bankruptcy shortly after its products were recalled, because the company could no longer meet expenses. On March 27, 1986, Los Angeles County prosecutors filed 60 misdemeanor charges against the president and vice-president of the company for alleged shortcuts and inadequate safety precautions that routinely occurred in the factory during cheese making [ 151. On May 20, 1986, the vice-president of the firm was sentenced to 60 days in jail, 2 years of probation, and was fined $9300 in connection with manufacturing and selling Listeria-contaminated cheese [ 141. In addition to investigative costs, which reportedly totaled $617,204, the company also is believed to be facing up to $700 million in lawsuits filed by some of the victims [225], making this one of the costliest and deadliest outbreaks of foodborne illness in U.S. history. After criticism for not recalling the contaminated Jalisco brand cheese sooner, state and federal officials issued a Class I recall for Mexican-style cheese produced by a second Los Angeles-area firm. Although this Listeria-contaminated cheese was supposedly prepared from raw milk as shown by the presence of alkaline phosphatase [ 101, the recall was subsequently downgraded to Class I1 (i.e., a situation in which use of the product may cause temporary or medically reversible adverse health consequences) after laboratory results confirmed that these cheeses contained L. innacua-a nonpathogenic Listeria species-rather than L. monocytogenes. Later investigators also showed that this cheese was prepared from properly pasteurized milk. Hence, one must conclude that falsepositive results were obtained with the phosphatase test, as described earlier in this chapter .

Foodborne Listeriosis

317

Vacherin Mont d'Or Soft-Ripened Cheese: Switzerland, 1983- 1987 Human listeriosis has been observed in Switzerland for many years [85], particularly in the Canton of Vaud, which borders France to the west and Lake Geneva to the south. The early scientific literature contains several reports of sporadic listerial infections that occurred in and around Lausanne, the population center of Vaud. Over 20 years ago, Piolino and de Kalbermatten [ 1831 reviewed five adult cases of listeriosis that were diagnosed at Vaudois University Hospital Medical Center in Lausanne (VUHC) between December 1964 and February 1967. Listeria monocytogenes serotype 4b was isolated from four of five patients who exhibited symptoms of meningoencephalitis. Although no common source of infection was demonstrated among these individuals, the authors suggested a possible role of domestic livestock in spreading listeriosis. In 1981, Yersin et al. [239] reviewed 10 adult cases of listeriosis (ages 35-76 years) that were diagnosed at VUHC between January 1974 and January 1980. (Ten cases of neonatalhfant listeriosis also were treated at this hospital during the same period.) All 10 adult patients suffered from one or more underlying illnesses, which increased their chance of developing listeriosis. During this 6-year period, two cases of septicemia, six of meningitis-encephalitis, and two of encephalitis were recorded, including five deaths (50% mortality rate). As in the previous study, none of the adult cases of listeriosis could be traced to an exact source of infection. Subsequently, Malinverni et al. [ 1481 noted that only 20 sporadic listeriosis cases were diagnosed at VUHC between 1974 and 1982, with a mean of three cases per year. Thus these figures reflect an endemic rate of approximately five listeriosis cases/ 1O6 population in the Canton of Vaud during this 9-year period. In sharp contrast to these findings, a cluster of 25 listeriosis cases (14 adults and 1 1 maternal/fetal) was observed at the same medical facility between January 1983 and March 1984 [147,148], with 15 additional cases being documented in surrounding hospitals (e.g., Geneva and Neuchatel) in western Switzerland during the same 15-month period [ 13,1471. This epidemic was somewhat atypical in that most adult listeriosis victims had been in good health before the outbreak. In addition, an unusually high incidence of brain-stem encephalitis was observed among patients. Eleven of 14 adults were treated at VUHC for meningitis and/or encephalitis, 5 of whom eventually died, giving a mortality rate of 45%. Septicemia was observed in the remaining three patients, two of whom were pregnant women. According to Bille [52] and Malinverni et al. [ 1471, 38 of 40 (95%) L. monocytogenes strains isolated from listeriosis victims during the epidemic period were of serotype 4b, whereas only 9 of 15 (60%) clinical isolates obtained during the previous 6-year epidemic period were serotype 4b. More important, 33 of 36 (92%) L. monocytogenes serotype 4b cultures were of two unique phage type configurations as compared with only 4 of 9 (44%) serotype 4b cultures obtained during the previous 6 years. This, in turn, suggested that ii common-source listeriosis outbreak had occurred in western Switzerland between January 1983 and March 1984. Unlike endemic cases of listeriosis treated between 1974 and 1982, most listerial infections recorded during the epidemic period were diagnosed during the winter months. However, listeriosis cases were uniformly distributed throughout the general population and were apparently unrelated to listeriosis in animals. Despite an in-depth investigation

318

Ryser

that included interviews with patients and a search for L. monocytogenes in several hundred food items, neither the source nor the mode of Listeria transmission could be found. Working under the assumption that a similar listeriosis outbreak was likely the following winter, public health officials initiated a case-control study using listeriosis cases that were diagnosed in French-speaking Switzerland between November 1, 1984, and April 30, 1985 [13,52]. Overall, 16 cases (7 adults and 9 motherhfant pairs) were identified and compared with 49 controls matched for age, sex, and underlying conditions. Fifteen of 16 (94%) patients were infected with L. monocytogenes serotype 4b, with 5 of 16 (3 1%) isolates belonging to the same phage type. Although these five cases suggest a possible epidemic focus, data obtained from questionnaires dealing with professional and home exposure as well as types of food consumed (e.g., milk products and raw vegetables) were inconclusive. In response to the 1985 listeriosis outbreak in California linked to consumption of Mexican-style cheese, Swiss officials initiated a series of surveys to determine the incidence of Listeria spp. in different dairy products, the results of which are summarized in Chapter 12. During one such survey of soft, semihard, and hard cheeses, Breer [62] isolated L. monocytogenes from 5 of 25 surface samples of Vacherin Mont d’Or, a soft, smear-ripened cheese that is only manufactured from October to March and consumed primarily in and around the Canton of Vaud. Subsequent test results indicated that all L. monocytogenes isolates from Vacherin Mont d’Or cheese belonged to serotype 4b and also demonstrated that two L. monocytogenes phage types isolated from this cheese were identical to most clinical strains isolated during the 1983- 1986 epidemic period. Investigators in Switzerland [52] then examined over 200 types of domestic and imported soft cheeses, 8- 10% of which contained L. monocytogenes. However, based on serotyping and phage typing, these strains as well as other food and dairy product isolates were distinctly different from those found on the surface of Vacherin Mont d’Or cheese. A subsequent review of hospital records indicated that 122 listeriosis cases involving 57 adults (Table 4) and 65 motherhnfant pairs were diagnosed in the Canton of Vaud between 1983 and 1987 [66] (epidemic rate of -50 cases/ 1O6 population/yr) as compared with only 28 cases between 1974 and 1982 (endemic rate of -5 cases/106 population/ yr) [37]. Interestingly, 84% of the cases that occurred during the epidemic period were identified between October and April. Thirty-four of 122 patients died, giving a mortality rate of 28%, with 18 of 57 (32%) adult cases proving to be fatal. Although the two previous case-control studies failed to uncover the source of this epidemic, a third case-control study conducted in 1987 demonstrated that 31 of 37 (84%) cases had consumed Vacherin Mont d’Or cheese as compared with only 20 of 51 (39%) controls [54]. In addition, investigators were able to isolate the epidemic strain of L. monocytogenes from a piece of Vacherin Mont d’Or cheese that had been partially consumed by one of the victims. Armed with this information, Swiss authorities halted production of Vacherin Mont d’Or cheese on November 20, 1987, and recalled the product throughout Switzerland [20,27,54]. Overall, 111 of 120 (93%) clinical isolates available from the epidemic period belonged to serotype 4b, with 98 of l l 1 (85%) serotype 4b strains matching the two epidemic phage types that were isolated from Vacherin Mont d’Or cheese. Several years later, clinical and cheese isolates were found to be identical based on multilocus enzyme electrophoresis [ 1711, pulsed-field gel electrophoresis [60,64], ribotyping [%I, restriction enzyme analysis [64,235], randomly amplified polymorphic DNA patterns [74], and pyrolysis mass

319

Foodborne Listeriosis

TABLE 4 Description of 57 Adult Listeriosis Cases Diagnosed i n Western Switzerland from 1983 to 1987

Manifestation ~

Characteristic Sex Males Females Age (years) Median Range >65 (%) Underlying illness (%)a S ymptoms Median onset time Fever (%) Vomiting/diarrhea (%) Meningismus (%) Altered mental state (%) Outcome Cured Neurological sequelae Fatal (%)

~~~

Septicemia (n = 12)

Meningitis (n = 23)

Meningoencephalitis (n = 22)

9 3

12 11

12 10

75 44-85 10 (83) 12 (100)

69 3 1-96 15 (65) 18 (78)

55 37-79 6 (27) 10 (45)

12 hours 12 (100) 4 (33)

2 days 21 (91) 11 (48) 18 (78) 19 (83)

3 days 17 (77) 11 (50) 14 (64) 13 (59)

9 -

15

3 (25)

7 (30)

6 8 8 (36)

1

Includes leukemia, cancer, alcoholism, immunosuppressive drug therapy, diabetes, and AIDS. Source: Adapted from Ref. 66.

spectroscopy [ 1011, thus confirming Vacherin Mont d’Or cheese as the infectious vehicle. Interestingly, this epidemic strain also is of the same phage type [60,64,1811, enzyme type [56], ribotype [56], and pulsed-field gel electrophoretic type [60,64] as strains isolated during the 1985 listeriosis outbreak in California [181]. However, two distinct DNA restriction endonuclease profiles [ 1721 were eventually identified among the Swiss epidemic strains corresponding to epidemic phage types I and 11, with Eioerlin et al. [60] also dividing the epidemic electrophoretic enzyme type into two major subtypes using pulsed-field gel electrophoresis. The fact that the Swiss and California epidemic strains are closely related to each other and are also phenotypically and genotypically similar to strains involved in major outbreaks traced to coleslaw, cheese, and p2t6 in Canada, Denmark, and France, respectively [65,110,126,163], raises some interesting questions as to why many of the most serious outbreaks in North America and Europe have been confined to this one particular strain. When Boerlin and Piffaretti [59] examined 181 Swiss L. rnonocytogenes isolates from clinical, veterinary, food, and environmental sources, the Swiss epidemic enzyme type comprised 26.5 and 24.2% of all strains collected during (1983-1987) and after (1988- 1989) the Swiss outbreak, respectively (Table 5). Furthermore, the epidemic enzyme type was widely distributed, with this strain being recovered from humans (clinical and fecal samples), animals (clinical and/or fecal samples from cows, sheep, and goats), meat/meat products, cheese/milk, and the environment (soil, silage). Since this particu-

Ryser

320 TABLE 5 Swiss Epidemic and Nonepidemic L. monocytogenes Enzyme Types Recovered from Various Sources within Switzerland During 1983-1989

Source of L. monocytogenes strain Enzyme type Epidemic (n = 1 ) Nonepidemic (n = 49)

Humans Animals Meat/Meat products (n = 40) (n = 43) (n = 49) 13 3O/2Oa

24 25/16

1 39/14

Cheese/Milk (n = 19)

Environment (n = 30)

4 15/13

241 I3

6

Number of nonepidemic straindnumber of nonepidemic enzyme types. Source: Adapted from Ref. 59. a

larly virulent strain is dominant in ruminants and since a similar strain was responsible for cheese-related outbreaks of listeriosis in California and Denmark, recovery of this L. rnonocytogenes subtype from foods has taken on added public health significance. However, the exact importance of this particular electrophoretic enzyme type as compared with others in foodborne listeriosis cannot yet be adequately assessed until more information is available regarding the distribution of different L. monocytogenes subtypes in nature along with the ability of these various strains to grow and/or survive in dairy products and initiate disease in both humans and laboratory animals. Most of the tainted cheese was marketed in Switzerland; however, small quantities were exported to other countries, including England and the United States. Hence, on November 25, 1987, health officials in England warned the general public against consuming Vacherin Mont d’Or cheese, which was available at a few delicatessens and specialty cheese shops in and around London [1,21]. Similarly, FDA officials in the United States became concerned after a major newspaper reported that five specialty shops in New York City and a chain of 37 stores in Connecticut had been distributing the cheese since November 1987 [20]. These recall efforts were largely successful, since all known listeriosis cases linked to consumption of this cheese were confined to Switzerland. Immediately after the recall, Swiss authorities began investigating possible routes by which Vacherin Mont d’Or cheese could have become contaminated. According to Bille [53,54], the cheese implicated in this outbreak was produced at 40 different factories located in western Switzerland. All contaminated cheese was reportedly prepared from Listeria-free cow’s milk. Following coagulation of milk, the resulting curd was dipped into wooden hoops and allowed to drain for 1-2 days. When thoroughly drained, the hooped cheeses were transported to 1 of 12 cellars (i.e., caves) located throughout western Switzerland and ripened for -3 weeks on wooden shelves during which time the cheeses were turned daily and brushed with salt water. Once ripened, the cheeses were packaged for sale and the wooden hoops were returned to the cheese factory. From this description, it is apparent that ample opportunity existed for contamination of Vacherin Mont d’Or cheese, particularly during ripening. In fact, the epidemic strain [5] was detected in 18.5% of surface (rind) samples from Vacherin Mont d’Or cheese at levels of 104-106 L. rnonocytogenes CFU/g and also on 6.8% of wooden shelves and 19.8% of brushes used in the ripening cellars. In all likelihood, this outbreak began several years earlier when L. monocytogenes entered one of the 40 cheese factories in raw milk from an infected dairy herd [59]. Although this outbreak was first detected in 1983, the epidemic strain was initially isolated from a listeriosis victim in 1977, which suggests that this outbreak may have been devel-

Foodborne Listeriosis

321

oping for at least 7 years. Investigations showed that nearly hall' of the 12 ripening cellars were contaminated with one or both epidemic strains of L. monocytogenes, thus suggesting cellar-to-cellar spread of the pathogen through production and distribution practices. This theory is strongly supported by the fact that cheeses produced at all 40 factories were normally transferred between different cellars for ripening and/or distribution. The practices of brushing cheeses with salt water, ripening cheese in wooden hoops, and returning these hoops to the cheese factory also were important factors in disseminating L. manocytogenes to different ripening cellars. Following the recall, all 40 factories in which Vacherin Mont d'Or cheese was manufactured were thoroughly cleaned and sanitized. More important, all wooden material (e.g., shelves, boxes, hoops) was removed from ripening cellars anti burned, The cellars were then thoroughly cleaned, sanitized, and refitted with metal shelves and easily sanitized equipment. Once this work was completed, experimental batches of Vacherin Mont d'Or cheese were produced during a 2-month period and examined for the epidemic strain of L. monocytogenes to assure government officials that the pathogen was eliminated from all ripening cellars. These clean-up efforts proved to be highly successful, with only two cases of listeriosis being reported in western Switzerland between January and September of 1988 [53]. Although both of these cases resulted from nonepidemic strains, multilocus enzyme electrophoresis later demonstrated that 45 of 145 (28%) human clinical and 44 of 116 (37%:) animal strains of L. monocytogenes isolated in Switzerland between 1988 and 1993 belonged to the previously identified epidemic enzyme type (601. After further analysis by pulsed-field gel electrophoresis, 34 of these 26 I ( 1 3%) humadanimal isolates matched the epidemic strain recovered from Vacherin Mont d"Or cheese, thus suggesting a continued presence of this strain in the natural environment. As a rr:sult of this outbreak which by one account cost an estimated $1.4 million [232], several steps have been taken to control and limit the extent of listeriosis in Switzerland [52,53]. First, health authorities are systematically screening high-risk foods for L. monocytogenes and have adopted a zero tolerance for the pathogen in 10-g samples. Second, physicians and laboratories are now required to notify health officials of every new case (i.e., clinical isolate) of listeriosis occurring throughout Switzerland. Finally, since 1990 the Swiss National Center for Listeriosis in Lausanne has been actively collecting human, animal, food, and environmental isolates and further characterizing these Listeria strains according to serotype, phage type, ribotype, enzyme type, DNA restriction pattern, and pulsed-field gel electrophoresis profile. These efforts will serve to identify the exact endemic rate of human listeriosis in the general population and lead to faster recognition of possible future listeriosis outbreaks as well as the vehicleis involved.

Blue-Mold/Hard Cheese: Denmark, 1989-1990 Listeriosis has been a reportable disease in Denmark since 198 1, with two apparent common source outbreaks being identified, the first of which occurred during 1985- I987 and included 35 cases of unknown origin [208]. Two years later, one specific phage type of L. monocytogenes serotype 4b was identified as being responsible for 26 of 69 (38%) listeriosis cases that were reported from March 1989 to December 1990 (Fig. 4) [131]. Although found throughout the country, epidemic cases were most frequently seen in suburban Aarhus, Denmark's second largest city. Twenty-three cases occurred in adults, 13 of whom suffered from underlying illnesses (i.e., leukernia, cancer, AIDS, diabetes, alcoholism), with three cases involving pregnant women. Prirnary manifestations included

322

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FIGURE4

Monthly distribution of epidemic and non-epidemic listeriosis cases in Denmark during 1989 and 1990. (Adapted from Ref. 131.)

meningitis ( IS cases) and septicemia ( 8 cases). Six of 23 adults died while hospitalized, giving a mortality rate of 26%. Evaluation of 90 food history questionnaires given to epideriiic/nonepideinic cases and matched controls showed a clear epidemiological link between consumption of Danish blue-mold cheese and cases of listeriosis, with one brand of cheese being cited in particular. Unfortunately. investigators were unable microbiologically to confirm blue-mold cheese as the vehicle of infection. However, one year earlier. the FDA issued a Class I recall for L. rizorzoc.?.togc.rzr.s-contaminated Danish blue cheese that had been shipped to the United States [ 23,251. Routine dairy inspections also indicated that the epidemic strain was present in the dairy environment as evidenced by recovery of this strain from eight different Danish hard cheeses. The fact that these extremely popular hard cheeses were consumed by more than 90% of both patients and controls makes these cheeses another plausible vehicle of infection. Inability to recover the epidemic strain from other packaged foods lends additional support for involvement of Danish blue and hard cheeses. Followstrain to be of international importance, since up studies showed this L. rizorzoc:\~to,~c’rzes the identical phage type also was responsible for two major foodborne outbreaks in Switzerland (Vacherin Mont d’Or cheese, 1983- 1987) and France (jellied pork tongue. 1992).

Brie de Meaux Cheese: France, 1995 A nationwide listeriosis surveillance program has been operating in France since 1987, with the French National Reference Center (NRC) (Pasteur Institute. Paris) being responsible for collecting. serotyping. and phage typing L. 1izorzo~~’togerie.s isolates from human. veterinary, and food sources [ 1261. Over 10,000 Listor-iir strains were received annually during I993 and 1994, so routine characterization is limited to serotyping and phage typ-

Foodborne Listeriosis

323

ing. with molecular typing methods such as pulsed-field gel electrophoresis and ribotyping being used only in epidemiological investigations. Using this surveillance system, three minor outbreaks involving less than 16 cases were detected from 1987 to 1992, including one in the Strasbourg area [ 143,196.198j. More important, three major foodborne listeriosis outbreaks were identified in 1992, 1993. and 1995. the most recent of which was traced to Brie de Meaux cheese. Between April 2 and 19 of 1995. the NRC received six L. rriorioc\’tos:eiies human isolatcs from hospitals in different regions of France which were soon identified as belonging to an unusual phage type that had been previously responsible for only 33 cases of listeriosis ( 1 [ < 1 % I to 8 [ 3 % ] cases annually) since 1987 [ 109,126). These findings prompted the NRC to inform the Ministry of Health on April 28 about a possible outbreak. A search of NRC records indicated that the same phage type had been recovered from 5 of 2200 food samples ( 1 delicatessen product, 4 Brie de Meaux cheeses) tested from January to April. Further characterization of these strains by pulsed-field gel electrophoresis indicated that the six patient and four cheese isolates were identical. Based on follow-up epidemiological investigations, all victims identified as of May 10 consumed Brie de Meaux cheese (a raw milk soft cheese) that had been cut and purchased at either supermarkets or small local markets [ 1091. Additional investigative work by the Ministry of Agriculture coupled with a case-control study soon implicated one particular brand of Brie de Meaux cheese. with this product being recalled from the market on May 18. Additional cheeses exported to Belgium were also recalled without incident shortly thereafter [ 2241. Isolation of the epidemic phage type and pulsed-field gel electrophoresis type from eight Brie de Meaux cheeses and several other cheeses ripened at the same facility confirmed the role of Brie de Meaux cheese in this outbreak [ 126,235). At the time of the May 18 recall. 20 epidemic cases were documented (Fig. 5 ) , with the victims residing in 8 of 22 French regions. Of these 20 cases, 1 I among pregnant women

FIGURE5 Distribution of epidemic and non-epidemic listeriosis cases in France from January to July, 1995. (Adapted from Ref. 126.)

324

Ryser

led to two spontaneous abortions, four premature deliveries, and two stillbirths, with the remaining nine cases involving immunocompromised adults (7 cases) and the elderly (2 cases). By the time this outbreak finally ended in July of 1995, 13 additional cases were confirmed by phage typing and pulsed-field gel electrophoresis, bringing the total number of cases to 33. Interestingly, this outbreak strain belonged to a new epidemic clone which was clearly distinct from the two L. monocytogenes strains responsible for the other major outbreaks in Canada (coleslaw 1981 ), California (Mexican-style cheese I985), Switzerland (Vacherin Mont d’Or cheese 1983- 1987), United Kingdom (pGt6 1987- 1989), Denmark (blue-mold/hard cheese 1989- 1990), France (jellied pork tongue I992), and France (pit6 1993) which either have been or will be discussed shortly. In 1997, the NRC also identified 14 listeriosis cases over a 4-month period that were directly traced to Pont I’EvEque cheese produced in Normandy [241]. The implicated cheese was manufactured from raw milk and contained L. monocytogenes serotype 4b at a level of > 1000 CFU/g. Although some of this cheese was exported to Sweden, no additional cases were reported.

Additional Reports of Cheeseborne Listeriosis Other than listerial infections associated with the aforementioned outbreaks in California and Switzerland, only two additional well-documented cases of nonfatal listeriosis have been directly linked to consumption of contaminated cheese, both of which occurred in England. The first case involved a healthy, nonpregnant 36-year-old woman who developed meningitis on January 9, 1986, 9 days after consuming a full-fat, soft French cheese [ 12,1041. According to Bannister [45], identical phage types of L. monocytogenes serotype 4b were isolated from the woman’s cerebrospinal fluid and a partially consumed package of cheese. However, the fact that six unopened packages of the same cheese failed to yield viable listeriae suggests that the cheese may have become contaminated in the refrigerator rather than during manufacture. In response to the 1987 outbreak of listeriosis in Switzerland and the discovery of L. monocytogenes in an increasing variety of foods, health officials in England began treating all reported listeriosis cases as possibly being foodborne. Follow-up questions about different foods consumed by victims led to discovery of a second cheese-related case of listeriosis in February of 1988 [ 1 171. According to official reports [30,44,158], a previously healthy, nonpregnant 40-year-old woman was admitted to a London hospital with meningitis following a 4-day bout with a “flu-like” illness. Identical phage types of L. monocytogenes serotype 4b were eventually isolated from the woman’s cerebrospinal fluid, stool, and an open package of Anari raw goat’s milk cheese (a Greek-style soft cheese) from which the victim had consumed -85 g 24 h before onset of symptoms. Four additional unopened packages of Anari cheese (from the same lot) purchased from a retail store yielded the same L. monocytogenes strain at levels of 3-5 X 10’ CFU/g. Consequently, this cheese was withdrawn from the market in February of 1988, with production not resuming until the summer of 1988. Following news of this cheeseborne listeriosis case, McLauchlin et al. [ 1581 examined the extent to which other dairy products produced by this manufacturer were contaminated with listeriae and attempted to identify the exact source of contamination. The Anari goat’s milk cheese responsible for the aforementioned case of listerial meningitis came from a one-man, off-farm dairy factory at which Halloumi, Cheddar, feta, soft chive, and Gjestost cheese as well as yogurt also were produced from goat’s milk. According to these

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325

investigators, 16 of 25 (64%) retail cheeses and 12 of 24 (50%) cheeses obtained directly from the factory over a period of 1 1 months yielded L. rnonocytogenes serotype 4b, with all goat’s milk cheese varieties except feta testing positive for the pathogen. Although 22 of 24 (92%) positive cheeses contained < 10 L. rnonocytogenes CFU/g, the two remaining cheeses that were purchased from a retailer 10 weeks before their sell date contained > 105 L. monocytogenes CFU/g, thus suggesting that the pathogen grew in the cheese during retail storage. This hypothesis was subsequently confirmed using naturally contaminated (<10 L. rnonocytogenes CFU/g) 2- to 3- day-old Anari and Ilalloumi cheeses that were periodically analyzed for numbers of listeriae during 8 weeks of refrigerated storage. Although no listeriae were detected in samples of raw goat’s milk. or yogurt obtained directly from the factory, L. rnonocytogenes serotype 4b was recovered from shelving within the factory, which in turn suggests that the cheese most likely became contaminated during the final stages of manufacture or packaging. Most important, phage typing indicated that 66 of 68 (97%) L. rnonocytogenes isolates recovered from various cheeses and factory shelving were identical to the strain isolated from the patient’s cerebrospinal fluid and stool. With the aforementioned evidence, there appears to be little doubt that this case of listerial meningitis resulted from consumption of Anari goat’s milk cheese in which L. rnonocytogenes likely grew to high numbers during retail storage. Although the only confirmed cases of cheeseborne listeriosis in the United States have been those associated with consumption of Jalisco brand Mexican-style cheese in 1985, cheese has been suggested as a vehicle of infection in several additional cases. These primarily unconfirmed reports include (a) isolation of Listeria from the blood of a 7-day-old California infant whose mother consumed a raw milk cheese 2 weeks before delivery [9]; (b) three cases of listeriosis in Arizona in which the victims consumed nonJalisco brand soft Mexican-style cheese; (c) a possible association between listeriosis and consumption of Italian cheese; (d) one case of listeriosis in California in which a woman delivered an aborted fetus after eating Monterey Jack cheese prepared from raw milk; (e) an alleged listerial abortion by a woman in New York who consumed contaminated feta cheese; (9 one case in which L. rnonocytogenes serotype 4b was isolated from a 3-yearold Washington state girl and cheese found in her family’s refrigerator [34]; (g) isolation of an identical L. monocytogenes strain (same phage type and electrophoretic enzyme type) from ii listeriosis patient in Philadelphia and from cheese that the victim reportedly consumed 1801; (h) one case involving a healthy woman from New Jersey who supposedly contracted listeriosis after consuming Ricotta cheese containing 10’- 1Oh L. monocytogenes CFU/g [ 1421; and (i) the report of a San Bernardino, California, woman who developed a fatal listerial infection after eating locally purchased soft Mexican-style cheese [ 102 1. Despite the recall of approximately 600 million pounds of French soft-ripened cheese in 1986 (see Chap. 12), no cases of listeriosis were linked to consumption of this cheese in the United States. However, several cases were documented in both Canada and England [45]. In one of these Canadian cases, L. rnonocytogmes serotype Ib of the same electrophoretic enzyme type was isolated from the blood of a 66-year-old man and opened packages of imported soft cheese that he consumed [95]. The same strain was later identified in unopened packages of cheese produced by the same manufacturer, thus confirming the role of cheese in this isolated case of listerial bacteremia. Additionally, ingestion of homemade fresh cheese by a pregnant Italian woman was hlamed for the listerial death of her infant three days after delivery [ 1681. Although the scientific literature contains one additional report of an AIDS patient in England who contracted meningitis after consuming Staffordshire cheese, the two non-phage typeable strains of L. rnonocytogenes

326

Ryser

serotype 1/2a recovered from the patient’s cerebrospinal fluid and cheese were subsequently found to exhibit different DNA restriction enzyme patterns, thus negating cheese as the vehicle of infection.

MEAT PRODUCTS Foods of animal origin have long been recognized as potential vehicles of infection, with meat-associated cases of salmonellosis and botulism being recorded in the scientific literature since the 1890s. Following confirmation of L. monocytogenes as a human and animal pathogen during the 1920s, listeriosis was subsequently identified as a zoonosis, a disease transmissible from animals to humans. Hence, when listerial infections in domestic livestock began to emerge with some regularity during the 1930s and 1940s, some individuals, including Wramby [238), who in 1944 first identified Listeria in raw meat, began to speculate that consumption of meat products could play a role in the spread of human listeriosis. Listeria-laden fecal material from asymptomatically infected livestock can readily enter the slaughterhouse environment and contaminate retail raw meats. Meat processors, veterinarians, and others who work closely with animal carcasses will also inevitably come in contact with L. monocytogenes as evidenced by recovery of this pathogen from the hands and gowns of Czechoslovakian line workers during the mid 1970s [86,87]. Although evidence is somewhat conflicting, most of the earlier studies previously reviewed by Ryser and Marth [205] also indicated that increased exposure to L. monocytogenes in the meat industry can lead to higher fecal carriage rates among workers. According to Elischerova and Stupalova [86,88], six clinically healthy Czechoslovakian meat workers who had an opportunity to consume crude and semicrude product during work were asymptomatic fecal shedders of L. monocytogenes. These same individuals also exhibited elevated 0 and H serum agglutination titers against L. monocytogenes serotype 1, which is compatible with oral transmission of Listeria via meat products. Since two of the three meatborne listeriosis outbreaks identified thus far have involved consumption of pit&-a ready-to-eat meat, fish, or vegetable product that is commonly marketed in Belgium, France, Germany, the Netherlands, and the United Kingdom and is consumed without reheating or further cooking, it is appropriate briefly to review the manufacture and safety-related issues surrounding this product. Preparation of meat pit& most often involves chopping pork liver with water, seasoning, salt, and sodium nitrite. This raw product is then either (a) cooked in a mold, decorated, sliced, and sold as loose or vacuum-packaged pit&;or (b) cooked in small hermetically sealed containers. The high water activity and pH of typical pi& provide an ideal growth environment for most bacteria, including L. monocytogenes, which has an estimated doubling time of 19 h in pit& stored at 7°C [76]. Hence, thorough cooking of the raw product, addition of preservatives, and proper packaging are all essential to preventing growth of listeriae to dangerously high levels in this food during 3 or more weeks of refrigerated storage. With this background information in mind, the role of pit6 in one of the largest listeriosis outbreak thus far reported will now be assessed. Numerous reports suggesting possible involvement of meat products in human listeriosis can be found in the scientific literature with over 60 primarily sporadic cases documented since 1955. However, the ability of meat products to serve as vehicles of listerial infection was not fully realized until the late 1980s when consumption of pit&was deemed responsible for over 350 cases of listeriosis in the United Kingdom. As a result of an

Foodborne Listeriosis

327

ongoing, nationwide listeriosis surveillance program in France, two additional meat-related outbreaks came to light in 1992 and 1993, with 279 and 39 cases being traced to Listeria-contaminated jellied pork tongue and pork pSt6 “rilletes,” respectively. These outbreaks will now be discussed in some detail followed by a review of the aforementioned sporadic cases of suspected meatborne listeriosis reported since the 1950s.

Piite: United Kingdom, 1987-1989 In the United Kingdom, a national voluntary reporting program for human listeriosis has been operating since 1967 under the direction of the Public Health Laboratory Service, Communicable Disease Surveillance Center (England, Wales, Northern Ireland), and the Communicable Disease Unit (Scotland), with the Listeria Reference Unit at the Central Public Health Laboratory (Colindale) being responsible for confirming and characterizing all strains received by serotype and phage type. As a result of these efforts, one cluster of 23 listeriosis cases ( 1 0 maternofetal and 13 adult) documented from early June to November of 1987 involved an unusual serotype of L. monocytogenes designated 4b(x) that was previously responsible for only 12 of 842 cases reported i n Britain between 1967 and 1986 [ 1571. Although all 23 clinical isolates were indistinguishable by phage typing and monoclonal antibody typing, subsequent food consumption profiles obtained from 17 cases failed to implicate a common food, brand, or supplier. From 1987 to mid 1989, a large upsurge in human listeriosis cases was observed in the United Kingdom [ 1551 (Fig. 6).

300

250

$

U)

1 200

I

%

0

b

a

E 150 3

z

-.I f’

\\\

\

O L_ _ ~ ~ - ~ _ _ _ 198384 85 86 87 88 89 90 91 92 93 94

_

J

Year

FIGURE6 Reported cases of human listeriosis in England, Wales and Northern Ireland, 1983-1994.

(Adapted from Ref. 155.)

Ryser

328

L. monocytogenes strains classified as serotype 4b phage type 6,7 and serotype 4b(x) were responsible for 366 of 823 cases reported during this period [ 1951, with these two strains being far less common before 1987 and after July 1989 (Fig. 7). Thereafter, the number of human listeriosis cases decreased sharply to pre- 1987 levels. During a routine food poisoning investigation in May of 1989, the Cardiff Public Health Laboratory identified high levels of L. monocytogenes in pit6 taken from one victim's refrigerator. This chance finding prompted an immediate survey of primarily imported pit6 sold from delicatessen display counters throughout southeast Wales between May and August of 1989 [164,165]. Overall, L. monocytogenes was recovered from 75 of 216 (35%) pit& tested at levels ranging from <20 (42 samples) to >104 (10 samples) CFU/g. More important, however, 32% of all positive samples harbored L. rnonocytogenes serotype 4b(x), which was responsible for the aforementioned cluster of 23 cases identified 2 years earlier. Results from this Welch survey prompted two actions in July of 1989: (a) a government health warning to vulnerable individuals about eating pit6 [78] and (b) a far more extensive survey [76] in which 1698 samples of pSt6 marketed in England and Wales were examined for listeriae. Overall, I86 ( 10%) samples contained L. monocytogenes at levels ranging from <200 (12 1 samples) to > 106(3 samples) CFU/g, with 37 of the I86 positive samples harboring > 103CFU/g. Investigators also noted that L. monocytogenes levels were generally higher in (a) pSt6 prepared from fish rather than meat, (b) loose slices rather than prepackaged pate, (c) samples marketed at >7"C, (d) pit6 tested at or

+

1385

4bX

1986

f

4b PT6,7

1987

1988

Other strains

1989

1990

Year

FIGURE7 Annual distribution of selected Listeria monocytogenes strains from hu-

man listeriosis cases reported in England, Wales, Scotland, Northern Ireland, and the Republic of Ireland, 1985-1990. (Adapted from Ref. 159.)

Foodborne Listeriosis

329

beyond the sell-by date, and (e) samples having standard bacterial plate counts of >106 CFU/g, with these findings supporting the reported ability of this pathogen to grow in p2t6 at near-refrigeration temperatures. More important, however, 5 1 of 107 (58%) piit6s produced in Belgium by manufacturer Y contained L. monocytogenes, with 12 of these samples yielding >103CFU/g. Follow-up investigations [ 1591 showed that 96% (48 of 50) of all L. monocytogenes isolates from manufacturer Y’s pGt6 belonged to either serotype 4b phage type 6,7 or serotype 4b(x), with these two strains being responsible for 30-54% of all human listeriosis cases reported during the epidemic period. (Fig. 7). In contrast, only 19% (6 of 31) of pitis from other producers contained these two strains, with cross contamination among pit& handled at delicatessen counters likely contributing to appearance of these otherwise rare strains. A subsequent epidemiological investigation revealed that 13 of 15 patients infected by these epidemic strains had consumed pit6 within 3 weeks of oriset compared with 6 of 17 patients infected with nonepidemic strains. Illness was strongly associated with piit6 consumption; however, pit6 samples were no longer available from the victim’s refrigerators to microbiologically confirm this food as the vehicle of infection. Nevertheless, all available evidence, along with the fact that the reported decline in listeriosis cases after mid 1989 coincided with both a government warning concerning pit6 consumption and removal of manufacturer Y’s pit6 from sale, clearly points to this particular brand of imported pit6 as being responsible for the outbreak observed from 1987 to mid 1989. A much lower incidence of L. monocytogenes in retail p2t6 samples tested in 1990 as compared with 1989, along with the virtual absence of both epidemic strains in pit6 and other foods examined after 1989 [ 1051, further support involvement of manufacturer Y’s pit6 in this outbreak.

Jellied Pork Tongue: France, 1992 As part of an ongoing Listeria surveillance program, the French National Reference Center (NRC) noted in May 1992 that 29 clinical L. monocytogenes serotype 4b isolates received during the previous 2 months belonged to an unusual phage type. Furthermore, this strain was previously responsible for only 6 (1%) to 27 (7%) listeriosis cases annually, thus suggesting a common source outbreak [ 126,1271. By the time this outbreak ended in December of 1992,279 phage typed cases (Fig. 8) involving 182 adults (53% with underlying illnesses), 5 children and 92 pregnant women were documented, including 63 deaths and 22 abortions [106,149], making this outbreak one of the largest thus far reported. Among the 73 live births, 7 newboms died, giving an infant mortality rate of 9.6%. Geographically, cases were reported from every region of the country except the island of Corsica, with as many as 10- 14 cases/million population being recorded in and around Limousin, Alsace, and the Rh6ne Valley. Following a nationwide alert in May, the French Ministries of Health, Agriculture, and Economy began investigating this outbreak. Initially, no correlations between development of disease and consumption of various meats, cheeses, and pit& was observed from 144 cases and 288 matched controls [ 106,1081. However, in a subsequent case-control study, 36 of 60 (60%) pregnant women who became ill recalled consuming jellied pork tongue as compared with only 5 of 82 (6.1%) healthy controls. Thus, jellied pork tongue was implicated as the vehicle of infection (odds ratio: 9.2) with product brand A (odds ratio: 14.8) identified in a later case-control study. Simultaneously, over 14,000L. monocytogenes isolates from food and related environmental sources were serotyped and phage typed by the NRC [ 1271, with the epidemic phage type eventually being identified in 135 delicatessen products, 40 cheeses, 40 meat/

330

Ryser

FIGURE8

Monthly distribution of epidemic and non-epidemic listeriosis cases in France during 1992. (Adapted from Ref. 126.)

nieat products. 3 1 milk s~implcs.and 10 cnvironiiicntal samples. Among the 279 cliniciil isolates. 249 strains exhibited the same pulsed-field gel clectrophoretic profile. with this epidemic strain also subsequently being confirmed in I 12 saniples of jellied pork tongue ;is \veil ;is i n 19. 13, and 1 1 samples of other meat products (i.e.. ham. pit&. sausage). cheeses. and miscellaneous foods. respectively. Furthermore. the epidemic strain was most closely associated Lvith brand A jellied pork tongue. with high numbers being recovered from st‘\re 11prc \io i i s 1y 11no pc ned con t ai ners and si x sam p 1c s s I iced at de 1i cate sse 11coii n tcrs . Seve r;i 1 e n v i ron me 11t ;I 1 sam p 1es from brand A * s m ;in 11fac t u ri ng frici 1i ty e \re n tu a11y y i e I dcd the epidemic strain [ 1271. with raw brine being identified iis the niost probable soiirce of contamination during iii:iii~ifiictiire [ 207 I. These findings and results from the earlier c;isecontrol studies contirni brand A jellied pork tongue ;is the primary \.ehicle of infection. w i t h ot he r cross con t am i 11;it ed foods at de 1i cat c sse 11 coil 11t er5 pre s i i 111;i b 1y ser\.i ng iis secondary \vehicles in approsiniately 19% of cases [ 1081. Support for the latter also comes fro111 ii s 11b s c q 11e 11t c ;i se - c on t ro 1 s t LIct y i n which ;i st at i st i c a1 assoc i at i o11 LV ;is demonst r;i t c d b e t ~ ~ eillness ii in patients who did not consume .jellied pork tongue and contact between brand A jellied pork tongue and other foods at the delicatessen counter [ I08 I . Furthermore. the epidemic strain was isolated from iitensils iised in slicing both brand A jellied pork tongue and other delicatessen meats [ 107.197 1. Based 011 phage typing. pulsed-tield gel electrophoresis. ribotyping. and multilocus e11z y me e I ec t ro phore s i s. t h i s c pi de 111i c s trai 11 is phc 11ot y pi cal I y and ge 110tJ.1~ i c all y s i 111i 1;ir to s t rai 11s re s po 11s i bl e for t he a f ’ore men t i oned chee se - re 1at ed 011 t b rcaks i 11 Cal i fo rn i ;i. Den111ark. id S\v i t ze 1.1 a11d. Th i s o bscr\.at i o n agai 17 con ti riii s that most 11i:i.j or 1is teri o s i s outbreaks appear to be caused b!, ;i small group ot closely related strains. Gi\.cn the low ;it t ;ic k r:i tc and w i de ?cograph i c al d i s t ri bu t i 011 of 1i s te ri osi s cascs. con t i 11ued o ngo i rig suri.eillance at the national Ic\.el is necessary for detection of future t’o,odbornc listeriosis 011t breaks.

Foodborne List eriosis

33 1

Pork Pate "Rillettes": France, 1993 A second markedly smaller meat-related outbreak was also documented i n western France

I year later. During late June and early July of 1993. 10 seemingly related cases of listcriosis were recorded at the Frcnch National Listeria Reference Center (NLRC) i n Paris. with all I0 clinical isolates belonging to an unusual phage type of L. /,io/ioc:\'togciic'.~ serotype 4a [ 1261. Since this epidemic strain was previously rcsponsible for only 2 ( < 1 % ) to 1 1 (3%) 1i ste ri osi s cases annual 1y si ncc 1 987. i n ve st i g at ors i m iii ed i ;I t e 1y 1au n c hed ;i c as e -c o 11 t ro 1 study in which ii pork pit& product (known locally ;is "rillettes" ) produced by onc manufacturer and sold through a single supermrirket chain was soon implicated as the \rehicle of i n fect i on. One e n v i ron men t al i soI ate i de n t i tied from the i m pl i cat ed man 11 frict iirer 4 iii on t h s earlier also matched the epidemic strain. These findings prompted a recall of the implicated product and a series of public warnings through the mass media. By the time this outbreak subsided in October. this single epidemic strain was responsible for 39 cases (i.e.. 15% of all human listeriosis cases reported from June to October) (Fig. 9). with approximately 80% of all listeriosis Lictims being identified :is iiiother/infiint pairs [ 1971. Three weeks after the recall. investigators isolated the epidemic strain from 49 opened/unopcned containers of implicated pork pit6 that were eithcr recalled from the s11perm ark e t . re t u 1-11ed by con s IIm e rs . o r retrieved fro171 v i c t i m ' s re fr i ge ra t or s. w i t h seven environmental swab samples from the implicated factory also yielding the outbreak strain [ 107.1261. These findings along with the fact that 38 of 39 clinical and SS of 56 pit&/ e n \iro n i n e n t a 1 i so1ates we re al so i dent i c ;i 1 based on pu 1sed-fi e 1d g e 1 e 1ec t ro ph ore si s con firmed pork pit6 as the infectious vehicle. According to follow-up DNA inacrorestriction analyses. this epidemic strain was closely related to those responsible for the aforeinentioned outbreaks that were epidemiologically linked to p i t i and pasteurized inilk i n England and Massachusetts. respectively [ 1261. However. only 0.2 and 0.1 c/r of - 17.400

-

FIGURE9

Monthly distribution of epidemic and non-epidemic listeriosis cases in France during 1993. (Adapted f r o m Ref. 126.)

Ryser

332

food isolates received at the NLRC since October of 1993 have matched the epidemic phage type and pulsed-field gel electrophoresis type, respectively, thus signaling the end of this most recent outbreak of meatborne listeriosis. Other than the three major outbreaks just discussed, the scientific literature primarily contains only circumstantial evidence linking or, in some instances, only suggesting involvement of meat products in cases of human listeriosis (Table 6). Beginning in 1955, consumption of contaminated pork (probably undercooked) was suggested as the possible cause of 27 listeriosis cases in the former Soviet Union [115,140]. The following year, Gudkova et al. [ 1161 isolated L. monocytogenes from the viscera of pigs on a Russian farm where several individuals contracted listerial infections, presumably after ingesting pork from an infected group of pigs. In 1960, Olding and Philipson [ 1751 investigated one adult and three perinatal cases of listeriosis that occurred within a three-block area of Uppsala, Sweden, during the previous 2 years. Although repeated attempts to isolate L. monocytogenes from water, milk, vegetables, and meat ended in failure, the fact that meat was the only food item obtained from the same source by all four individuals suggests the possible involvement of unspecified meat products in this apparently common-source outbreak. In the only other early recorded incident involving meat products from domesticated animals, ground meat from a dead calf was suspected of transmitting L. monocytogenes to the wife of a Dutch farmer in the early 1960s [ 1351. Although involvement of meat in this case of listeriosis appears plausible, the remainder of the suspected meat was sterilized during canning, thus eliminating any hope of confirming the causative agent. During a review of listerial infections in Canada over a 21-year period, Bowmer et al. [61] uncovered one case in which a pregnant woman in Newfoundland delivered an infant who died 1 month later from listerial meningitis. Ten days before the infant became ill, the mother recalled skinning, cooking, and eating two previously frozen hares that were brought from New Brunswick, thus suggesting rabbit meat as a possible vehicle of infection. Although less commonly consumed, it appears that rabbit meat also may serve

TABLE 6 Human Listeriosis Cases in Which Consumption of Meat Products Was Suggested as a Possible Source of Infection Area USSR USSR Sweden The Netherlands Newfoundland/Canada United States Philadelphia, PA Italy United States Spokane, WA San Francisco, CA Victoria, Australia

Year

I955 I956 1958-59 early 1960s 1963 1986-87 1987 1988 1988- 1990 1988- I990 1989 1989 1990- I995 1990- 1995 1990- 1995

Number of cases

Possible vehicle of infection

Reference

27 19 4

Pork Pork Meat Ground veal/beef Rabbit Uncooked hot dogs Salami Cooked pork Pork sausage Ground beef Cooked ground beef Cooked Cajun pork sausage Prepackaged sliced meat Sliced ham Sliced meats

115, 140 116 175 I35 61 214 31 70 182 182 34 36 222 222 222

1

1 Unknown Unknown I 1 1 1 1 1 1

1

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as a potential source of L. monocytogenes, as evidenced by a long history of listerial infections among rabbits [ 113,114,215,2331.In fact, the first type-strain of L. monocytogenes was isolated by Murray et al. [167] in 1924 from the blood of infected rabbits. Several European scientists have expressed some concern about the incidence of L. monocytogenes in rabbit meat, along with possible risks of consuming such potentially contaminated products. Despite such circumstantial evidence suggesting that consumption of contaminated meat products can lead to cases of human listeriosis, the possible involvement of meat products in listerial infections received little if any further attention before 1981, primarily because listeriosis had not yet been associated with any foods other than raw milk. However, this situation changed after three major listeriosis outbreaks were positively linked to consumption of contaminated coleslaw, Mexican-style cheese, and Vacherin Mont d’Or soft-ripened cheese in 1981, 1985, and 1987, respectively. Several factors, namely, (a) the long-time association of L. monocytogenes with domestic: livestock, (b) the ability of L. monocytogenes to grow at refrigeration temperatures, and (c) questions from public health authorities prompted numerous studies on the incidence and behavior of this pathogen in raw and processed meat products (see Chap, 13) and also led to increased surveillance and reporting of listeriosis cases. After CDC officials in Atlanta began receiving information about scattered cases of listeriosis occurring throughout the United States, Schwartz et al. [2 141 initiated a retrospective epidemiological study to identify food products that might be associated with sporadic cases of listeriosis. According to their 1988 report which appeared in the: British journal, Lancet, an active L. monocytogenes surveillance program was established in Missouri, New Jersey, Oklahoma, Tennessee, Washington, and Los Angeles County, California, in January of 1986. During the following 18 months (12 months in Los Angeles County), 154 listeriosis cases were identified among 34 million people with approximately one third and two thirds of the patients being classified as newborn infants and elderly or immunocompromised adults, respectively. Overall, 82 of these 154 individuals agreed to participate in a retrospective case-control study in which patients responded to a series of questions concerning demographic characteristics, underlying illnesses, medication, exposure to other sick individuals or animals, excavation work, and dietary history. The latter included questions pertaining to consumption of raw fruits and vegetables, poultry, eggs, and dairy products as well as raw, processed, and pickled meats. After comparing their answers with those from 239 controls (individuals without listeriosis) that were matched to the cases in terms of age and underlying illness, individuals who consumed uncooked frankfurters and undercooked chicken were 6.1 and 3.2 times more likely to contract listeriosis, respectively, than those who did not consume these products. Overall, epidemiological evidence from this study suggested that consumption of these foods accounted for 30 of 154 (20%) listeriosis cases reported in the surveillance area with 1 in 1200-6000 and 1 in 1500-7500 individuals likely to contract listeriosis after consuming uncooked frankfurters and undercooked chicken, respectively [32]. Given that about 1600 cases of listriosis occurred annually in the United States during the late 1980s, these investigators speculated that 255 and 102 of these cases were attributable to eating uncooked frankfurters and undercooked chicken, respectively. Although this case-control study identified uncooked frankfurters and undercooked chicken as risk factors in sporadic cases of listeriosis, it is important to stress that such epidemiological investigations cannot establish causality. Furthermore, one must also remember that lack of an association with other foods does not necessarily mean that con-

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sumption of such products poses no risk of listeriosis. Several shortcomings of this retrospective case-control study were echoed by the scientific community [26], including, (a) omission of questions concerning cooking methods and consumption of foods such as seafood that until recently have seldom been associated with listeriosis, (b) limited ability to identify risk factors when exposure was very common or very rare, and (c) difficulty in obtaining accurate diet histories with the possibility of cases more clearly recalling what they consumed before their illness than controls. Nonetheless, results from numerous microbiological surveys (see Chaps. 13 and 14), along with a report by the American Meat Institute indicating that 5- 10% of prepackaged frankfurters produced in the United States were contaminated with L. monocytogenes [22], support the possibility of contracting listeriosis from consuming uncooked frankfurters or undercooked chicken as was suggested in the case-control study by Schwartz et al. [214] and a similar case-control study [212] reported by the CDC several years later. During their work, these researchers [31,2141 also identified another processed meat product consumed without further cooking, namely salami, as a possible risk factor in a 1987 listeriosis outbreak in Philadelphia that claimed 14 lives. However, CDC officials again lacked the bacteriological data to positively link consumption of the salami to illness. In a subsequent case-control study [ 1821, L. monocytogenes strains of the same electrophoretic enzyme type were recovered from two patients and two unopened packages of pork sausage and ground beef that were epidemiologically linked to illness. However, inability to recover and test these products from patient’s refrigerators prevented CDC investigators from positively confirming the vehicle of infection. As mentioned earlier, piX, jellied pork tongue, and pork pgt6 “rilletes” were responsible for three major meat-related listeriosis epidemics in England and France, including two of the largest outbreaks of foodborne listeriosis recorded worldwide (see Table 1). However, it must be stressed that as of July 1998, no American-produced raw, cooked, or otherwise processed meat product has been conclusively proven as the vehicle of infection in any case of human listeriosis. Although it is important to remember that such a causal relationship can only be shown conclusively by isolating the identical L. monocytogenes strain from the patient, product consumed, and unopened packages of the implicated food, numerous North American and European surveys have uncovered low to moderate levels of L. monocytogenes in a wide range of commercially available raw, processed, and ready-to-eat meat products (see Chap. 13). Even before Schwartz et al. [31,2141 announced preliminary results from their study, the meat industry [29] maintained that susceptible individuals who consume Listeria-contaminated dry sausage, frankfurters, luncheon meats, and other packaged pasteurized products are at low to moderate risk of contracting listeriosis. Since 1988, eight isolated cases and one small outbreak of listeriosis have occurred worldwide where meat products were suspected as the most likely vehicle of infection (see Tables 1 and 5 ) . In the first such case, a previously healthy Italian man contracted nonfatal meningitis several days after consuming cooked homemade pork sausage that was later shown to contain -3 X 106L. monocytogenes CFU/g [70]. According to investigators, the clinical and sausage isolates were both identified as belonging to serotype 4, the most common serotype encountered in clinical cases of listeriosis. Unfortunately, the exact source of contamination was never determined; however, antiquated sausage-making practices and storage of sausage at ambient rather than refrigeration temperature were cited as major contributing factors in this isolated case of listerial meningitis. Nevertheless, although numbers of listeriae present in this sausage were probably more than sufficient

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to induce illness, some caution still must be used in evaluating the role of sausage in this case, since both isolates were never characterized beyond serotype. Four of these unconfirmed cases of possible meatborne listeriosis have been recorded in the United States and include (a) a 76-year-old man from Spokane, Washington, who died from an L. rnonocytogenes serotype 4b infection after consuming cooked ground beef; however, only serotype l a was recovered from the ground beef thus making it an unlikely source of infection [34], (b) a case-control study in which identical L. monocytogenes electrophoretic enzyme types were recovered from two patients as well as retail packages of pork sausage and ground beef [212], and (c) an incident in which L. monocytogenes serotype 4b was isolated from cooked Cajun pork sausage that was consumed by an elderly San Francisco man who developed a nonfatal case of listeriosis [36]. Approximately 1000 pounds of this sausage were subsequently recalled from the market after investigators recovered L. rnonocytogenes serotype 4b from similar unopened packages. Even though the patient and sausage isolates were not further classified, isolation of the same L. monocytogenes serotype from unopened packages of sausage and the ability of investigators presumably to trace the source of contamination to natural sausage casings imported from China [83] provides reasonably convincing evidence that Cajun pork sausage was directly responsible for this case of foodborne listeriosis. The remaining listeriosis cases (Table 6) as well as an outbreak which included six stillbirths or mid term miscarriages among 11 pregnant women (Table 1 ) were identified in Australia during routine surveillance programs with processed meats and piit6 cited as possible vehicles of infection [ 139,162,2341 based on incomplete laboratory andlor epidemiological findings. Continued surveillance of listeriosis cases by CDC officials uncovered a direct link between consumption of contaminated turkey frankfurters and listerial meningitis in an Oklahoma breast cancer patient [47] (to be discussed shortly) and also led to a nationwide recall of the product [35] along with radical changes in the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) policy regarding the presence of L. rnonocytogenes in cooked, ready-to-eat, or otherwise processed meat and poultry products. In the light of this information, some public health officials are now advising highrisk individuals (i.e., pregnant women, immunocompromise:d adults, and the elderly) to thoroughly reheat previously cooked and chilled meat and poultry products before consumption. Hence, the proven ability of L. rnonocytogenes to grow and/or survive in many refrigerated raw, processed, and ready-to-eat foods, including meat and poultry products, together with extensive food histories now being obtained from many listeriosis victims in the United States, make it highly probable that meat products, particularly frankfurters and ready-to-eat meats, will be positively linked to cases of human listeriosis in the future.

POULTRY PRODUCTS Shedding of L. rnonocytogenes in fecal material from both clinically and subclinically infected domestic fowl 1791 appears to place poultry workers at a somewhat higher than normal risk of contracting superficial listerial infections, particularly conjunctivitis. This probable association between handling infected poultry and contracting conjunctivitis is partially based on a 1951 report by Felsenfeld [96], who, 7 years earlier, identified listerial conjunctivitis in two employees who dressed poultry in Illinois. On further investigation, L. rnonocytngenes was isolated from the spleens of five birds that were not dressed in the same shop but came from an area in Illinois in which avian listeriosis was previously observed, thus suggesting poultry as the probable source of infection. Although reports

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of listerial conjunctivitis can be found in the early scientific literature, including several cases in which patients had contact with birds suffering from undetermined illnesses [ l 111, the 1951 report by Felsenfeld [96] remains one of the few instances where avian listeriosis was linked to listerial conjunctivitis in humans. A search of the early scientific literature has uncovered only two reports indicating that contact with infected poultry may lead to the systemic infections for which L. rnonocytogenes is best known. In 1958, Gray [ 1111 cited numerous instances in which Central European women gave birth to Listeria-infected infants following contact with sick or dead birds; however, evidence for the link between listeriosis and contact with infected poultry was only circumstantial. Similarly, Embil et al. [89] identified a woman in Nova Scotia, Canada, who gave birth to an infected infant who died of listeriosis 1 h after delivery. Although the mother reportedly prepared poultry for sale in a family-owned store during the previous 8 months, researchers again failed positively to link this listeriosis case to contact with raw poultry by not isolating the pathogen from raw chickens sold at the store. Given the preceding evidence, Kampelmacher [135] suggested as early as 1962 that consumption of contaminated poultry might lead to cases of human listeriosis. Although this view also was voiced 10 years later by Mir6 and Ralovich [159a], transmission of L. monocytogenes from contaminated poultry was not documented until November 1988 [137]. As was true for meat products, failure positively to link consumption of contaminated poultry to human listeriosis was until recently primarily related to difficulties in isolating L. monocytogenes from poultry and other foods containing a complex microflora and to a generalized lack of concern about foodborne listeriosis. Following the two major cheese-associated outbreaks in 1985 and 1987, public health officials in the United States and England implemented active/semiactive surveillance programs to obtain more accurate data on the incidence of listeriosis in the general population. Attempts also were made to trace the source of reported infections to consumption of dairy products and other foods such as poultry which at the time had not yet been linked to listeriosis. As a result of these efforts, three cases of listeriosis were positively linked to consumption of poultry products, which, in turn, has led to inclusion of poultry in the list of foods that may pose a potential threat of listeriosis to susceptible individuals. These three recently recognized cases will now be reviewed in some detail. Worlung in England, Kerr et al. [33,137] identified the first case of listeriosis clearly linked to consumption of contaminated poultry. According to their November 1988 report, a 3 1-year-old pregnant woman with a 24-h history of flu-like symptoms was admitted to a hospital and subsequently delivered an aborted 23-week-old fetus. On further investigation, the woman reportedly consumed a heated chicken dish prepared from cooked-andchilled chicken 5 days before onset of symptoms, with the remaining chicken being refrigerated and consumed 3 days later in a salad. Thus the woman had a maximum incubation time of only 4 days before onset of symptoms as compared with the more typical 730 days for listeriosis. Following bacteriological analysis, an identical phage type of L. monocytogenes serotype 4 was found in samples of chicken and fetal liver. Other foods in question were tested, with no evidence of Listeria contamination, thus confirming chicken as the vehicle of infection. Considerable research and regulatory activity, prompted by reports suggesting that 12-25% of cook-chill poultry products marketed in England may be contaminated with L. rnonocytogenes, uncovered a second case of poultry-associated listeriosis early in 1989. According to this report [134], L. rnonocytogenes serotype 1/2a was cultured from the

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blood of a 52-year-old immunocompromised woman who was receiving steroids for systemic lupus erythematosus. Three to 5 days before onset of vomiting and diarrhea, the hospitalized woman and her 29-year-old son shared some ready-cooked chicken nuggets which he had purchased at a fast-food restaurant. Detailed questioning later revealed that he experienced a short-lived illness with diarrhea and vomiting on the same night that his mother became sick. Subsequently, the son’s stool sample yielded L. innocua as well as L. rnonocytogenes serotypes 1/2a and 1/2c, with the DNA homology pattern of the serotype 1/2a isolate being identical to that of the L. rnonocytogenes strain originally isolated from the woman’s blood. Although L. innocua and an L. rnonocytogenes strain of unreported serotype and DNA homology pattern were recovered from uncooked chicken nuggets, these investigators failed to detect L. rnonocytogenes in a subsequent lot of cooked chicken nuggets obtained from the same source. Nonetheless, infection in both the woman and her son presumably was acquired from commercially cooked chicken nuggets of the fast-food variety, which, although served hot, were most likely undercooked, thus allowing L. monocytogenes to survive in sufficient numbers to cause illness. Although this is only the second case of poultryborne listeriosis recorded in England, similar cases have likely gone undetected because of inadequacies in reporting and difficulties encountered in linking these illnesses to consumption of poultry or any other food. These two cases of poultryborne listeriosis and a recent survey of listeriosis cases in Scotland which included identification of possible food-related risk factors associated with the disease [68] prompted the Public Health Laboratory Service (PHLS) to conduct a national case-control study in England and Wales which attempted to correlate consumption of high-risk foods (i.e., poultry, pitt5, cheese, prepared salads, delicatessen items) with human listerial infections [ 1181. A total of 124 cases diagnosed from July 1990 to January 1992 were identified from both the national voluntary reporting laboratory system and the PHLS Listeria reference laboratory and matched to 459 controls according to age, sex, underlying illness, and pregnancy status. After obtaining dietary histories, undercooked and readycooked chicken consumed either hot or cold were statistically related to development of listeriosis in both pregnant and nonpregnant individuals. Additional epidemiological studies are needed in England, the United States, and elsewhere to expand the number of reported foodborne listeriosis cases and generate a more comprehensive list of foods that pose a significant public health threat. Despite the controversial nature of many epidemiological studies, such efforts have already played an important role in identifying possible risk factors associated with foodborne listeriosis. As you will recall from our aforementioned discussion of meatborne listeriosis, undercooked chicken was identified as a high-risk vehicle of infection by CDC officials during several case-control studies [ 182,212,2141 conducted in conjunction with an active listeriosis surveillance program in Oklahoma and five other states. In two sporadic cases traced to turkey frankfurters and sliced turkey ham [ 1821, L. rnonocytogenes isolates from unopened packages of the same product brand belonged to the same electrophoretic enzyme type as the patient isolate, thereby implicating turkey frankfurters and sliced turkey ham as the source of infection. During the first of these surveillance programs, CDC officials learned of a breast cancer patient in Oklahoma who had been infected with L. rnonocytogenes and hospitalized for listerial septicemia and meningitis in December 1988 [35,47]. In an attempt to identify the vehicle of infection, investigators went to the woman’s home, obtained foods from her refrigerator, and eventually isolated Listeria from various products, including an opened package of turkey frankfurters that contained > 1.1 X 103L. rnonocytogenes CFU/g. A

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swab sample from the refrigerator’s interior also yielded the pathogen. Although CDC investigators initially concluded that the woman had contaminated the food herself, public health officials from Oklahoma began examining the same brands of retail products from the woman’s refrigerator that were positive for L. monocytogenes. Interest soon focused on turkey frankfurters after officials learned that the woman consumed one turkey frankfurter daily after 45-60 s of heating in a microwave oven. Four months later, the same strain and isoenzyme type of L. monocytogenes serotype 1/2a recovered from this patient and the opened package of turkey frankfurters was also identified in five of seven unopened packages of identical product purchased from nearby stores [243a], thereby confirming turkey frankfurters as the vehicle of infection in the first poultryborne listeriosis outbreak recorded in the United States. Once the USDA-FSIS was notified of this case by the CDC on April 14, 1989, government officials prompted the Texas manufacturer to issue an immediate recall for approximately 600,000 pounds of turkey frankfurters that were marketed by retail and institutional establishments in 23 states [35]. Joint investigations initiated by the CDC and USDA-FSIS 1 day later eventually showed that six of seven retail lots of product produced over a 37-day period contained the implicated L. monocytogenes strain at a most probable number (MPN) level of <0.3 CFU/g. Furthermore, environmental testing of the production facility showed that only 2 of 40 samples taken before sausage peeling harbored L. monocytogenes as compared with 12 of 14 samples taken after peeling, with the implicated strain also being recovered from a conveyor belt attached to the peeler. These findings suggest that the factory was experiencing an ongoing contamination problem at a single point during the sausage peeling process. Although large quantities of product contained relatively few listeriae, the ability of L. monocytogenes to grow to hazardous levels in such processed poultry products during refrigerated storage indicates that special precautions must be taken to eliminate this pathogen from the food processing environment. This incident has since prompted the USDA-FSIS to toughen its regulatory policy regarding Listeria-contaminated poultry and meat products (see Chap. 13).

EGGS AND EGG PRODUCTS Acquiring listeriosis through consumption of contaminated eggs and egg products has been considered for nearly 40 years; however, unlike poultry products, no such cases have been firmly documented. Geurden and Devos [ 1031, who in 1952 isolated L. monocytogenes from a necrotic lesion in the oviduct of an infected hen, were first to suggest that eggs might serve as a possible vehicle of infection. Nonetheless, these authors also admitted that a 35-year-old man who consumed raw eggs from this infected chicken flock showed no signs of listeriosis. Three years later, Urbach and Schabinski [227] found that guinea pigs who were fed artificially infected eggs in which L. monocytogenes had grown to very high levels soon died of listerial septicemia, thus suggesting that humans also might develop listeriosis by consuming contaminated eggs. During the same year (1955), Dedii [77] reported an interesting case in which a pregnant woman, who owned nine chickens, gave birth to a Listeria-infected infant. Although 26 eggs from these chickens were negative for L. monocytogenes, the H and 0 agglutination titers of the chickens increased dramatically during the 4-week period in which the eggs were collected, thus suggesting recent exposure to this pathogen. Subsequent attempts to isolate L. monocytogenes from 200 eggs laid by experimentally infected hens ended in failure. However, the fact that L. monocytogenes was detected in feces and

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nasal secretions from these birds suggests that externally contaminated eggs may constitute a potential source of infection and a potential source for cross contamination of other foods if the eggs are handled improperly. As is true for meat and poultry workers, listerial infections among apparently healthy individuals employed in the egg industry are extremely rare. In fact, a search of the literature uncovered only one documented case from 1965 in which a 39-year-old male egg factory worker became infected with L. rnonocytogenes and subsequently died of meningitis [ 135a1. Other than the fact that 29.1 and 10.6% of the egg factory workers carried L. rnonocytogenes in their feces 4 and 16 months after the man’s death, respectively, no further evidence was reported to incriminate eggs in this fatal case of listerial meningitis. Hence, at this time it appears that healthy egg factory workers need not take any special precautions to guard against listeriosis. Despite the inability to link listeriosis to consumption of eggs or egg products, the recognized ability of L. monocytogenes to grow in egg products [ 1001 along with emergence of this organism as a bonafide foodborne pathogen may change this picture in the future. This prediction is supported by the fact that CDC officials [2 131 identified a possible cluster of listeriosis cases in Los Angeles County in which 6 of 33 cases and 4 of 101 matched controls consumed raw eggs (odds ratio = 6.4) over a 5-month period spanning 1986- 1987. Although epidemiological investigations can never conclusively prove causality, the possible associations discovered in such studies will likely prompt public health authorities seriously to consider eggs and other foods (e.g., seafood, fruits) as potential vehicles of infection, which, in turn, may lead to their implication in future cases of listeriosis.

SEAFOOD PRODUCTS Despite the recent discovery of L. rnonocytogenes in a wide range of raw and processed fish and seafoods, including finfish (i.e., smoked salmon, cod, trout), mussels, oysters, shrimp, crabmeat, lobster tails, and surimi, most attempts directly to link consumption of such products to cases of human listeriosis have proven unsuccessful. Nonetheless, a few scattered reports attesting to possible involvement of seafoods in listerial infections along with three reports of cases positively linked to consumption of fish, smoked mussels, and artificial crabmeat have found their way into the scientific literature. Two early reports from New Zealand include (a) a 1971 observation that two pregnant women delivered Listeria-infected infants after presumably consuming raw fish sometime during their pregnancies [49], and (b) a cluster of 22 perinatal listeriosis cases between January and November of 1980 in which food histories suggested, at best, a weak association between consumption of contaminated shellfishh-awfish and development of listeriosis [ 1441. In 1980, Vilde et al. [228] published a report suggesting that a 48-year-old immunocompromised French woman had contracted listeriosis after consuming contaminated oysters. More recently, Arriold and Coble [39] identified two miscarriages among Australian women (one case in Victoria and the other in New South Wales) in which smoked salmon was implicated as the vehicle of infection, with Tan et al. [222] suggesting possible involvement of smoked salmon and salmon cheese spread in two additional Australian cases of listeriosis. In one of the largest suspected seafood-related outbreaks thus far reported, 8 of 36 previously healthy adults attending a June 1989 party in New York City developed a predominantly mild form of listeriosis characterized by fever, nausea, vomiting, diarrhea, and musculoskeletal distress [ 1941. However, two cases of bacteremia caused by the epi-

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demic strain (an unusual enzyme type of L. monocytogenes serotype 4b) also occurred among expectant mothers, with one pregnancy ending in a miscarriage. Although epidemiological evidence most strongly implicated shrimp as the vehicle of infection, shrimp from the party was unavailable for testing, and investigators also were unable to detect Listeria in shrimp purchased 6 weeks later from the party’s supplier. Thus, as already implied, a causal link between consumption of seafood and listeriosis was never clearly proven in any of these cases. The first convincing evidence for direct involvement of fish or seafood in human listeriosis is that of a 54-year-old Italian woman who in 1988 or 1989 contracted nonfatal meningitis 3-4 days after consuming undercooked fish from which L. monocytogenes was subsequently isolated [92]. The fact that L. monocytogenes isolates from the patient’s cerebrospinal fluid and a leftover portion of the fish were of serotype 4 and were identical based on phage typing and DNA restriction analysis confirmed fish as the vehicle of infection in this case of listerial meningitis. According to the investigators, survival and transmission of the pathogen was most likely the result of undercooking, since the fish was eaten and refrigerated after steaming. However, the mode by which this fish became contaminated could not be determined. In August of 1991, the Tasmanian Health Department was notified about an otherwise healthy 37-year-old woman and her 10-year-old son who developed malaise, chills, fever, headache, vomiting, and diarrhea 3 days after eating 90 g of smoked mussels imported from New Zealand [160,161]. On culturing, opened and unopened packages of smoked mussels yielded L. monocytogenes at a level of 1.6 X 107CFU/g (i.e., oral infective dose of 9 X log CFU), with contamination traced to three batches in which the product’s shelf life was inadvertently overestimated by at least 3 months. Despite two public warnings and withdrawal of the implicated samples from sale, one additional case was reported a month later in an 83-year-old woman who developed similar symptoms of gastroenteritis. Although results from strain-specific typing studies are not available, the fact that all three victims became ill within 3 days of consuming smoked mussels clearly points to this heavily contaminated product as the most probable source of infection. Working in New Zealand, Brett et al. [63] also identified two perinatal cases of listeriosis in Aukland during November and December of 1992 in which the women gave histories of consuming one particular brand of smoked mussels. During follow-up investigations, an unopened package of smoked mussels obtained from one victim’s refrigerator yielded a strain of L. monocytogenes serotype 112 that was indistinguishable from both clinical isolates based on phage typing and pulsed-field gel electrophoresis. On further searching, this unusual strain was identified in retail packages of smoked mussels marketed in Aukland, New Zealand, and Brighton, England, as well as in environmental swab samples from the mussel processing factory, thereby confirming smoked mussels as the vehicle of infection and the factory environment as the source of contamination. Although no additional cases were confirmed in New Zealand, export of these mussels to England combined with a reported association between mussel consumption and listeriosis in England [118] suggests that these mussels may have been responsible for additional cases abroad. Given a 1995 report from Tasmania [219] in which L. monocytogenes was recovered from 15.4% of premarket raw blue mussels and Pacific oysters collected in the wild and from farms, public health concerns regarding shellfish safety need to be reexamined. In 1997, Farber [94] also identified two cases of foodborne listeriosis among healthy adults in Ontario, Canada. Samples of imitation crab meat, canned black olives, macaroni/

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vegetable salad, spaghetti sauce with meatballs, and mayonnaise taken from the victim’s refrigerator all yielded L. monocytogenes, with imitation crab meat containing 2.1 X 109 CFU/g. On further investigation, clinical strains and isolates from both opened and unopened packages of crab meat were identified as serotype 1/2b and were also indistinguishable by both randomly amplified polymorphic DNA analysis and pulsed-field gel electrophoresis, thereby confirming imitation crab meat as the vehicle of infection. The aforementioned listerial infections along with other recently reported cases of foodborne listeriosis that have been positively linked to consumption of dairy as well as ready-to-eat meat and poultry products suggest it would be naive to assume that L. monocytogvnes poses any less danger to public health when present in cooked and/or ready-to-eat seafood than in other foods. Hence, FDA officials have maintained a policy of zero tolerance for L. monocytogenes in all ready-to-eat foods and have (mid 1998) issued numerous Class I recalls involving well over 45,000 pounds of contaminated cooked/ready-to-eat seafood. These recalls and the fact that CDC officials now include various shellfish and finfish in food history questionnaires given to listeriosis victims in five states as well as Los Angeles County [24,28,212,214] make it likely that additional listeriosis cases will be positively linked to fish and seafood in the future.

FOODS OF PLANT ORIGIN Evidence for transmission of listeriosis through foods of plant origin probably dates back to 1922 when investigators in Iceland described a “listeriosis-like” illness in silage-fed animals [ 1 131. This apparent relationship between consumption of improperly fermented silage and listeriosis in ruminants was clarified in 1960, and now numerous reports of silage-related listeriosis outbreaks in sheep and cows can be found in the scientific literature. Although additional cases of animal listeriosis associated with consumption of other types of animal feed have been primarily limited to scattered reports involving cattle that grazed on Ponderosa pine needles in western Canada, one 1977 outbreak of listeriosis in chinchillas was attributed to a batch of meal containing beet pulp [97]. However, L. monocytogenes was never isolated from the incriminated feed. Given this link between animal listeriosis and consuinption of contaminated plant material, i1 is reasonable to suspect that raw vegetables (e.g., cabbage, lettuce) and fruits are responsible for a certain percentage of listeriosis cases appearing in the human population, as was first suggested by Blendon and Szatalowicz [58] in 1967. Evidence for involvement of raw fruits in human listeriosis is currently limited to one 1984 unpublished report frorn Connecticut suggesting a possible link between listeriosis and consumption of unwashed strawberries, blueberries, and/or nectarines and one recent case of neonatal listeriosis in Italy which was traced to olives and confirmed by DNA fingerprinting [71a]. In contrast, the ability of certain segments of the population to succumb to listeriosis after consuming contaminated raw vegetables has been well documented over the past 15 years. In 1986, Ho et al. [122] published results from an earlier epidemiological investigation in which raw vegetables were suggested as a possible vehicle of infection in an outbreak of listeriosis that occurred among patients in eight Boston-area hospitals during September and October of 1979. However, it is noteworthy that 1 year before these findings were published, Schlech et al. [210] published their landmark report describing the first confirmed North American outbreak of foodborne listeriosis in which 41 Canadians became ill in 1981 after consuming coleslaw contaminated with L. monocytogenes. Hence,

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it seems unlikely that the listeriosis outbreak in Boston would have been reported if Schlech et al. [210] had not published their results first. In the outbreak described by Ho et al. [ 1221, 23 patients admitted to Boston-area hospitals acquired systemic listerial infections during September and October of 1979, with L. monocytogenes isolates from 20 of 23 (87%) patients identified as serotype 4b (Fig. 10). In contrast, only 19 listeriosis cases were identified at the same eight hospitals during the 26-month period immediately preceding the outbreak. Unlike the previously described foodborne outbreaks, which included pregnant women, neonates, and adults, all 20 outbreak-related cases of listeriosis from which L. monocytogenes serotype 4b was isolated were adults ranging in age from 46 to 89 years, with half the victims being immunocompromised as a result of cancer, chemotherapy, or steroid treatment, Overall, 18 of 20 (90%) and 8 of 20 (40%) patients suffered from listerial bacteremia and meningitis, respectively. Although 5 of 20 (25%) patients died, two of these deaths were related to underlying illnesses rather than listeriosis. A series of epidemiological studies revealed that cases were more likely than controls (i.e., listeriosis patients treated at the same eight hospitals during the 26-month period preceding the outbreak) to have (a) become infected with L. monocytogenes serotype 4b rather than another serotype, (b) acquired listeriosis during hospitalization, (c) exhibited gastrointestinal symptoms, and (d) received antacids or cimetidine before onset of illness. More important, results from food histories revealed that cases were more likely than controls to have consumed tuna, chicken salad, and cottage cheese as well as hard cheese. Although it was at first difficult to develop a scenario by which this apparent commonsource outbreak could have resulted from consumption of three seemingly unrelated foods obtained from different distributors, hospital kitchen records revealed that all three foods

Type other than 4b

... ... .., ... ..< ... ... ..< ... ... ... .... .< ... ... ... ... . . I

,,.. .. ... ... I . .

.

.

I

4

-R

0

2

1977

1978

3

4

5

6 7

8

n

9 10 11 I21

1979

FIGURE10 Number of listeriosis cases in eight Boston-area hospitals that resulted f r o m infection with serotype 4b, non-serotype 4b, and untypeable strains of L. monocytogenes between July 1977 and December 1979. (Adapted f r o m Ref. 122.)

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were commonly used in salads containing lettuce, celery, and/or tomatoes. Thus, in light of the 1981 Canadian listeriosis outbreak involving coleslaw., these researchers postulated that the Boston outbreak was the result of victims having consumed raw lettuce, celery, and/or tomatoes that were contaminated with listeriae. However, since no attempt was made to isolate L. monocytogenes from these vegetables during the outbreak, the exact source of infection will always remain in question.

Coleslaw: Canada, 1981 Two years after this presumed vegetableborne outbreak of listeriosis in Boston, Schlech et al. [210] positively identified coleslaw as the vehicle of infection in a major outbreak of listeriosis that occurred in the Maritime Provinces of Canada. According to their report, 34 perinatal and 7 adult cases of L. rnonocytogenes serotype 4b infection were diagnosed between March 1 and September 1 of 1981 in Nova Scotia, New Brunswick, and Prince Edward Island as compared with 22 cases in the 26-month period immediately preceding the outbreak (Fig. 11). Perinatal cases were characterized by acute febrile illness in pregnant women followed by spontaneous abortion (5 cases), stillbirth (4 cases), live birth of a seriously ill infant (23 cases), or live birth of a healthy infant (2 cases). Detailed clinical and laboratory findings concerning 15 of these cases diagnosed at Grace Maternity Hospital in Halif'ax, Nova Scotia, were subsequently reported by Evans et al. [90]. Six cases of meningitis and one case of pneumonia/septicemia were diagnosed in seven nonimmunocompromised adults (six men and one nonpregnant woman) who were 2 1-8 1 years old. Fifteen of 34 (44%) perinatal and 2 of 7 (29%) adult victims died, giving an overall mortality rate of 41% [141]. Two case-control studies were subsequently initiated 1.0 assess possible risk factors for acquisition of listeriosis. The first study examined medical, residential, occupational, travel, and educational histories as well as exposure to animals and information concerning gardening and outdoor activities, whereas the second case-control study involved a general

'' 7

Perinatai cases

14

12

I0 8 U

8 6 4

2

0

I

1979

I

1980

I

198 1

I

FIGURE11 Number of perinatal and adult cases of listeriosis recorded in the Maritime Provinces of Canada from January 1979 t o December 1981. (Adapted from Ref. 210.)

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history of foods consumed during the 3 months before onset of illness. Analysis of results from the first case-control study failed to implicate a common environmental source for this outbreak. Although initial data collected from food histories also failed to implicate any particular food, results from a second questionnaire that included names of several food items found in the refrigerator of a man who developed pneumonia and septicemia indicated that cases were more likely than controls to have consumed both coleslaw and radishes. Multivariate analysis later showed that ingestion of radishes was associated with consumption of coleslaw rather than illness. However, repeated interviews with cases and controls who had previously denied eating coleslaw revealed that 100% of the cases but only 40% of the controls remembered consuming the product during the 3-month period before the outbreak. Armed with this information, investigators visited the home of one of the patients, sampled foods from the refrigerator, and then isolated L. rnonocytogenes from coleslaw but no other foods obtained from the patient’s refrigerator. The strain isolated from coleslaw was soon identified as serotype 4b, the same serotype isolated from all 34 victims. To prove that the patient had not inadvertently contaminated the coleslaw, investigators obtained two unopened packages from two different Halifax-area supermarkets and recovered L. rnonocytogenes serotype 4b from each package. Audurier et al. [43] later reported that 28 of 3 1 (90.3%) L. monocytogenes serotype 4b isolates obtained from blood, cerebrospinal fluid, and/or placental material between January and September of 1981 were of the same phage type. Any remaining doubt concerning the role of coleslaw in this outbreak was eliminated when clinical and coleslaw isolates were later shown to be of the same electrophoretic enzyme type [40,411, pulsed-field gel electrophoretic type [65], ribotype [ 1101, restriction enzyme pattern [235], randomly-amplified polymorphic DNA pattern [74], and DNA macrorestriction pattern [ 1261 with this strain being closely related both phenotypically and genotypically to those responsible for outbreaks in the United States (Mexican-style cheese 1985) and Switzerland (Vacherin Mont d’Or cheese 1983- 1987) [126]. Thus Schlech et al. [209,210] can be credited with providing the first concrete evidence that consumption of contaminated food, in this instance coleslaw, can cause listeriosis in humans. After coleslaw was confirmed as the infectious vehicle, Schlech et al. [210] attempted to determine the route by which the coleslaw became contaminated. Investigators soon traced the tainted coleslaw to a regional manufacturer whose product had been distributed exclusively in the Maritime Provinces of Canada. Although repeated microbiological testing of environmental samples from the coleslaw factory failed to uncover the contamination source, review of factory records along with recent data on animal listeriosis revealed that the manufacturer had received at least 2250 kg of cabbage from a farmer who also raised sheep. Furthermore, the farmer lost two sheep in his flock to listeriosisone in 1979 and one in 1981. A review of the farmer’s agronomic practices indicated that cabbage was routinely fertilized with both raw and composted sheep manure obtained from animals that were presumably fecal carriers of L. rnonocytogenes. Moreover, the final October cabbage crop was held in a large cold-storage shed until early spring. Such storage practices, which somewhat resemble cold enrichment, may have led to an increase in numbers of L. rnonocytogenes in the cabbage. Although none of the implicated cabbage crop (other than that which was previously manufactured into coleslaw and contaminated with the epidemic strain of L. rnonocytogenes) was available for testing, and none of the farm environmental samples, including raw sheep manure, yielded Listeria, the aforementioned circumstantial evidence still supports an indirect link between sporadic cases of ovine listeriosis on a cabbage farm and cases of listeriosis in humans consuming a Listeria-

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contaminated product. The fact that (a) crops grown in Canada, the United States, and industrialized European nations are routinely fertilized with material other than raw manure, (b) 82% of all L. monocytogenes strains isolated during one large survey of raw vegetables marketed in the United States were of serotype la [ 1201, and (c) most L. monocytogenes strains isolated from animal sources, including feces, in North America are of serotype 4 rather than 1 support transmission of L. monocytogenes from raw sheep manure to cabbage. Hence, raw manure should not be used to fertilize vegetables that will be consumed without cooking.

Additional Reports of Listeriosis Associated with Products of Plant Origin Despite the proven link between coleslaw and development of listeriosis in the Canadian outbreak just discussed, consumption of raw vegetables, fruits, and other types of produce has not generally been deemed to constitute a major public health threat 12121. This lack of concern resulted because consumption of raw produce has until recently been infrequently associated with most forms of foodborne disease. However, after Mexican-style and Vacherin Mont d’Or soft-ripened cheese were directly linked to two separate listeriosis outbreaks (see earlier discussion in this chapter), public health officials around the world became increasingly interested in the possibility that nondairy foods such as meat, poultry, seafood, and raw produce could transmit L. monocytogenes to humans. As of May 1997, only one additional small outbreak and three sporadic cases have been documented in which consumption of a product of plant (or microbial) origin was directly linked to human listeriosis. According to a 1996 report by Simpson [2 161, a hospital laboratory on the Texas-Mexican border cultured L. monocytogenes serotype 4b from five patients (three newborn infants, one pregnant woman, and one immunocompromised adult) over a 5-week period, with two additional cases being identified over the following 2 months. Although 23 food samples initially collected from the victim’s houses were negative for listeriae, frozen broccoli and cauliflower were implicated in a follow-up casecontrol study. Investigators eventually recovered L. monocytogenes serotype 4b from opened and later unopened packages of frozen vegetables. Using pulsed-field gel electrophoresis, the food and seven patient isolates proved to be identical, thereby confirming commercially frozen broccoli and cauliflower as infectious vehicles in this unusual outbreak of listeriosis. In November 1988, Ken et al. [ 1371 reported that a 29-year-old pregnant woman in England miscarried after a 2-week history of pyrexia and rigors. An identical phage type of L. monocytogenes serotype 4b was subsequently isolated from placental swabs and maternal/fetal blood samples as well as a 3-month-old bottle of vegetable rennet (may have been microbial rennet) that was discovered in the woman’s refrigerator, thus implicating vegetable rennet which was presumably used to produce a custard-like product. In another of these isolated cases, an 80-year-old previously healthy man in Finland developed a nonfatal septicemic infection 1 day after consuming previously cooked but not reheated homemade salted mushrooms that were later found to contain 3.8 X 106L. monocytogenes CFU/g [133]. Listeria isolates from the patient’s blood and a portion of the uneaten mushrooms belonged to serotype 4b and the same phage type, thus confirming salted mushrooms as the vehicle of infection. According to the investigative team, the implicated homegrown mushrooms were washed, cooked, salted to a level of 7.5% NaC1, and then stored in a cold cellar for 5 months before consumption. Since this product, which was presumably contaminated after cooking, had a pH of 5.9 with visible mold

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growth on the surface, the pathogen probably grew in these mushrooms during the 5 months of storage. Although the exact source of contamination was never determined, it is noteworthy that the man’s wife did not become ill after consuming thoroughly reheated mushrooms from the same container. Hence, it appears that heavily contaminated vegetables also can be rendered safe by thorough cooking. The final case of listeriosis directly linked to products of plant origin is markedly different in that consumption of alfalfa tablets, a dry product in which L. rnonocytogenes is presumably unable to grow, was directly responsible for a fatal case of listerial meningoencephalitis in a 55-year-old immunocompromised Canadian man [95]. As in the two cases just discussed, L. rnonocytogenes strains of serotype 4b isolated from the victim’s blood and cerebrospinal fluid as well as the remaining alfalfa tablets were all of the same electrophoretic enzyme type, which in 1990 confirmed alfalfa tablets as the vehicle of infection in the only listeriosis case thus far linked to ingestion of a nearly completely dry product. However, since Czojka and Batt [74] recently found that these isolates yielded slightly different randomly amplified polymorphic DNA patterns, the exact source of infection now appears to be somewhat open to debate. Although not directly linking consumption of raw vegetables to listerial infection, considerable circumstantial evidence exists for involvement of vegetables in human listeriosis. In the first of these reports [43], a 74-year-old man contracted L. rnonocytogenes serotype 1/2c septicemia and meningitis 1 1 days after repair of his perforated duodenal ulcer in a London-area hospital. During the week before onset of illness, the patient consumed hospital food supplemented with high protein puddings and sandwiches containing various fillings, including cheese; however, hospital personnel failed to maintain an exact record of foods eaten. In conjunction with a survey to determine the incidence of listeriae in hospital-prepared foods, investigators isolated L. rnonocytogenes serotype 1/2a and L. innocua from 1 of 15 and 2 of 15 samples of washed English round lettuce but failed to detect Listeria spp. in 40 other food samples consisting primarily of dairy products and other raw vegetables. Most evidence suggests that listeriosis has an incubation period of 1 to several weeks; however, three of four patients in England with confirmed cases of foodborne listeriosis exhibited incubation periods of 5 1 week. Hence, although the incubation period observed in this 74-year-old man is compatible with a hospital-acquired listerial infection, isolation of different L. rnonocytogenes serotypes from the man and washed English round lettuce appears to preclude direct involvement of lettuce in this particular case of listeriosis. Nevertheless, since L. rnonocytogenes was recovered from washed lettuce but from no other hospital-prepared food, consumption of lettuce still appears to be a possible risk factor in development of hospital-acquired listeriosis. During the previously discussed 1986- 1987 case-control study in which consumption of uncooked frankfurters and undercooked chicken was epidemiologically associated with listeriosis, Schwartz et al. [214] isolated the same serotype and enzyme type of L. rnonocytogenes from five Hispanic listeriosis patients who resided in Los Angeles County. Of the four patients who voluntarily enrolled in the case-control study, all consumed lettuce as well as chicken and whole milk. Unfortunately, because matched controls consumed these products at rates similar to those of cases, it was impossible to link consumption of lettuce as well as chicken or whole milk to listeriosis. More recently, routine followup investigations in Victoria, Australia, have provided some circumstantial evidence for possible involvement of lettuce (one cluster of eight cases) [222], coleslaw (one case) [222], raw vegetables/mayonnaise vegetable dip (one case) [201], and raw vegetables (one case) [201] in sporadic cases of listeriosis. In the United States, active surveillance of a

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large multistate population uncovered a small outbreak of listerial gastroenteritis during 1994 in which commercially prepared potato salad containing low levels of L. monocytogenes serotype 1/2a was the presumed vehicle of infection [221]. According to Salamina et al. [206]. a similar listeriosis outbreak occurred 1 year earlier near Bologna, Italy, with 18 of 39 (46%) young (median age 28 years), previously healthy, nonpregnant adults becoming ill after attending a party. Fourteen of 18 individuals developed listerial gastroenteritis characterized by diarrhea, fever, headache, vomiting, and abdominal pain (median onset time 18 h), with the remaining four patients complaining of flu-like symptoms (median onset time 43 h). Although four individuals with acute gastroenteritis were hospitalized, two of whom also developed septicemic infections, all patients were released 314 days after antibiotic therapy. Analysis of the foods consumed showed that rice salad (prepared from boiled rice, Swiss cheese, pickled vegetables, hard-boiled eggs and frozen vegetables [i.e., peas, carrots]) was the most likely vehicle, since only those individuals who ate the salad became ill. Unfortunately, none of the rice salad remained after initial microbiological testing eliminated E. coli, Salmonella, B a c i h s cereus, and Stuphylococcus aweus as possible causes. However, overwhelming evidence from the food-intake survey, coupled with recovery of the epidemic strain of L. rnonocytogenes serotype 1/2b (confirmed by both enzyme and phage typing) from several other foods consumed at the party, reported temperature abuse of the rice salad as further evidenced by a total aerobic plate count >109 CFU/g and probable cross contamination of other foods in the kitchen points to rice salad as the most probable vehicle of infection. In 1997, Italy was the site of another apparent foodborne outbreak of listerial gastroenteritis, this time the largest to date involving 1594 cases, 123 of which required hospitalization (24 1). Preliminary epidemiological findings suggest a possible link to consumption of sweet corn. Although such epidemiological investigations alone can never prove causality, future studies similar to those just described will continue to play a vital role in elucidating possible relationships between listeriosis and consumption of vegetables as well as fruit, dairy, meat, poultry, seafood, and other foods not yet thought to be associated with this disease.

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Ryser, E.T., and E.H. Marth. 1991. Listeria, Listeriosis and Food Safety. 1st ed. New York: Marcel Dekker. 206. 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. 1 17:429-436. 207. Salvat, G., M.T. Toquin, Y. Michel, and P. Colin. 1995. Control of Listeria monocytogenes in the delicatessen industries: the lessons of a listeriosis outbreak in France. Intern. J. Food Microbiol. 25:75-8 1. 208. Samuelsson, S., N.P. Rothgardt, A.C. Christensen, and W. Fredericksen. 1990. An epidemiological study of human listeriosis in Denmark 1981 - 1987 including an outbreak November 1985- March 1987. J. Infect. 20:25 1-259. 209. Schlech, W.F. 1984. New perspectives on the gastrointestinal mode of transmission in invasive Listeria monocytogenes infection. Clin. Invest. Med. 7:32 1-324. 210. 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. N. Engl. J. Med. 308:203-206. 21 1. Schmidt, V., and A. Nyfeldt. 1938. Ueber Mononucleosis Infectiosa und Meningoencephalitis. Acta Oto-Laryngol. 26:680-688. 212. Schuchat, A., K.A. Deaver, J.D. Wenger, B.D. Plikaytis, I,. Mascola, R.W. Pinner, A.L. Reingold, C.V. Broome, and the Listeria study group. 1992. Role of foods in sporadic listeriosis---I. Case-control study of dietary risk factors. JAMA 267:204 1-2045. 213. Schwartz, B., D. Hexter, C.V. Broome, A.W. Hightower, R.B. Hirschhom, J.D. Porter, P.S. Hayes, W.F. Bibb, B. Lorber, and D.G. Faris. 1989. Investigation of an outbreak of listeriosis: New hypothesis for the etiology of epidemic Listeria monocytogenes infections. J. Infect. Dis. 159:680-685. 214. Schw;irtz, B., C.V. Broome, G.R. Brown, A.W. Hightower, C A . Ciesielski, S. Gaventa, B.G. Gellin, L. Mascola, and the Listeriosis Study Group. 1988. Association of sporadic listeriosis with consumption of uncooked hot dogs and undercooked chicken. Lancet 2:779-782. 215. Seeliper, H.P.R. 1961. Listeriosis. New York: Hafner. 216. Simpson, D.M. 1996. Microbiology and epidemiology in foodborne disease outbreaks: the whys and why nots. J. Food Prot. 59:93-95. 217. Sipka, M., B. Stajner, and S. Zakula. 1973. Detection of Listeria in milk. Wien tierarztl. Monatsschr. 60(2/3):50-52. 218. Slade, P.J., and D.L. Collins-Thompson. 1988. Enumeration of Listeria monocytogenes in raw milk. Lett. Appl. Microbiol. 6: 12 1 - 123. 219. Soontharanont, S., and C.D. Garland. 1995. The occurrence of Listeria in temperate aquatic habitats. Proceedings of XIIth International Symposium on Problems of Listeriosis, Perth, Western Australia, October 2-6, pp. 145- 146. 220. Souef, P., N. Le, and B.N.J. Walters. 1981. Neonatal listeriosis-a summer outbreak. Med. J. Austral. 2: 188- 191. 221. Swaminathan, B., C. Dalton, P. Mead, P.S. Hayes, W.F. Bibb, and A. Schuchat. 1995. Update on listeriosis in the United States. Proceedings of XIIth International Symposium on Problems of Listeriosis, Perth, Western Australia, October 2-6, p. 489. 222. Tan, A., H. Li, S. Heaton, and J.R.L. Forsyth. 1995. Probing epidemiological associations of Listeria monocytogenes with PFGE techniques. Proceedings of XIIth International Symposium on Problems of Listeriosis, Perth, Western Australia, October 2-6, pp. 191-194. 223. Tappero, J.W., A. Schuchat, K.A. Deaver, L. Mascola, and J.D. Wenger. 1995. Reduction in the incidence of human listeriosis in the United States--effectiveness of prevention efforts? JAMA 273: I 1 18- 1 122. 224. Tjomb, P. 1995. La "Listeria" a frappe le Brie de Meaux. Rev. Econ. Technol. Indust. Aliment. 539: 13. 205.

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Todd, E. 1988. Cost of foodborne listeriosis. Proc. X International Symposium on Listeriosis, Pecs, Hungary, Aug. 22-26, Abstr. P4 I . 226. Tulzer, G., R. Bauer, W.D. Daubek-Puza, F. Eitelberger, C. Grabner, E. Heinrich, L. Hohenauer, M. Stojakovic, and F. Wilk. 1987. A local epidemic of neonatal listeriosis in Austriareport of 20 cases. Klin. Padiar. 199:325-328. 227. Urbach, H., and G.1. Schabinski. 1955. Zur Listeriose des Menschen. 2. Hyg. 141:239-248. 228. Vilde, J.L., A. Huchon, M. Mignon, H. Scherrer, E. Bergogne-Berezin, and J. Pierre. 1980. Infection d’allure typique due i Listeria monocytogenes apris absorption d’huhres. Nouvelle Presse Med. 9:3281. 229. Vizcaino, L.L., M.-J. Cubero, and A. Contreras. 1988. Listeric abortions in ewes and cows associated to orange peel and artichoke silage feeding. Proc. X International Symposium on Listeriosis, Pecs, Hungary, Aug. 22-26, Abstr. P29. 230. Vlahovic, S.M.,D. Pantic, M. Pavicic, and J.H. Bryner. 1988. Transmission of Listeria monocytogenes from mother’s milk to her baby and to puppies. Lancet 2:1201. 23 1 Vries, J., and R. Strikwerda. 1957. Ein Fall klinishcher Euter-Listeriose beim Rind. Zbl. Bakteriol. Abt. I. Orig. 167:229-232. 232. Waites, W.M., C.E.R. Dodd, and K.J. Bolton. 199 I . Microbial food poisoning: problems and solutions. Br. Food J. 93:4-9. 233. Watson, G.L., and M.G. Evans. 1985. Listeriosis in a rabbit. Vet. Pathol. 22:191-193. 234. Watson, C., and K. Ott. 1990. Listeria outbreak in Western Australia. Commun. Dis. Intell. 24:9- 12. 234a. 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 in turkey franks: evaluation of a production facility. J. Food Prot. 53:1015-1019. 235. Wesley, I.V., and F. Ashton. 199I . Restriction enzyme analysis of Listeria monocytogenes strains associated with food-borne epidemics. Appl. Environ. Microbiol. 57:969-975. 236. WHO Working Group. 1988. Foodborne listeriosis. Bull. WHO 66:421-428. 237. Wramby, G.O. 1944. Ovn Listerella monocytogenes bokteriologi ach our forekomst av listerellainfection has djur. Skandinavisk Veterinar Tidskrift 34:278-290. 238. Wramby, G.O. 1944. Unpublished data. 239. Yersin, B.R., M.P. Glauser, and F. Regli. 198I . Infections i Listeria monocytogenes chez I’adulte-Etude de 10 cas et revue de la littkrature. Schweiz. Med. Wochenschr. 1 1 1 :15961602. 240. Yousef, A.E., and E.H. Marth. 1988. Behavior of Listeria monocytogenes during the manufacture and storage of Colby cheese. J. Food Prot. 5 1 : 12- 15. 241. Goulet, V. 1998. Personal communication. 225.

Incidence and Behavior of Listeria monocytogenes in Unfermented Dairy Products ELLIOTT. RYSER Michigan State University, East Lansing, Michigan

INTRODUCTION Recognition of raw milk as a potential source of Listeria monocytogenes led to speculation that consumption of such milk was at least partly responsible for the previously described listeriosis outbreak in post-World War I1 Germany. After this listeriosis epidemic, only scattered reports of individuals drinking raw milk, along with assurances that raw milk was being properly pasteurized, virtually eliminated the threat of any further outbreaks of milkborne listeriosis. Consequently, research in this area also subsided. However, in 1983, concerns about the possibility of milkborne listeriosis were rekindled when consumption of pasteurized milk was epidemiologically linked to an outbreak of listeriosis in Massachusetts. Two events, namely, publication of an article in the New England Journal of Medicine detailing this outbreak in Massachusetts and a report in June of 1985 that as many as 300 people in California had acquired listeriosis after eating Mexican-style cheese contaminated with L. monocytogenes, caused considerable concern in the United States about the presence of Listeria in dairy products. This problem subsequently took on international proportions with the 1987 report of another cheese-related outbreak in which consumption of tainted Vacherin Mont d’Or soft-ripened cheese was directly linked to numerous cases of listeriosis in Switzerland. Despite considerable progress, such epidemics continue to plague the dairy industry, with pasteurized chocolate milk and Pont 359

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1’EvSque cheese being responsible for the most recent dairy-related outbreaks of listeriosis in the United States and France, respectively. In response to questions raised by milk producers, dairy processors, health officials, and the general public, a plethora of work has been conducted worldwide since 1983 to determine the incidence and behavior of L. rnonocytogenes in unfermented (raw milk, pasteurized milk, chocolate milk, cream, butter, ice cream, other frozen dairy desserts) as well as fermented (cheese, yogurt, cultured milk) dairy products. The incidence and behavior of L. rnonocytogenes in unfermented dairy products will be dealt with in this chapter; similar information about fermented dairy products appears in Chapter 12.

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

Raw Cow‘s Milk As you will recall from the discussion of animal listeriosis in Chapter 3, dairy cattle can intermittently shed L. monocytogenes in their milk as a consequence of listerial mastitis, encephalitis, or a Listeria-related abortion. Although milk from animals showing obvious signs of listeriosis is unlikely to reach consumers, the scientific literature contains numerous accounts in which mildly infected and apparently healthy dairy cattle have shed L. monocytogenes intermittently in their milk for many months. Thus it appears that such asymptomatic carriers of listeriae pose the greatest threat to public health. The 1983 listeriosis outbreak in Massachusetts that was supposedly associated with drinking a particular brand of pasteurized milk raised numerous questions about milk safety. The well-publicized outbreak of 1985 in which consumption of contaminated Mexican-style cheese was directly linked to at least 40 deaths in California prompted additional concerns about the safety of dairy products manufactured in the United States. Since raw milk is a potential source of L. monocytogenes, this fact together with recalls of Listeriacontaminated pasteurized dairy products (i.e., milk, chocolate milk, ice cream) and imported soft-ripened cheeses prompted nearly 70 surveys worldwide to determine the extent of Listeria contamination in raw milk. Results of these surveys, which will now be described in some detail, have been summarized in Tables l and 2. The first large-scale survey of raw milk for Listeria spp. was prompted by the 1983 listeriosis outbreak in Massachusetts. During the 3-week period immediately following the outbreak, Fleming et al. [ 1 101 and Hayes et al. [ 1291 examined 121 raw milk samples collected from milk trucks (40 samples), milk cooperatives (72 samples), and bulk tanks from four farms on which bovine listeriosis was diagnosed (9 samples), as well as 14 milk socks (used to remove debris but not leukocytes from milk). All samples were analyzed for

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TABLE 1 Incidence of Listeria spp. in Raw Milk Produced in the United States and Canada Location USA

California Massachusetts Massachusetts, Vermont Minnesota Nebraska Ohio, Kentucky, and Indiana Pennsylvania Tennessee Wisconsin

Total Canada Alberta Manitoba Ontario Total

Number of samples

Number of positive samples (%)

L. inonocyogenes

L. innocuu

L. ri.eist'iirneri

Others

Ref.

200 100 121 939

14 (7.0) 0 15 (12.4) 15 (1.6)

19 (9.5) 4 (4.0) ND ND

0 0 ND ND

0 1 (1.0y ND ND

141 141 129 87

300 84 200 350

9 (3.0) 0 8 (4.0) 13 (3.7)

77 (25.7) 6 (7.1) 10 (5.0) 27 (7.7)

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

0 0 0 3 (0.9)a

144 161 139 141

251 1 292 50 55

79 (3.1) 12 (4.1) 0 0

ND ND ND 0

ND ND ND

88 177 89 20 1

5 197

165 (3.2)

143 (11.1)

12 (0.9)

4 (0.3)

426 252 256 445 3 15 1694

8 (1.9) 4 (1.6) 4 (1.6) 6 (1.3) 17 (5.4) 39 (2.3)

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

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

ND ND ND

ND, not determined (omitted from total). Two L. ivnnovii and one L. seeligeri

ND ND ND 0

0

0 0 0 (0)

106 200 80 97 190

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362 TABLE 2 Incidence of Listeria spp. in Raw Milk Collected Outside of North America Location Europe Czechoslovakia

Denmark Finland France

Ireland Italy

Netherlands Poland

Number of positive samples (%)

Number of samples

L. monocytogenes

L. innocua

L. welshimeri

Others

Ref.

177 123 1,227,053 256 59 1409 561 337 51 635 80 50 589 50 290 142 98 85 50 40 32 137 134 81

6 (3.4) 4 (3.3) 278 (0.02) 13 (5.1) 1 (1.7) 85 (6.0) 21 (3.8) 14 (4.2) 10 (19.6) 2 (0.3) 3 (3.8) 2 (4.0) 29 (4.9) 4 (8.0) 8 (2.8) 0 2 (2.0) 0 0 0 0 6 (4.4) 2 (1.5) 6 (7.4)

ND ND ND ND ND ND ND 5 (1.5) 10 (19.6) ND ND ND 20 (3.4) ND 16 (5.6) 1 (0.6) ND 0 1 (2.0) 0 2 (6.3) ND ND ND

ND ND ND ND ND ND ND 0 18 (35.3) ND ND ND ND ND 0 0 ND 0 0 0 ND ND ND ND

ND ND ND ND ND ND ND 0 9 (17.6) ND ND ND ND ND 2 (0.7) 0 ND 0 0 0 ND ND ND ND

151 152 167 167 167 43 49 120,121 120,121 115, 197 174 174 173 133 83 192 138 117 199 148 112 68 136 178

L. monocytogenes in Unfermented Dairy Products Switzerland Turkey United Kingdom Great Britain England/Wales Scotland Northern Ireland Total Elsewhere Australia Brazil Costa Rica Egypt Iran Japan Mexico Morocco New Zealand South Africa Taiwan Total

4046 340 77

14 (0.4) 2 (0.6) 14 (18.2)

ND ND ND

ND ND ND

ND ND ND

66 66 187

350 2009 361 640 560 540 176 113

13 (3.6) 102 (5.1) 13 (3.6) 90 (14.1) 14 (2.5) 14 (2.6) 27 (15.3) 6 (5.3)

ND ND ND ND 7 (1.3) ND 18 (10.2) ND

ND ND ND ND 0 ND 0 ND

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

118 157 126 108 107 107 127 119

14,678

527 (3.6)

80 (3.4)

18 (0.8)

27 (1.2)

ND 4 (2.7) 21 (9.5) ND 10 (4.5) 20 (8.5) ND ND 2 (1.3) 7 (7.0) ND 10 (14.1) ND ND

ND 0 2 (0.9) ND 3 (1.4) 0 ND ND 0 2 (2.0) ND 1 (1.4) ND ND

ND 0 1 (0.4) ND 1 0 ND ND 0 0 ND 7 (9.9) ND ND

74 (6.5)

8 (0.7)

9 (0.8)

169 150 220 20 220 236 190 120 150 100 30 71 982 80 2738

ND, not determined (omitted from total). L. seeligeri. L. grayi.

a

363

1 (0.6) 0 11 (4.8) 0 0 7 (3.0) 4 (2.1) 0 6 (4.0) 0 3 (10.0) 0 67 (6.8) 5 (6.3) 104 (3.8)

95 130 155 73 63 93 172 196 186 142 94 194 204 74

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L. rnonocytogenes using the multiple two-stage enrichment procedure. Although investigators at the U.S. Centers for Disease Control and Prevention (CDC) isolated the epidemic serotype along with other serotypes of L. rnonocytogenes from 15 of 121 (12.4%) (including 1 of 9 bulk tank samples) [ 1281 and 2 of 14 (14%) raw milk and milk sock samples, respectively, the epidemic phage type was never detected. Between October 1984 and August 1985, U.S. Food and Drug Administration (FDA) officials surveyed 650 raw milk samples that were collected from bulk tanks in Massachusetts, Vermont, California, and the tristate area of Kentucky, Ohio, and Indiana. The samples were examined for Listeria spp. using the original FDA method [141]. Low levels of various Listeria spp., including L. rnonocytogenes, were detected in raw milk samples obtained from all states except California. Overall, 82 of 650 (12.6%) samples contained Listeria spp., with L. rnonocytogenes being detected in 27 of 650 (4.2%) samples. Of the 27 L. rnonocytogenes strains isolated from raw milk, I6 were serotype 1, 10 were serotype 4, and 1 was nontypable. In addition, only 2 of the 27 L. rnonocytogenes strains proved to be nonpathogenic to mice, and both were of serotype 4. In 1988, Donnelly et al. [87] reported using an automated flow cytometric procedure to analyze 939 samples of raw milk obtained from 54 farms in California. Unlike the FDA study just described [ 1411, string samples (milk pooled from 25 to 40 cows), combination samples (milk pooled from -200 cows), and samples of raw milk from bulk tanks were tested for L. rnonocytogenes. Using this novel method of analysis, L. monocytogenes was detected in 15 of 939 ( I .5%) samples of raw milk. Researchers in Minnesota [ 1611 and Wisconsin [89,201] failed to detect L. rnonocytogenes in raw milk during three small surveys. However, this organism was found in 4.0 and 2.8% of raw milk samples obtained from bulk storage tanks and tank trucks in Nebraska [ I401 and Pennsylvania [88], respectively, with approximately equal numbers of L. rnonocytogenes isolates being classified as serotype 1 , 4, or nontypable (non-serotype 1 or 4) in the latter study. More recent findings indicate a relatively stable incidence for L. rnonocytogenes in bulk tank raw milk, with 3.0 and 4.1 % of such samples from Minnesota [ 1441 and Tennessee [177], respectively, being positive. In the Minnesota survey, Listeria-positive samples also tended to have higher bacterial and somatic cell counts, which are indicative of less stringent sanitation and mastitis control practices. The overall findings from Table 1 indicate that L. rnonocytogenes was present in 0- 12.4% of the U.S. raw milk samples examined. When the results are averaged, 3.2% of all raw milk processed in the United States can be expected to contain low levels (i.e., <10 CFU/mL) of L. rnonocytogenes at any given time with the incidence of L. innocua being appreciably higher at 1 I . 1 %. Hence, it is imperative that all milk processors take special precautions to prevent the spread of L. monocytogenes from raw milk to production, packaging, and other sensitive areas within the factory. Turning to the incidence of Listeria in Canadian raw milk (see Table l), Farber et al. [97] and Slade et al. [ 1901, respectively, isolated L. monocytogenes from 6 of 445 (1.3%) and 17 of 315 (5.4%) raw milk samples obtained from bulk tanks located throughout the province of Ontario. Davidson et al. [80] also reported that a similar percentage of raw milk samples collected from four local dairies and 48 farms in Manitoba contained L. rnonocytogenes. In the study by Slade et al. [ 1901, 14 of 17 (82%) L. rnonocytogenes isolates belonged to serotype 1, with the remaining three strains being classified as serotypa 4. Although subsequent work [ 1091 demonstrated that none of these L. rnonocytogenes strains harbored plasmid DNA, 8 of 22 (36%) L. innocua strains did carry plasmids ranging in size from 10 to 44 MDa. Variable plasmid profiles among these isolates further suggests

-

’

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365

that contamination of raw milk on the farm is an ongoing process. Whereas these authors did not quantitate L. monocytogenes in any of the samples examined, Slade and CollinsThompson [ 1891 reported that positive raw milks from bulk tanks in southwestern Ontario always contained <5 L. monocytogenes CFU/mL when samples were analyzed by direct plating and a most probable number (MPN) enrichment procedure. Thus the positive raw milk samples encountered in three Canadian studies likely contained only low levels of L. monocytogenes. In two subsequent surveys, L. monocylogenes was identified in 1.6 [200] and 1.9% [106] of all raw milk bulk tank samples examined in the province of Alberta. However, both surveys also indicated substantially higher contamination rates for commingled raw milk before processing with milk in 5 of 72 (6.9%) tank trucks [ 1061 and in 4 of 15 (26.6%) milk silos [200] testing positive for L. monocytogenes. As was true for the three United States surveys [ 1411just discussed, L. innocua also was the most common Listeria sp. isolated from Canadian raw milk, with 8.2 and 9.7% of the samples being reported as positive. Overall, 2.3 and 9.1% of all Canadian raw milk samples contained L. rnonocytogenes and L. innocua, respectively, as compared with 3.2 and 1 1.1 % of raw milk samples examined in the United States. Thus the incidence of listeriae in raw milk from both countries appears to be similar. Public concern and economic hardships brought about by several recalls of French Brie cheese imported into the United States prompted French scientists to begin surveying raw milk for L. monocytogenes. Results from three such surveys [43,49,120] (see Table 2) conducted between 1986 and 1988 indicated that 85 of 1409 (6.0%), -21 of 561 (3.8%), and 14 of 337 (4.2%) raw milk samples from French bulk tanks were positive for L. monocytogrnes, with L. innocua being detected in about one third as many samples in the latter study [120]. During January of 1986, 51 raw milk samples were submitted to the French Central Laboratory of Food Hygiene. Both L. monocytogenes and L. innocua were found in 19.6% of these samples. The unusually high incidence of listeriae observed in this survey, as compared with other studies described thus far, may be the result of nonrandom sampling or seasonal variations, the latter of which will be discussed shortly. Frequent isolation of L. monocytogenes from dairy products prompted additional surveys of the raw milk supply throughout Europe and later elsewhere, with primary focus being given to countries with sizeable dairy industries (see Table 2). Results from two comprehensive year-long surveys in the United Kingdom [ 1 181 and Scotland [ 1071 revealed an incidence of L. monocytogenes in raw milk similar to that observed in the United States and Canada, with actual Listeria populations in positive samples also being estimated to be extremely low. As in North America, L. monocytogenes strains belonging to serotype 1/2 appear to predominate in European raw milk [ 107,108,126,1781. Although four subsequent surveys from the United Kingdom yielded similar findings [ 107,119, 126,1571, L. monocytogenes contamination rates of 14 to 15% have been reported from both Scotland [ 1081 and Northern Ireland [ 1271, thus suggesting considerable local variability. Harvey and Gilmour [127] also found that 33% of raw milk samples collected from processing centers harbored L. monocytogenes, which again emphasizes the impact of commingling milk. However, levels of listeriae in such milk again appear to be quite low, with actual numbers of L. monocytogenes typically being < 10 CFU/mL in positive samples from Scotland [ 1081. In 1987, Beckers et al. [68] reported culturing L. monocytogenes from 6 of 137 (4.4%) raw milk samples obtained from farms in the Utrecht region of The Netherlands. As was true for raw milk tested in the United States and Canada, milk samples from The

366

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Netherlands again contained < 100 L. monocytogenes CFU/mL. Similar findings have been reported from most other European surveys, with 3-5% of raw milk samples harboring low levels of L. monocytogenes. However, two nationwide surveys conducted in Switzerland during and after the outbreak involving Vacherin Mont d’Or cheese indicated a far lower incidence, with only 0.4 and 0.6% of the raw milk samples being positive for L. monocytogenes [66]. Several years earlier, Terplan [ 1971 detected L. monocytogenes in only 2 of 635 (0.3%) raw milk samples obtained from farm bulk tanks in Wurtemburg, Germany. Subsequent attempts to isolate this pathogen from 448 quarter-milk and 30 separator sludge samples failed, along with attempts to culture this organism from raw milk samples obtained from tank trucks and storage tanks. More recently, findings from an exhaustive survey in Denmark of over 1 million raw milk samples demonstrated an L. monocytogenes contamination rate of only 0.2%, with several additional small-scale Italian surveys yielding negative results. Thus, when the Danish results are excluded, 3.6% of all European raw milk would be expected to contain low levels of L. monocytogenes as compared with 3.0% of the raw milk produced in North America. In response to the aforementioned North American and European surveys, investigators in Australia, New Zealand, the Middle East, South America, Africa, and the Orient also have begun assessing the incidence of L. monocytogenes contamination in their own raw milk supplies. In the first of these surveys, workers in New Zealand [ 1941 collected and analyzed 71 raw milk bulk tank samples between August 1986 and March 1987 for listeriae using both warm and cold enrichment. Although L. monocytogenes was apparently absent from this milk as well as from milk examined in a later Australian survey, isolation of other Listeria spp. from these samples strongly suggests that such milk is unlikely to be completely free of L. monocytogenes. Although similar negative findings have been obtained from surveys conducted in Brazil [73] and Costa Rica [63], Arias et al. [63] also cited another survey in which 20% of hand-milked samples harbored L. monocytogenes. Working in Japan, Takai et al. [196] reported the absence of Listeria spp. from 120 raw milk samples, whereas L. monocytogenes was recovered from 6 of 150 (4.0%) samples in a subsequent Japanese survey [186]. Given these findings along with additional reports of L. monocytogenes in raw milk from Egypt [93,1], Iran [172], Turkey [124], Morocco [94], and South Africa [204], it is now clear that milkborne listeriosis constitutes a worldwide threat. However, confirmation of dairyrelated listeriosis cases in such developing countries will likely remain very difficult given their lack of resources and understandable preoccupation with far more immediate health concerns. Examination of data summarized in Tables 1 and 2 indicates that 3.2, 2.3, and 3.6% of the raw milk produced in the United States, Canada, and Europe, respectively, can be expected to contain low levels of L. monocytogenes, with similar contamination rates also likely occurring in other parts of the world. Except for the European samples, L. innocua was isolated from raw milk more frequently than L. monocytogenes, with the former found in 11.1, 9.1, and 3.4% of the samples analyzed for all Listeria spp. in the United States, Canada, and Europe, respectively. This observation also is supported by additional survey findings from Australia, Costa Rica, Egypt, and New Zealand. Although L. innocua is nonpathogenic, isolation of this organism and other listeriae from dairy products and dairy processing facilities is taken very seriously in the United States, since nonpathogenic listeriae and L. monocytogenes are assumed to occur in similar environmental niches. Use in the United States of L. innocua as a potential indicator of L. monocytogenes is supported by data from the raw milk surveys just described (see

L. monocytogenes in Unferrnented Dairy Products

367

Table 1) in that isolation of listeriae other than L. morzocytogenes from dairy products and processing facilities suggests that the factory environment may be contaminated with raw milk; that is, a product that can be expected to contain L. monocytogenes 3-4% of the time. Although long theorized, the spread of identical L. nzonocytogenes strains from the farm environment into the raw milk supply and ultimately to dairy processing facilities now has been confirmed using various strain-specific typing methods, including restriction fragment length polymorphism [ 1271 and automated ribotyping [64]. In addition to assessing the general incidence of listeriae in raw milk, several of the surveys just described also dealt with seasonal variations in the incidence of L. monocytogenes and L. innocuci in raw milk produced in the United States [88,139,141] and Canada (971. However, since all of the aforementioned studies differ in numbers and sizes of samples analyzed as well as the Listericl isolation procedures employed, it is difficult to make any definitive statement concerning the seasonal occurrence of Listerin in raw milk [ 107,190]. Nonetheless, several distinct trends can be observed from selected data in Figure 1. First. the overall incidence of L. rnonocytogenes in raw milk was highest in spring (5.8%) followed by winter (4.5%), fall (2.8%), and summer (2.5%). Second, a somewhat similar seasonal variation also can be observed for the incidence of L. innocun in raw milk, with the highest overall percentage of positive samples again occurring in winter (1 1.7%) followed by spring (10.2%). summer (6.0%), and fall (4.2%). These findings again suggest that L. innocirn can be used as a potential indicator for the presence of L. inonocytogenes. Although not fully understood, current herd management and feeding practices may be at least partly responsible for seasonal differences observed in isolation rates for L. inonocytogenes and possibly L. iiinocun. During cold winter months, silage comprises a major component of the diet. While investigating a listeriosis outbreak, Donnelly [85]

FIGURE1 Seasonal variation in the incidence of L. monocytogenes and L. innocua in raw milk from the United States [88,129,139,1411, Canada 1971, and England/Wales [ 1571.

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368

observed that 8 of 44 Holstein cows fed Listeria-contaminated silage shed the organism in their milk. Furthermore, milk from these animals was free of L. monocytogenes 1 month after feeding of contaminated silage ceased. Ruminants that ingest contaminated silage may either succumb to infection or carry L. monocytogenes asymptomatically ; however, if the animal lives, the organism can be shed for many months in feces and in milk from lactating animals. Extended survival of L. monocytogenes in fecal material, soil, and grass can perpetuate the infectious cycle shown in Figure 2, particularly when animals are wintered in cramped quarters. Once dairy cattle resume grazing on pastures during late spring, summer, and early fall, L. monocytogenes becomes dispersed over a wide area, which, in turn, weakens the infectious cycle by decreasing the likelihood that animals will come in contact with contaminated material. Seasonal differences in the incidence of Listeria spp. in raw milk also may be related to breeding practices. Dairy cattle typically bear their young in late winter or early spring. During winter gestation, dairy cattle develop a weakened immune system as a direct result of pregnancy, which, in turn, makes these animals more susceptible to listerial infections and abortions. These events can then culminate in the shedding of L. monocytogenes in milk and fecal material. Increased environmental stress and changes in habitat that occur during winter, along with increased difficulties in providing proper herd hygiene, all can serve to decrease the natural defense system in dairy cattle, which again increases the likelihood for listerial infections. Once an asymptomatic animal begins shedding L. monocytogenes in feces, the organism is likely to spread quickly to other animals that are housed in close proximity to the shedder. In this way, confinement of dairy cattle may play an important role in increasing the number of animals that shed L. monocytogenes in their milk during late winter and early spring.

Survival and/or growth

/

Survival in s o i l and grass

Ingestion by ruminants

confinement

4

L. monocytogenes excreted in feces

L.

monocytogenes secreted in milk

FIGURE2 Infective cycle for maintaining L. rnonocytogenes in ruminants.

L. monocytogenes in Unfermenfed Dairy Producfs

369

Raw Ewe‘s and Goat’s Milk Surveys in Europe, Australia, and the United States also have demonstrated that raw milk from ewes and goats can occasionally contain L. monocytogenes (Table 3) with incidence rates ranging from 0-2.2% (average of 1.6%) and 0-4.0% (average 2.4%), respectively. These contamination levels are similar to those reported for cow’s milk. Working in Vermont, Abou-Eleinin et al. recently recovered Listeria spp. from 35 of 445 (7.9%) bulk tank samples of raw goat’s milk using three different enrichment methods. Seventeen samples contained L. monocytogenes and 8 contained both L. monocytogenes and L. innocua. Listeria contamination rates were higher in winter (14.3%) and spring (10.4%) than in fall (5.3%) and summer (0.9%) with similar observations reported for cow’s milk [ la]. Overall, 62.6% and 37.4% of those L. monocytogenes that were further characterized belonged to serovars 1 and 4, respectively. Automated ribotyping of selected isolates also indicated five distinct ribotypes, two clinically important ribotypes of which were eventually traced back to the farm environment.

Pasteurized Milk and Other Unfermented Dairy Products The dairy industry has long been considered as the most regulated food industry in the United States. The FDA was given responsibility under the Food, Drug and Cosmetic Act and the Public Health Service Act to assure the public that this country’s milk supply is both uniformly safe and wholesome. Dairy sanitation laws and regulations, including microbiological criteria for some dairy products, enforced by the FDA and state agencies are based almost exclusively on the Public Health Service/FDA Grade .4Pasteurized Milk Ordinance. In the United States, pasteurized milk and other unferniented dairy products prepared from pasteurized milk, including ice cream, butter, and nonfat dry milk, generally have

TABLE 3 Incidence of L. rnonocytogenes in Raw Ewe’s and Goat‘s Milk Type of milk Ewe

Location

Number of samples

Number of samples positive (%)

England/ Wales Italy Italy Spain Turkey

56 40 34 1052 302

1 (1.8) 0 0 23 (2.2)a

1484

24 (1.6)

450 480 24 25 1445 69

17 (3.8)” 4 (0.8) 0 1 (4.0) 37 (2.6) 1 (1.4)

2493

60 (2.4)

Total Goat

Total a

United States England/Wales Italy Portugal Spain Australia

0

Also 21 L. innocua, 4 L. welshimeri, 3 L. seeligeri, 3 L. grayi, and 2 I,. ivanovii 8 L. monoi’vtogenes and L. innocua, and 26 L. innocua.

Ref. 126 71 76 175 78 la 126 71 79 116 125

370

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earned a reputation for being both safe and nutritious, with dairy products accounting for <1.5% of all foodborne illnesses [67]. Pasteurized milk is responsible for 5 5 % of all reported dairy-related illnesses [ 1501. Despite these impressive findings, public confidence in the safety of pasteurized milk began to erode in July of 1982 following an outbreak of yersiniosis in Tennessee, Arkansas, and Mississippi [ 1951 and again in 1983 when the CDC claimed that consumption of pasteurized milk was responsible for 49 cases of listeriosis in Massachusetts. In 1985, the dairy industry was dealt another blow when at least 16,000 culture-confirmed cases of salmonellosis were associated with drinking a particular brand of pasteurized milk produced in the Chicago area [183]. These three outbreaks, along with the previously discussed listeriosis outbreak in California linked to consumption of contaminated Mexican-style cheese, prompted the FDA to take corrective action in the form of a large-scale testing program commonly referred to as the FDA Dairy Initiative Program [134] (Fig. 3). This program, begun in April of 1986 in cooperation with individual state agencies and members of the National Conference on Interstate Milk Shipments, was designed to examine every interstate milk shipment (IMS) pasteurization facility in the United States for potential safety problems related to pasteurization, postpasteurization contamination, cleaning and sanitizing regimens, equipment maintenance, and educationaUtraining programs for dairy factory personnel. As part of the FDA Dairy Initiative Program, the agency also established the Microbiological Surveillance Program, which was designed to detect L. monocytogenes, Salmonella spp., Yersinia enterocolitica, Campylobacter jejuni, and C. coli (Campylobacter omitted in 1987) as well as Staphylococcus aureus, which was added in 1987 for dry and fluid milk [16]. Dairy products tested under this program included fluid milk, nonfat dry milk, cream, butter, ice cream, ice milk, and other dairy commodities over 1

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

I

!

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

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

I

I

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

April 1986:

~

October 1986:

October 1987:

Continue sampling except omit Cam 1444 samples analyzed during f

lobacter e

1

Continue sampling remainder of inventory and the following composite samples - Fluid milk: Listeria, Staphylococcus aureus and Yersinia; Dry milk: Listeria, Salmonella, and Scaphylococcus aureus.

-I

FIGURE3 FDA Dairy Initiatives Program. IMS, interstate milk shipment. (Adapted from Ref. 134)

L. monocytogenes in Unfermented Dairy Products

371

which the FDA has jurisdiction. Analysis of cheeses (except cottage cheese) was covered under a series of separate programs, which will be discussed in Chapter 12 concerning fermented dairy products. Under provisions of this program, FDA inspectors collected 30 retail-sized containers of as many as five different products available from dairy factories at the time of inspection. Duplicate 25-g or 25-mL samples obtained after combining 30 retail-sized samples per product were then analyzed for L. monocytogenes and other listeriae using the original and later versions of the FDA procedure. Since raw milk containing L. monocytogenes will enter every dairy factory in the United States from time-to-time, it is logical to assume that finished products also may become contaminated with this pathogen. During the first 2 years of the FDA Dairy Initiative Microbiological Surveillance Program (Table 4), L. monocytogenes was isolated from 2 of 350 samples of pasteurized whole milk and 5 of 415 samples of chocolate milk, which suggests that approximately 0.67% of the pasteurized milk available in the United States could contain this pathogen unless factories take corrective action to reduce this value. In contrast, L. monocytogenes was isolated from 3 of 99, 23 of 659, and 30 of 351 samples of ice milk, ice cream, and novelty ice cream, respectively. Only two of the positive ice cream samples were analyzed quantitatively for L. monocytogenes. One contained an average of 15 L. monocytogenes CFU/g, whereas the other sample contained between 1 and 5 CFU/g. Thus during the time of this survey, approximately 5% of frozen dairy products manufactured in the United States presumably contained low levels of L. monocytogenes. Furthermore, L. innocua was isolated from three of four product categories (see Table 4) which also contained samples positive for L. monocytogenes. Since both organ-

TABLE 4 Incidence of Listeria spp. in Unfermented Dairy Products Manufactured in the United States during 1986 and 1987

Product Whole milk Lowfat milk Skim milk Chocolate milk Cream Half and half Ice milk Ice cream Novelty ice cream Butter Nonfat dry milk Casein/Milk protein hydrolysate Other products Total

Number of samples tested

Number of positive samples (%)

-

L. rnonocytogenes

L. innocua

350 182 98 415 52 42 99 659 35 1 30 44 15 171

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

0 2 (1.10) 0 1 (0.24) 0 0 0 12a(1.82) lob (2.85) 0 0 0 0

2518

63 (2.50)

25 (0.99)

L. seeligeri also detected in 7 of 659 (1.06%) samples. L. gruyi also detected in I of 351 (0.28%) samples. Dairy blend whey, eggnog. Source: Refs. 62 and 135.

372

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isms likely occupy similar niches in the natural environment and dairy processing facilities, isolation of L. innocua from a dairy product should raise immediate concerns about the possible presence of L. monocytogenes. Greater success in isolating L. monocytogenes from chocolate rather than whole milk is likely related to the organism’s ability for enhanced growth in this product as compared with other fluid dairy products. Reasons for increased growth of Listeria in chocolate milk will be discussed shortly in conjunction with the behavior of listeriae in autoclaved fluid dairy products. The higher incidence of L. monocytogenes in frozen rather than fluid dairy products coincides with the relatively complex handling of ice milk, ice cream, and particularly ice cream novelties during manufacture and packaging. This, in turn, suggests that these products are most likely contaminated after pasteurization through either direct or indirect contact with listeriae within the dairy factory environment. This hypothesis is supported by frequent isolations of L. monocytogenes from many areas within dairy factories, including floors, ceilings, drains, and coolers. In addition, this organism also has been found in air and condensate and on various pieces of equipment, including conveyor belts. A detailed discussion of the incidence of L. monocytogenes in food processing facilities, including dairy factories, can be found in Chapter 17. Inability of the FDA to detect L. monocytogenes in skim and low-fat milk as well as half and half, cream, and butter may have resulted from the separation processes used for adjusting milk-fat content that these products undergo. Such centrifugal separation processes tend to decrease levels of listeriae, particularly if leukocytes containing the organism are still present after initial clarification of the milk. Failure to isolate L. monocytogenes from nonfat dry milk and casein/ protein hydrolysates may be partly related to the heat treatments necessary to manufacture these products. This theory is supported by the work of Doyle et al. [90], who demonstrated that populations of L. monocytogenes decreased 1 9 0 % during conversion of skim milk into nonfat dry milk via spray drying. However, failure to isolate L. monocytogenes from dried dairy products also may result from the generally recognized inability of the FDA method to detect cells of L. monocytogenes that have been sublethally injured during thermal processing. Thus the methodology employed to detect L. monocytogenes will predetermine whether or not the organism can be isolated from a particular food. According to the Food, Drug and Cosmetic Act of 1938, a food may be considered adulterated and therefore unfit for human consumption if the product contains poisons or other harmful substances (e.g., pathogenic microorganisms) at detrimental concentrations. Although the oral infective dose for L. monocytogenes is presently unknown, evidence from the California listeriosis outbreak involving Mexican-style cheese suggests that the number of L. monocytogenes cells needed to induce this life-threatening illness may be quite low-perhaps as few as several hundred to a few thousand total cells for certain segments of the population. Although not directly applicable to the human population, several independent studies involving immunocompromised mice have demonstrated LDSo values (the dose of cells which is lethal to 50% of a given population) in the range of approximately 10 [ 1231 and 4 to 480 [77,193] L. monocytogenes cells when the pathogen was administered orally and intraperitoneally, respectively. Consequently, because of a moral obligation to the public, the FDA has adopted and is continuing to uphold a policy of ‘ ‘zero tolerance” regarding presence of L. monocytogenes in ready-to-eat foods. In accordance with Title 21 of the United States Code of Federal Regulations, Section 7.40 [ 1 1 I], the FDA can request that firms voluntarily recall any product that contains

L. monocytogenes in Unfermented Dairy Products

373

or is suspected of containing L. monocytogenes. These recalls can be classified into one of three categories: Class I, Class 11, or Class 111. A Class I recall, which is the most serious, is defined by the FDA as “a situation in which there is a reasonable probability that the use of or exposure to a violative product will cause rserious adverse health consequences or death.” Thus far, all recalls issued for products contaminated with L. monocytogenes have been categorized as Class I. In the unlikely event that a firm fails to comply with the FDA’s request to recall a product containing L. monocytogenes, FDA officials can (a) initiate a seizure request in the U.S. District Court to have the product removed from commerce (Title 21 Code of Federal Regulations, Section 334) or (b) obtain a legal injunction to halt production and distribution of the contaminated product (Title 21 Code of Federal Regulations, Section 332). In addition, the FDA also can take criminal action against individuals of a company who are responsible for commercial distribution of a contaminated product. Adoption of the FDA Dairy Initiative Microbiological Surveillance Program in April of 1986 fostered the beginning of a series of recalls issued for milk and unfermented dairy products contaminated with L. monocytogenes (Table 5). Just 1 month after the surveillance program began, a California firm voluntarily recalled an unknown quantity of ice milk mix contaminated with L. monocytogenes. During the same month, approximately I million gallons of fluid dairy products that comprised milk, chocolate milk, half-andhalf, whipping cream, ice milk mix, ice milk shake mix, and ice cream mix also were recalled in Texas [ 1 1,121. However, other than this single incident, the remaining recalls have been primarily confined to frozen dairy products such as ice cream, ice cream novelties, ice milk, and sherbet, with only four additional recalls thus far being traced to nonfroZen dairy products (i.e., butter). In July of 1986, a large recall of Listeria-contaminated ice cream bars received considerable media attention, which in turn did much to enhance the state of hysteria concerning the presence of L. monocytogenes in dairy products IS]. The following month, another nationwide recall was issued for frozen dairy products. As a result of this recall, approximately 1 million gallons of products possibly contaminated with L. monocytogenes and including ice cream (1 32 flavors), ice milk ( I 6 flavors), sherbet (9 flavors), and gelatida products (6 flavors) were reportedly buried at a Minnesota landfill site. One year later, a similar recall was issued for contaminated ice cream, ice milk, and sherbet manufactured in Iowa [26,30]. By the end of 1987, well over 500 Listeria-contaminated dairy products were voluntarily recalled in the United States at a total cost to the dairy industry of well over $70 million [29,149]. Although corrective measures instituted by the dairy industry in response to governmental pressure reduced both the extent of Listeria contamination in processing facilities and the number of Class I recalls issued for L. monocytogenescontaminated dairy products during 1988 and 1989 [49], recalls involving frozen desserts and more recently butter continue to plague the industry. During the 6-year period from 1990 to 1995, 17 of 39 (44%) dairy-related L. monocytogenes recalls involved unfermented dairy products, with Listeria being responsible for 3 I % of all dairy-related recalls issued for any reason [ 1841. Unfortunately, product losses are also being substantially underreported, since, under certain circumstances, manufacturers can retrieve their own product without issuing a formal Class I recall. In one such instance, a manufacturer of Listeriacontaminated ice cream was not required to issue a formal recall, because the product had not yet reached the consumer [34]. Additional cases also likely occurred in which contaminated products moved only as far as the company’s warehouse and were recalled internally by the manufacturer.

374

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TABLE 5 Chronological List of Voluntary Class I Recalls Issued in the United States During 1986 and 1987 for Milk and Unfermented Dairy Products Contaminated with L. monocytogenes

Type of dairy product

Month/ year of recall

State of manufacture

~~~~~~~~~~

~

Ice milk mix

5/86

California

Milk (2 and 112% fat), chocolate milk, chocolate milk (2% fat), half and half, whipping cream, vanilla and chocolate ice cream mix, ice milk shake mix, and ice cream mix Ice cream bars Ice cream, sherbet, glacee Ice cream (1 32 favors), ice milk (16 flavors), sherbet (9 flavors), gelati-da products (6 flavors)

5/86

Texas

7/86 7/86 8/86

Virginia Wisconsin Minnesota

8/86

Iowa

Ice cream and ice cream novelties-bars, drumsticks, slices, sundaes Ice cream Ice cream Ice cream Ice cream, ice milk, sherbet

9/86 12/86 12/86 1187

Wisconsin New York West Virginia Iowa

Distribution

Quantity

Ref.

~

9,ll

Arizona, Nevada, California Texas

Unknown -1 million gal

11,12

Eastern United States Minnesota, Wisconsin Illinois, Indiana, Iowa, Kentucky, Michigan, Minnesota, Missouri, North Dakota, Ohio, South Dakota, Wisconsin Illinois, Iowa, Minnesota, Missouri, North Dakota, Wisconsin Wisconsin New York West Virginia, Ohio Arkansas, Delaware, Illinois, Iowa, Kansas, Maryland, Minnesota, Missouri, Nebraska, Oklahoma, Pennsylvania, South Dakota, Wisconsin

Large but unknown 8000 gal 1 million gal

5 8 10,13

-

Unknown Unknown -835 gal 450 gal -1 million gal

6 7 25,30 15,26 15,19,28,30

375

L. monocytogenes in Unfermented Dairy Products

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

4/87 7/87

New York California

New York, Pennsylvania California, Oregon

-316 gal -60,000 gal

27,30 20,40

7/87

Maryland

20,400 boxes

21,28

Ice cream, ice milk

7/87

Nebraska

-30,000 gal

17

Ice cream sundae cones

8/87

Florida

Unknown

22

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

8/87 9/87 9/87

Kentucky Maryland Ohio

Connecticut, Florida, Maryland, New Jersey, New York, North Carolina, Ohio, Pennsylvania, Virginia Colorado, Iowa, Kansas, Nebraska, North Dakota, South Dakota, Wyoming Alabama, Arizona, British West Indies, Florida, Louisiana, Mississippi, North Carolina, Ohio, Puerto Rico, South Carolina, Tennessee, Virginia, West Virginia Florida, Puerto Rico Nationwide Nationwide

-956 gal Unknown Unknown

23 24

Michigan, Ohio, Pennsylvania, West Virginia Georgia, North Carolina Ohio

5 1,780 bars

18

Unknown >1083 gal

32,35 31

Connecticut, New York, Massachusetts Connecticut Connecticut, New York, Massachusetts

5.6-8.4 gal

37

30 gal 1700 pies

36 33

10187

Ohio

Chocolate ice cream Ice cream, sherbet, ice milk Ice cream

2/88 7/88

Georgia Ohio

8/88

Connecticut

Ice cream Ice cream pies

8/88 9/88

Connecticut Connecticut

14

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376

TABLE 5 Continued Type of dairy product Ice Ice Ice Ice Ice

cream cream bars cream cream bars cream bars

Monthlyear of recall

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

State of manufacture Pennsylvania New York Connecticut Wisconsin New Mexico

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

6/90

Ohio

8/90 5/91

Ohio Tennessee

Ice cream, ice milk

619 1

Illinois

Butter Butter Butter, butterine

619 1 619 1 3/92

North Carolina, Tennessee North Carolina, Tennessee Wisconsin

10192 6/93

Wisconsin Ohio

Chocolate milk Whipping cream Butter

7/94 8/94 8/94

Wisconsin Wisconsin California

Butter Ice cream bars

9/94 9/94

Wisconsin Wisconsin

Ice milk, ice cream Ice cream bars

Distribution Pennsylvania New York Connecticut Wisconsin Alabama, Illinois, Pennsylvania, Texas Indiana, Kentucky, Michigan, Ohio Indiana, Kentucky, Ohio Alabama, Georgia, Kentucky, Tennessee, Virginia, West Virginia Illinois, Indiana, Wisconsin Pennsylvania Pennsylvania Arizona, California, Florida, Illinois, Maryland, Massachusetts, Michigan, Minnesota, Mississippi, Missouri, New Jersey, Ohio, Wisconsin Wisconsin Ohio, Kentucky, New England Michigan, Wisconsin Michigan, Wisconsin Maine, Michigan, New Hampshire, Wisconsin Pennsylvania, Wisconsin Michigan, Wisconsin

Quantity

Ref.

215 gal 128 gal Unknown -365 gal 1000 gal

-

38 41 42 44 46

Unknown

48

-778 gal Unknown

47 45

>2275 gal

51

-18,165 lb -18,428 lb Unknown

50 50 52

-1 125 gal 34,752 bars

53 54

-

Unknown Unknown 36 Ib

168 168 55

Unknown 167 gal

168 56

L. monocytogenes in Unfermented Dairy Products Ice cream novelties

10195

Ohio

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

10195

Ohio

Ice cream

12/95

California

Ice cream Frozen yogurt

12/95 12/95

Michigan Ohio

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

1/96 7/97 7/97 10197

Ohio Michigan Pennsylvania Texas

Ice cream sandwiches Ice cream

1/98 3/98

California North Carolina

377 Illinois, Indiana, Iowa, Kentucky, Maryland, Michigan, Ohio, North Carolina, Pennsylvania Georgia, Indiana, Kentucky Michigan, North Carolina, Ohio, Pennsylvania, South Carolina, Tennessee, Virginia Arizona, Hawaii, California, New Mexico, Nevada Michigan Connecticut, Delaware, Florida, Maine, Maryland, Massachusetts, Michigan, New Hampshire, New Jersey, New York, Ohio, Pennsylvania, Rhode Island, Vermont, Virginia Ohio Michigan Pennsylvania Arkansas, California, Florida, Georgia, Illinois, Kansas, Maryland, New York, Ohio, Oregon, Texas, Washington State California Delaware, Florida, Georgia, Kentucky, Maryland, North Carolina, Pennsylvania, South Carolina, Tennessee, Virginia, West Virginia

Large but unknown

57

-420,000 gal

60

>500 gal

61

Unknown -12,381 gal

61 58

2,864 gal Unknown -42 gal 29,814 cases

59 205 206 207

Unknown 130,000 gal

208 209

378

Ryser

Despite millions of gallons of frozen dairy products that have been recalled both formally and internally, it must be stressed that only one case of listeriosis has been positively linked to consumption of a contaminated frozen dairy product in Belgium [4] (see Chapter 10). On this basis, the International Ice Cream Association and the Milk Industry Foundation have contended that a Class I recall may be too harsh a response for a frozen dairy product containing presumably very low levels of L. monocytogenes. However, until the oral infectious dose and relative risk for susceptible individuals can be firmly established, the FDA is likely to maintain its zero tolerance policy for L. monocytogenes and continue requesting recalls of products containing this pathogen at any detectable level. The U.S. government has developed one of the most stringent policies regarding presence of L. monocytogenes in ready-to-eat foods, whereas most other countries have adopted more relaxed policies (i.e. not >100 CFU/g or ml), particularly where consumption of contaminated products that have not yet been firmly linked to cases of listeriosis is concerned. As an example of the latter attitude, the Canadian government has decided to confine all formal recalls to only those foods that have been linked to major outbreaks of listeriosis, namely, coleslaw, soft cheese, and pasteurized milk, with the role of pasteurized milk in foodborne listeriosis still being highly debated [ 1331. Hence, no recalls were issued when researchers at the Health Protection Branch of Health and Welfare Canada (analogous to the U.S. FDA) identified L. monocytogenes in 1 of 394 (0.25%) and 1 of 51 (2.0%) samples of ice cream and ice cream novelties, respectively [96], during their own federal inspection program. Although subsequent investigations were presumably conducted to identify (a) the source of contamination, (b) proper corrective measures, and (c) possible links to human illness, Canadian officials maintained that recalling the two contaminated lots would be inappropriate without proof that consumption of Listeriacontaminated ice cream can lead to listeriosis. Many individuals and most manufacturers will undoubtedly argue in favor of the more relaxed Canadian position. When one considers the numerous recalls of Listeria-contaminated ice cream in the United States, that worldwide only one case of listeriosis has been positively linked to ice cream containing unusually high numbers of listeriae, the inability of L. monocytogenes to grow in this product during frozen storage and the normal exposure rate of the human population to listeriae, it appears that the risk of contracting listeriosis from contaminated ice cream is extremely low. Although current scientific data mandate the immediate removal of fluid dairy products and cheeses that support growth of L. monocytogenes, it appears that a scientifically valid argument can be made against recalling certain dairy products in which listeriae will not proliferate such as ice cream and dried goods which, if contaminated, typically contain very low numbers of listeriae as postpasteurization contaminants. As a result of several large recalls of French Brie cheese and a listeriosis outbreak in Switzerland that was traced to consumption of Vacherin Mont d’Or soft-ripened cheese, European scientists have logically focused their attention on the incidence of listeriae in cheese. However, numerous recalls of unfermented dairy products in the United States also have heightened public health concerns about the presence of listeriae in pasteurized dairy products manufactured outside of North America. In one of the first European surveys of finished products reported in 1988, researchers in Germany [ 1971 failed to isolate Listeria spp. from pasteurized milk (39 samples), nonfat dry milk (1 1 samples), caseidcaseinate (30 samples) and various dried products, including baby food (Table 6). During the same year, investigators in Hungary [ 1001 and The Netherlands [69] also failed to recover L. monocytogenes from samples of pasteurized milk, with similar negative findings being obtained from most other subsequent surveys

L. monocytogenes in Unfermented Dairy Products

379

of properly pasteurized milk and cream produced in Europe, Australia, the Middle East, and North Africa (Table 6). However, L. monocytogenes was eventually demonstrated in 11 of 1039 (1.1%), 4 of 115 (3.5%), and 1 of 95 (1.1%) pasteurized milk samples examined in the United Kingdom [ 119,126,1821 for a combined contarnination rate of 1.3%, with these findings generally being similar to those observed in the United States. According to Garayzabal et al. [113], 21.4, 89.2, 10.7, and 3.6% of pasteurized milk samples from one particular milk processing facility in Madrid contained I,. monocytogenes, L. grayi, L. innocua, and L. welshimeri, respectively. These same authors [ 114,1761 previously reported similar Listeria contamination rates for raw milk entering the same processing facility. Furthermore, after pasteurization these same samples had a total mesophilic aerobic plate count of 2.5 X 107CFU/mL, which is well above the maximum allowable limit of 1 X 104CFU/mL for properly pasteurized milk in the United States. Hence, improper pasteurization caused by leaking pasteurizer plates, as suggested by Northolt et al. [ 1561, and/or postpasteurization contamination from the factory environment appear to be most likely responsible for the unusually high incidence of listeriae in “pasteurized” milk samples from this particular dairy factory. Although results from these aforementioned surveys of pasteurized milk, cream, and dried products are very encouraging, the isolation methods used in these studies were generally unable to detect sublethally injured listeriae. Hence, the true incidence of listeriae in pasteurized milk, cream, and dried products may well be somewhat higher. To enhance recovery of injured cells, the International Dairy Federation has recommended that such dairy products undergo preenrichment in a nonselective medium (i.e., buffered peptone water) before primary enrichment in various selective broths and plating on Listeria-selective media [37,198]. Further details concerning recovery of sublethally injured listeriae can be found in Chapter 7. Results from a 1989 International Dairy Federation survey [ 1331 indicated that public health issues regarding the presence of listeriae in pasteurized milk were clearly spreading beyond the continental boundaries of Europe and North America, with the many aforementioned surveys from Table 6 attesting to these concerns. More recently, the safety of several additional dairy products, including flavored milks, chocolate milk, ice cream, and butter has attracted international attention with the FDA Initiatives Program, the many Class I recalls of Listeria-contaminated dairy products, and fears of international trade embargoes fueling these concerns. Following the 1987 discovery of L. monocytogenes in Australian ricotta cheese, New Zealand and Australian officials instituted Listeria-monitoring programs for caseidcaseinate products as well as high-moisture cheese, pasteurized milk, ice cream, and milk powders. Results from one 10-month survey begun in April 1988 [202] revealed the presence of L. monocytogenes in 1 of 206 (0.48%) samples of pasteurized flavored/unflavored milk processed in and around Melbourne. Subsequent identification of heat-labile alkaline phosphatase in the contaminated product (pasteurized milk to which a pasteurized flavored syrup was added) suggested that improper pasteurization was most likely responsible for the presence of L. monocytogenes in the final product. However, unsatisfactory storage of the flavored syrup also may have contributed to contamination. In keeping with Listeria policies developed in the United States and Canada, Australian officials withdrew the affected product from the marketplace and prohibited the sale of all subsequently produced product until 12 consecutive lots of Listeria-free pasteurized flavored milk could be produced from the same product line. As in the United States, recent foreign surveys also have shown a higher incidence of L. monocytogenes in chocolate milk (1 1.6%), ice cream (2.0-13.9%), and butter (3.86.7%) as compared with pasteurized milk and dried products which are seldom contami-

Ryser

380 TABLE 6 Incidence of Listeria spp. in Pasteurized Dairy Products Produced Outside the United States and Canada Country of origin

Product Milk

Australia Brazil Czechoslovakia Germany Hungary Italy Morocco Netherlands Poland Turkey United Arab Emirate

Number of samples

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

Number of positive samples (%)

L. monocytogenes

L. innocua

L. welshimeri

Other

Ref.

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

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

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

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

125 65 155 73 151 152 197 131 100 117 199 94 69 178 187 122

L. monocytogenes in Unfermented Dairy Products

Chocolate milk Flavored milk Ice cream

Cream

Butter Nonfat dry milk Caseidcaseinate Dry infant formula

United Kingdom England/Wales Scotland Northern Ireland Hungary Australia Australia Costa Rica England/Wales Turkey Australia England/Wales Hungary Morocco Hungary Italy Germany Germany Germany

ND, not determined. a Seven non-L. monocytogenes isolates. One non-L. monocytogenes.

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

381

11 (1.1) 4 (3.5) 1 (1.1) 7 (11.6) 1 (0.5) 23 (13.9) 1 (2.0) 0 5 (10.0) 0 0 0 0 1 (6.7) 5 (3.8)

0 0 0

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

ND ND ND 0

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

ND ND ND 0 ND ND ND ND 0 0 ND ND ND Ib ND 0 0 0

126 182 119 171 202 65 154 118 75 125 126 131 94 131 169 197 I97 197

382

Ryser

nated (Table 6). The increased incidence of listeriae in ice cream and butter is clearly the result of postpasteurization contamination during handling and packaging as evidenced by the highest contamination rates in ice cream bars and novelties. The fact that Listeria spp. are more commonly found in chocolate milk, as opposed to unflavored milk, is also not surprising given that the added ingredients can serve as an additional source for listeriae.

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

Raw Milk Despite longtime recognition of L. monocytogenes as a raw milk contaminant, relatively few studies assessing the behavior of this organism in raw milk can be found in the literature. In 1958, Dedie [82]found that L. monocytogenes survived 210 days in naturally contaminated raw milk stored in an ice chest. Thirteen years later, Dijkstra [84]reported results from a much longer storage study in which 36 samples of naturally contaminated raw milk (obtained from cows that experienced Listeria-related abortions) were held at 5°C and examined for viable L. monocytogenes over a period of 9 years. Although 4 of 36 (1 1%) samples were free of L. monocytogenes within 6 months, the pathogen was still detected in 16 of 36 (44%) samples following 2 years of refrigerated storage. The number of samples from which listeriae could be isolated continued to decrease, with 9 of 36 (25%) samples being positive after 4 years of storage. However, the pathogen was still present in 4 of 36 (1 1%) raw milk samples after 8-9 years of storage. These early findings emphasize the importance of establishing proper cleaning and sanitizing programs for all phases of milk production. If routinely used, such programs will likely prevent this organism from finding an appropriate niche within the farm or dairy factory environment and greatly reduce the threat of this pathogen surviving long term. The studies just described adequately demonstrate that L. monocytogenes can persist in raw milk for long periods; however, until several outbreaks of “milkborne” and cheeseborne listeriosis were reported in the 198Os, little attention had been given to the potential for growth of L. monocytogenes in raw milk. In 1988, Northolt et al. [ 1561 examined the behavior of listeriae in samples of freshly drawn raw milk that were inoculated to contain approximately 500 L. monocytogenes CFU/mL and incubated at 4 and 7°C. As shown in Figure 4, Listeria populations decreased approximately 4- and 8.5-fold in raw milk during the first 2 days of incubation at 4 and 7”C, respectively. These authors suggested that naturally occurring bacterial substances

L. monocytogenes in Unfermented Dairy Products

lo4

10

383

I

L

0

Raw Milk

L l L L

2

4

6

Days

FIGURE4 Growth of Listeria rnonocytogenes strains in raw milk incubated a t 4 and 7°C (enumerated on Trypaflavine Nalidixic Acid Serum Agar). (Adapted from Ref. 156.) in raw milk (i.e., lactoperoxidase and lysozyme) may have partially inhibited growth of listeriae during the first 2 days of incubation. However, in a Canadian study which will be discussed shortly [98], no such decrease was observed when incubated samples of naturally contaminated raw milk were surface plated on FDA Modified McBride Listeria Agar. Hence, a more likely explanation is that the plating medium Trypaflavine Nalidixic Acid Serum Agar used by Northolt et al. [ 1561 was less than ideal for recovering listeriae, as also was observed during concurrent work with pasteurized milk. Although L. monocytogenes failed to grow in raw milk samples incubated at 4°C for up to 7 days, Listeria populations increased approximately 10-fold during this period when the incubation temperature was raised to 7°C. Following 3 days of incubation at 4 and 7OC, Listeria populations began doubling every 3.5 and 1.0 day, respectively. Two years later, Wenzel and Marth [203] reported that populations of L. monocytogenes strain V7 remained constant in inoculated raw milk during 5 days of storage at 4 and 7"C, with numbers of listeriae also being unaffected by the presence of a commercial raw milk lactic acid bacteria inoculant designed to suppress the growth of primarily gram-negative psychrotrophic bacteria. Since L. monocytogenes failed to grow during 3-5 days of incubation at 7"C, it appears that the 3-day period during which raw milk is sometimes held in farm bulk tanks is insufficient to allow growth of the organism. However, the temperature of raw milk in farm bulk tanks will fluctuate every time freshly drawn raw milk at 37°C is commingled with bulk tank milk at -4°C from previous milkings. In 1985, Oz and Farnsworth [I591 found that raw milk in farm bulk tanks attained temperatures of 30-3 1OC, 10- 14"C, I2"C, and 9°C when freshly drawn raw milk was added after the first, second, third, and fourth milking periods, respectively. Moreover, 6 h were generally needed for the milk to cool to 4°C after each milking period. In view of these findings, it appears that temperatures obtained after adding warm milk to farm bulk tanks may be sufficient to allow at least limited growth of L. monocytogenes, particularly when raw milk from early millungs

384

Ryser

enters the bulk tank. Although the temperature of bulk tank milk will eventually decrease to -4"C, exposure to temperatures as high as 9°C when raw milk is trucked to processing facilities during summer [99] also may lead to some multiplication of the pathogen. Discovery of a naturally infected cow in Canada that shed freely suspended and phagocytized cells of L. monocytogenes in milk (maximum of 104CFU/mL in milk from one of four quarters of the mammary gland) continuously for nearly 3 years provided Farber et al. [98] with a unique opportunity to study growth of L. monocytogenes in naturally rather than artificially contaminated raw milk during extended storage. When raw milk from this cow was analyzed for numbers of L. monocytogenes,no appreciable growth of the pathogen was observed during the first 3 days and 1 day of incubation at 4 and IO'C, respectively (Fig. 5). The delay in onset of growth was less than 1 day at 15°C. Immunological staining of milk smears indicated that some multiplication of L. monocytogenes had occurred within macrophages after I and 2 days of incubation at 15 and 10°C, respectively, with 1 0 4 0 % of the macrophages containing 1-20 intracellular listeriae. Nonetheless, as previously noted by Doyle et al. [91], rapid deterioration of macrophages shortly thereafter was followed by appearance of freely suspended listeriae in milk with few intact macrophages remaining after 5 days regardless of incubation temperature. Following the lag phase, L. monocytogenes entered a period of logarithmic growth, with generation or doubling times of 25.3, 10.8, and 7.4 h being calculated for raw milk samples held at 4, 10, and 15OC, respectively. Although maximum L. monocytogenes populations were approximately 2 X 107CFU/mL after 10, 7, and 3 days of incubation at 4, 10, and 15"C, respectively, the highest achievable population in raw milk was independent of

7.0 -

m-

4°C

t- 10°C

.0

2

4

6

8

15OC

10

12

14

FIGURE5 Growth of L. monocytogenes in naturally contaminated raw milk during incubation at 4, 10, and 15°C. (Adapted from Ref. 98.)

L. monocytogenes in Unfermented Dairy Products

385

incubation temperature (Fig. 6). As in the previous study by Northolt et al. [156], these findings again stress the importance of maintaining raw milk at 1 4 ° C during storage and transport to milk processing facilities. Investigations dealing with behavior of listeriae in raw milk have not been limited to cow's milk. Reports of ovine listeriosis in Europe prompted Ikonomov and Todorov [132] to examine the behavior of L. monocytogenes in raw ewe's milk inoculated with the pathogen. Their results show that L. monocytogenes remained viable for long periods and persisted in the milk even after coagulation at 10 and 20°C. In 1987, a pregnant woman in the United States reportedly aborted after consuming feta cheese contaminated with L. monocytogenes. Since feta and other cheeses such as Roquefort, Manchego, Gjeost, and Chachcaval are traditionally manufactured from ewe's or goat's milk, interest in the behavior of listeriae in these milks as well as in ethnic-type cheeses manufactured from these milks has increased over the last several years.

Pasteurized and Intensively Pasteurized Milk In addition to defining the growth pattern of L. monocytogenes in artificially contaminated raw milk (Fig. 4), Northolt et al. [ 1561 also examined behavior of this organism in pasteurized (72"C/I 5 sec) and intensively pasteurized whole milk (Fig. 6). Although L. monocytogenes failed to grow in raw milk incubated at 4°C (Fig. 4), Listeria populations in pasteurized milk increased nearly 10-fold during 7 days of incubation at the same temperature.

I/ 4

"C

&ITS?'-Pasteurized Milk 0

2

Days

4

6

0

2

4

6

Days

FIGURE6 Growth of L. monocytogenes in high-temperature, short-time (HTST)-pasEnumerated teurized and intensively pasteurized milk incubated at 4 and 7°C. -: on Trypafiavine Nalidixic Acid Serum Agar,---: Enumerated on Nutrient Agar. (Adapted from Ref. 156.)

Ryser

386

i-

91-

8

-+- - - - - - - - -

7 6

-

a 5 -

----A

SkimMilk Whole Milk

Chocolate Milk

Days

FIGURE7 Growth of L. rnonocytogenes strain California in fluid dairy products at 4°C. (Adapted from Ref. 180.) The organism also grew markedly faster in pasteurized than in raw milk when both products were incubated at 7°C. In contrast to their data for raw and pasteurized milk, lag times for L. rnonocytogenes were reduced considerably when the organism was grown in intensively pasteurized milk incubated at 4 and 7°C. Furthermore, numbers of listeriae in intensively pasteurized milk increased approximately 100-fold following 3 and 6 days of incubation at 7 and 4OC, respectively. When L. rnonocytogenes was later grown in ultrahigh temperature (UHT) sterilized milk, Rajkowski et al. [ 1711 reported generation times of 4.7, 1.7, 1.0, and 0.9 h for samples incubated at 12, 19, 28, and 37"C, respectively. Hence, these findings suggest that the growth rate for L. rnonocytogenes in milk is directly related to the degree of heat applied to milk. Further work is needed to define more clearly the effect of competing microorganisms on growth of listeriae in raw and pasteurized milk as compared with intensively pasteurized and UHT-sterilized milk with biochemical changes that occur in milk during thermal processing (i.e., protein denaturation, enzyme inactivation, carmelization) also likely influencing listeriae growth in these products.

Autoclaved Milk, Cream, and Chocolate Milk Except for the two studies just described [98,156] and an initial attempt by Pine et al. [ 1661 to follow growth of L. rnonocytogenes in inoculated samples of pasteurized milk, all remaining work dealing with behavior of Listeria in fluid dairy products has been done using autoclaved samples. Although using such sterile products as growth media for listeriae offers several major advantages, including the ability to accurately quantitate both stressed and unstressed listeriae on nonselective plating media in the absence of other

L. monocytogenes in Unfermented Dairy Products

387

microbial competitors, readers should keep in mind that growth rates for L. monocytogenes are likely to be somewhat faster in autoclaved than in pasteurized or especially in raw milk products. Nevertheless, L. monocytogenes clearly can grow to dangerously high levels in all three types of milk during extended refrigeration. In 1987, Rosenow and Marth [180] published results from a definitive study in which autoclaved (12 1"Cl15 min) samples of whole, skim, and chocolate milk as well as whipping cream were each inoculated separately with four strains of L. monocytogenes (Scott A, V7, V37CE, or California), incubated at 4, 8, 13, 21, or 35"C, and examined for numbers of listeriae at suitable intervals by surface plating appropriate dilutions on Tryptose Agar. Growth rates of L. monocytogenes were generally similar in all four products at a given temperature and increased with an increase in incubation temperature. At 4"C, listeriae began growing after an initial delay of approximately 5- 10 days depending on the bacterial strain and type of product (see Fig. 7). All four strains generally attained maximum populations of 2 1O7 CFU/mL after 30-40 days of incubation, with little change in numbers occurring after 30-40 days of additional storage. Overall, chocolate milk supported development of the highest Listeria populations followed by skim milk, whole milk, and whipping cream. Generation times for growth at 4°C ranged between 28.16 and 45.55 h. Average generation times for L. monocytogenes in all four products are shown in Table 7. Although these results clearly demonstrate the ability of L. monocytogenes to reach potentially hazardous levels in fluid dairy products held at 4"C, more recent data suggest that slow growth of this organism can even occur in milk held at 0°C. Thus the only way to avoid a public health problem with fluid dairy products is to prevent L. monocytogenes from entering such products before, during, and after manufacture. Increasing the incubation temperature from 4 to 8°C decreased the lag period to 1.5-2 days (Fig. 8) and nearly tripled the growth rate for L. monocytogenes in all four products (Table 7) [ 180,1811. After 10- 14 days of incubation, the growth curves at 4 and 8OC were similar, with highest Listeria populations again being found in chocolate milk. Theoretical calculations based on these data indicate that Listeria populations could increase from 10 to 4.2 X 106organismdqt (947 mL) of milk during 10 days of storage at 8°C (46"F), a temperature that commonly occurs in some home and commercial refrigerators. These findings, which since have been confirmed by Siswanto and Richard [188] using skim milk, raise additional safety concerns about reclaiming and reprocessing returned products that have likely undergone some degree of temperature abuse. As is true for 8"C, 13°C (55°F) also represents a temperature that dairy products occasionally encounter during transportation and storage. Following a 12-h lag period, all

TABLE7

Generation Times for L. rnonocytogenes in Autoclaved Samples of Various Dairy Products Generation time (h) at Product

4°C

8°C

13°C

2 1"Cb

350Cb

Whole milk Skim milk Chocolate milk Whipping cream

33.27 34.52 33.46 36.30

13.06 12.49 10.56 11.93

5.82 6.03 5.16 5.56

1.86 1.92 1.72 1.80

0.692 0.693 0.678 0.683

Average generation times for four strains of L. monocytogenes. Strain V7 only. Source: Adapted from Ref. 180.

a

Ryser

388 9.

-

8.

-

7.

-

6.

-

5.

-

4.

-

3.

-

A-.

Chocolare milk

FIGURE8 Growth of L. monocytogenes strain California in fluid dairy products at 8°C. (Adapted from Ref. 180.)

four Listeria strains grew nearly twice as fast at 13°C as at 8°C (see Table 7) and generally attained levels of 2 106CFU/mL in all four products by the third day [ 1801. These generation times are somewhat longer than those observed by Farber et al. [98] when naturally contaminated raw milk was incubated at 4 (25.3 h), 10 (10.8 h), and 15°C (7.4 h). L. monocytogenes also attained maximum populations that were approximately 1 0-fold lower in raw than in sterile milk, which in turn suggests possible depletion of essential nutrients by raw milk contaminants or production of substances inhibitory to growth of the pathogen. Maximum Listeria populations of 1 O9 CFU/mL were again observed in chocolate milk, with numbers generally being 10-fold lower in skim milk, whole milk, and whipping cream [ 1801. Increasing the incubation temperature to 2 1"C doubled the growth rate (see Table 7) and led to maximum Listeria populations of I Ox- 10' CFU/mL within 48 hs. As expected, L. monocytogenes grew most rapidly at 35"C, with populations of 1Ox- 10' CFU/ mL being observed after only 24 h of incubation. In another study examining the influence of temperature and milk composition on growth of listeriae, Donnelly and Briggs 1861 found that five L. monocytogenes strains began growing in inoculated samples of autoclaved (121 "C/ 10 min) whole, skim, and reconstituted nonfat dry milk (1 1 % total solids) after approximately 24-48, 2-24, 4-12, and 0.5-4.0 h of incubation at 4, 10, 22, and 37OC, respectively. Although growth rates for all Listeria strains were primarily determined by the incubation temperature, two strains of L. monocytogenes serotype 4b grew considerably faster in whole rather than skim or

-

L. monocytogenes in Unfermented Dairy Products

389

reconstituted nonfat dry milk during incubation at 4 and 10°C. These observations led Donnelly and Briggs [86] to suggest a possible relationship between levels of milkfat and the growth rate of L. monocytogenes in milk during refrigerated storage. Furthermore, these authors suggested that enhanced psychrotrophic growth in whole milk may be related to a listerial lipase produced by both P-hemolytic strains of L. monocytogenes serotype 4b. Unlike both of these strains, the three remaining L. monocytogenes strains of serotypes 1 and 3 failed to exhibit enhanced growth in whole milk at 10°C and had little if any hemolytic activity on McBride Listeria Agar containing sheep blood. In contrast to what might be expected from the study just described, Rosenow and Marth [ 1801 failed to observe any significant difference in growth rates among four strains of L. monocytogenes (two serotype 4b, two serotype 1) when they were incubated in autoclaved samples of whole and skim milk at 4, 8, 13, 21, and 35°C. The pathogen also attained lower maximum populations in whipping cream than in whole, skim, or chocolate milk at all incubation temperatures. In support of these findings, Marshall and Schmidt [ 1451 failed to observe enhanced growth of L. monocytogenes strain Scott A (serotype 4b) in whole rather than skim milk during 8 days of incubation at 10°C. Finally, in a study to be discussed in greater detail in Chapter I2 [ 1851, four strains of L. monocytogenes (three serotype 4b and one serotype 1) frequently attained higher maximum populations in whey samples that were defatted by centrifugation, filter sterilized, and incubated at 6°C than would be expected to occur in autoclaved skim milk, whole milk, or whipping cream after prolonged incubation at 8°C. Thus, although some L. monocytogenes strains are lipolytic as reported by Marshall and Schmidt [ 1461, one must presently conclude that psychrotrophic growth of L. monocytogenes is not generally enhanced by the normal level of milk fat found in fluid milk. Recognizing the vital importance of carbohydrates in microbial metabolism, researchers at the CDC [ 1661 attempted to define growth of Listeria spp. in terms of sugar utilization. An initial experiment using aerobically incubated broth media indicated that five strains of L. monocytogenes and one strain each of L. Jnnocua, L. seeligeri, and L. ivanovii utilized only the glucose moiety of lactose, whereas single strains of L. grayi and L. murruyi utilized both the glucose and galactose of lactose. Overall, maximum cell populations, as determined by optical density, were directly proportional to the concentration of glucose ( S O . 125%) in the growth medium. However, marked differences were observed in the ability of L. monocytogenes and L. innocua to utilize lactose, with three strains of L. monocytogenes (isolated from Mexican-style cheese in connection with the 1985 listeriosis outbreak in California) being unable to grow in a medium containing lactose as the only carbohydrate. Although these observations agree with several reports [102,145,146] indicating that the pH of fluid milk is unaffected by L. monocytogenes growth, Quinto et al. [I701 did report a sharp pH decrease in such milk after 16 and 24 days of incubation at 14 and 7"C, respectively, with these differences most likely being related to strain variation. Growth of L. monocytogenes in autoclaved samples of whole and skim milk was generally similar to that previously observed by Rosenow and Marth [ 1801, with maximum populations of 5 5 X IO* CFU/mL developing after extended incubation at 5 and 25°C. Except for L. seeligeri, the behavior of L. innocua and L. ivanovii did not differ markedly from that of L. monocytogenes in these samples (Fig. 9). However, as noted by Northolt et al. [ 1561, higher maximum populations and increased survival rates were again observed when these organisms were grown in autoclaved rather than pasteurized whole milk. Examination of milk by gas-liquid chromatography indicated that lactic, acetic, isobutyric,

Ryser

390 L. rnonocvloaeneg

+

Lseeliaeri

m o

C

ivanovil

\ innocua

A

0

A

e o

9.0

8.0

7.0

6.0 0

4

8 12 16 20 24 Days

FIGURE9 Growth of Listeria spp. in pasteurized (open symbols) and autoclaved whole milk (solid symbols) incubated at 5°C. (Adapted from Ref. 166.) isovaleric, and 2-hydroxy isocaproic acids were formed during incubation. Since this milk initially contained -81 -85 mg of glucose/L, the aforementioned acids likely resulted, at least in part, from fermentation of glucose. Considerably lower populations of L. monocytogenes as well as L. innocua, L. grayi, and L. murrayi also developed in glucose oxidasetreated (an enzyme that degrades glucose) rather than untreated milk during both aerobic and anaerobic incubation, and so it is evident that glucose is one of the major substrates for growth of listeriae in milk. However, when incubated anaerobically in glucose oxidase-treated milk, two lactose-negative L. monocytogenes isolates from Mexican-style cheese still attained final populations of 10' CFU/mL; thus suggesting the involvement of other as yet unidentified growth factors. In the aforementioned study by Rosenow and Marth [ 1801, maximum populations of L. monocytogenes were typically about 10-fold higher in chocolate milk than in other fluid dairy products. To explain the enhanced growth .of L. monocytogenes in chocolate milk, several investigators at the University of Wisconsin examined the effect of major chocolate milk constituents (i.e., cocoa, sugar, and carrageenan) on growth of this organism in autoclaved skim milk and laboratory media. Rosenow and Marth [ 1791 found that

-

L. monocytogenes in Unfermented Dairy Products

391

growth of L. monocytogenes at 13°C was only slightly enhanced in skim milk containing 5% cane sugar, and that the organism attained higher final populations when commercial cocoa power (1.3%) and carrageenan stabilizer (0.5%) were used in place of cane sugar (Fig. 10). Carrageenan also enhanced the growth rate of L. monocytogenes in the presence of cocoa; however, the organism attained similar maximum populations regardless of the presence or absence of carrageenan. These findings suggest that carrageenan may be more important in increasing contact between cocoa particles and Listeria than as a source of nutrients. Highest final populations and shortest generation times were observed when L. monocytogerzes was grown in skim milk containing cocoa, sugar, and carrageenan. In addition, maximum Listeria populations obtained in skim milk containing all three ingredients (see Fig. 10) were similar to populations observed in initial work with commercially produced chocolate milk (see Figs. 7 and 8). Subsequently, Pearson and Marth [ I621 examined growth of L. monocytogenes strain V7 at 13°C in skim milk containing various concentrations of cocoa, sugar, and carrageenan. Since some Listeria strains can utilize sucrose, it is not surprising that L. monocytogenes developed significantly higher final populations (see Fig. 1 1 ) and had shorter generation times (5.05 vs 5.17 h) as the concentration of cane sugar (sucrose) in skim milk was increased from 0 to 12%. (Peters and Liewen [ 1651 also reported that addition of 7% sucrose to ultrafiltered (concentrated) skim milk caused rnaximum L. monocytogenes populations to increase rather than decrease.) A near-linear relationship between increasing sugar concentration and maximum attainable populations of L. monocytogenes also was observed for all but one combination of sugar, cocoa, and carrageenan tested; that is,

10

8

7

rl

8b

--

2%milk(m)

- ------

2%m + sugar (s)

-- -A

2%m + cocoa (c) + can.

- - - - - -+ 2 % m + c + s + c a r r .

m

r

2 O

n20

~ 40

'

60 l

80 '

l 100

'

120 '

140 '

1160

'

180 I

200 '

FIGURE10 Growth of L. rnonocytogenes strain V7 in 2% fat milk with added sugar, cocoa, and carrageenan (carr.) at 13°C. (Adapted from Ref. 179)

l

Ryser

392

8.90 8.80

8.70 8.60

8.50 8.40

0

3

6

9

12

Cane Sugar (O/., w/v) URE 11 Maximum L. monocytogenes population in skim milk alone (m), skim skim milk + cocoa (O), and skim milk cocoa + carrageenan milk carrageenan (A), (+) with 0,6.5, and 12.0% cane sugar after 36 h of incubation at 13°C. Any two points differing by 20.07 loglo CFU/mL are significantly different (P < .05). (Adapted from Ref. 162.)

FI

+

+

12% sugar and 0.03% carrageenan (see Fig. 11). Although addition of 0.03% carrageenan significantly lengthened generation times and decreased maximum populations compared with those observed in skim milk without carrageenan, L. monocytogenes achieved highest populations in skim milk containing 0.75% cocoa with or without carrageenan, which in turn indicates that the apparent ability of cocoa to stimulate growth of this organism in skim milk containing 0- 12% sugar is independent of carrageenan. Since cocoa contains only trace amounts of fermentable carbohydrates, these authors theorized that cocoa enhanced growth of L. monocytogenes in skim milk by providing increased levels of peptides and amino acids, particularly valine, leucine, and cysteine, which are reportedly essential for growth. Additional work showed that agitation, combined with the presence of cocoa, sugar, and/or carrageenan in skim milk, enhanced growth of the pathogen at 30°C when compared with growth in the same medium that was incubated quiescently. However, growth of Listeria in skim milk alone was better without rather than with agitation. Thus agitation most likely increased the availability of extractable nutrients from cocoa, which in turn led to enhanced growth of the pathogen. In 1968, anthocyanins in cocoa were reported to inhibit growth of salmonellae in laboratory media; however, the inhibitory effect of cocoa could be neutralized with casein [72]. These early findings prompted Pearson and Marth [164] to investigate the effect of cocoa with and without casein on growth of L. monocytogenes strain V7. Using Modified Tryptose Phosphate Broth containing 0.2% tryptose, addition of 0.75- 10% cocoa increased the generation time for L. monocytogenes at 30°C (1.02-1.12 h) as compared with samples without cocoa (0.94 h). However, the pathogen generally attained higher populations when grown in media with (1.1 - 1.5 X 109CFU/mL) rather than without (6.4 X 108CFU/mL) cocoa. Interestingly, when the same medium was inoculated to contain

L. monocytogenes in Unfermented Dairy Products

393

-

1 OS L. monocytogenes CFU/mL and agitated, the pathogen decreased to nondetectable levels in samples containing 5-10% cocoa after 15-24 h of incubation at 30°C. Nonetheless, the organism readily grew in the presence of 0.75% cocoa and attained higher maximum populations in media with ( I .9 X 109CFU/mL) rather than without (7.6 X 108CFU/ mL) cocoa during agitated incubation at 30°C. As previously reported for salmonellae, the presence of 1.5 or 3.0% casein neutralized the inhibitory effect of cocoa toward L. monocytogenes, with the pathogen exhibiting shorter lag phases and higher maximum populations in media containing both casein and 5.0% cocoa rather than cocoa alone and incubated quiescently at 30°C. However, results obtained during agitated incubation of cultures containing 5% cocoa were far more dramatic, with L. monocytogenes populations of 2.9 X 109rather than <10 CFU/mL developing in samples with rather than without 2.5% casein. Hence, these findings suggest that the behavior of L. monocytogenes in laboratory media containing cocoa partially depends on the concentration of one or more inhibitory substances than can be neutralized by casein and that are more readily extracted during agitated rather than quiescent incubation. Next, Pearson and Marth [I631 determined if theobromine and caffeine (i.e., two methylxanthine compounds in cocoa that reportedly possess different degrees of antimicrobial activity) were responsible for the previously observed antilisterial activity of cocoa. Overall, addition of 2.5% theobromine to both Modified Tryptose Phosphate Broth and autoclaved skim milk with and without 0.5% caffeine did not markedly influence the behavior of L. monocytogenes during incubation at 30°C. This suggests that theobromine is not responsible for suppressing or enhancing growth of listeriae in chocolate milk. Unlike theobromine, addition of 0.5% caffeine to Modified Tryptose Phosphate Broth doubled or tripled the length of the organism's lag phase, nearly doubled the organism's generation time, and led to maximum Listeria populations approximately I 0-fold lower than those obtained in caffeine-free media. Similar trends also were observed when autoclaved skim milk instead of Modified Tryptose Phosphate Broth served as the growth medium. Thus, although caffeine in cocoa may contribute to inhibition of L. monocytogenes in a broth medium, failure of casein in skim milk to neutralize the inhibitory effect of cocoa indicates that caffeine also is not responsible for inhibition of listeriae as observed in the previous study. Such efforts to identify Listeria-active components within cocoa should be continued to better understand the behavior of this pathogen in chocolate milk.

Sweetened Condensed and Evaporated Milk To our knowledge, Listeria spp. have not yet been isolated from commercially produced sweetened condensed milk (i.e., a nonsterile concentrated fluid milk product containing approximately 64% sucrose or glucose in the water phase, 8.5% milk fat, and 28% total milk solids) or evaporated milk (an unsweetened commercially sterile concentrated fluid milk product containing approximately 7.9% milk fat and 25.9% total milk solids). However, given the widespread incidence of Listeria in food processing facilities, it is conceivable that listeriae could enter both of these products as postprocessing contaminants. Such concerns prompted Farrag et al. [103] to examine the fate of three L. monocytogenes strains in samples of commercially produced sweetened condensed and evaporated milk that were inoculated to contain three different levels (- 1 03- 1O7 CFU/mL) of the pathogen. Regardless of initial inoculum, Listeria populations in sweetened condensed milk decreased 5 1.2 and 1.6-3.4 orders of magnitude following 42 days of storage at 7 and 2 1"C, respectively. This behavior was not surprising, since addition of sugar to this product

394

Ryser

during manufacture reduces its water activity (a,) to -0.83, which is well below the minimum a, value of 0.90 reported for growth of L. monocytogenes. Unlike sweetened condensed milk, the relatively high a, value for evaporated milk (-0.986) allowed profuse growth of listeriae, with lowest inoculum levels increasing approximately 4 orders of magnitude after 7 and 14 days of incubation at 21 and 7OC, respectively. In addition, no decrease in numbers of listeriae was noted during continued incubation at either temperature. Thus, since L. monocytogenes can survive >42 days in sweetened condensed milk and grow rapidly in evaporated milk, special precautions should be taken to prevent listeriae from entering these products during packaging, storage, and subsequent use.

Ultrafiltered Milk Ultrafiltration, a mechanical process by which milk is filtered under pressure and concentrated, results in major compositional changes in the finished product when compared with the starting material. During ultrafiltration, 94- 100% of the milk proteins and proteinbound vitamins (i.e., vitamin BI2and folic acid) remain in the retentate along with milk fat, whereas lactose is equally divided between the retentate and permeate. Increased use of ultrafiltered milk in cheesemaking prompted El-Gazzar et al. [92] to investigate the growth characteristics of L. monocytogenes in 2X and 4X retentate as well as the corresponding permeate obtained from ultrafiltered pasteurized milk. When samples were inoculated to contain about 104CFU/mL of L. monocytogenes strain V7 or CA and incubated at 4"C, growth of both organisms was enhanced 10- to 100-fold in retentate as compared with unfiltered skim milk. Increasing the concentration of ultrafiltered (UF) skim milk retentate from 2X to 5X also resulted in faster growth, with the pathogen attaining a population of 106CFU/mL in 5X and 2X retentate after approximately 7 and 10-12 days of refrigerated storage, respectively. L. monocytogenes also grew to dangerous levels in permeate with maximum levels of 10' CFU/mL as compared with 10' CFU/mL in retentate following 30 days of incubation. When identical tyndalized samples were incubated at 32 and 4OoC, both Listeria strains grew similarly in skim milk and retentate, with populations of 10' CFU/mL generally being reported after 24 h of incubation. However, as was true for samples incubated at 4OC, maximum numbers of listeriae were again 10to 100-fold lower in permeate than in retentate and unfiltered skim milk. Hence, the same care should be given to production of unfiltered milk to prevent contamination and subsequent growth of Listeria in the product during cold storage.

GROWTH OF L. MONOCYTOGENES IN MIXED CULTURES Except for several early works assessing the behavior of L. rnonocytogenes in raw and pasteurized milk, all studies described thus far have dealt with growth of L. monocytogenes in the absence of competitive microorganisms. Although results from these studies have been of great value to the dairy industry, one should remember that pasteurized dairy products are not sterile. Psychrotrophic bacteria, belonging to the genera Pseudomonas and Flavobacteriurn are typically present in raw milk and, like L. rnonocytogenes, can grow in milk at refrigeration temperatures both before and after milk is pasteurized. Although readily destroyed during pasteurization, these organisms universally appear in pasteurized dairy products as postpasteurization contaminants, often at levels > 100 CFU/mL. Since L. monocytogenes is thought to enter dairy products primarily after pasteurization, products that contain low levels of listeriae (probably
L. monocytogenes in Unfermented Dairy Products

395

higher populations of other psychrotrophs. Although the ability of psychrotrophic pseudomonads to stimulate growth of nonpathogenic as well as pathogenic bacteria in dairy products has been recognized for more than 25 years; data concerning the behavior of L. monocytogenes in mixed cultures is of far more recent origin. After initial work [I011 with Tryptose Broth demonstrated that growth of L. monocytogenes was slightly inhibited by the presence of Pseudomonas jluorescens (see Chapter 6), Farrag and Marth [66] examined associative growth of L. monocytogenes (strains Scott A, CA, and V7) with P. juorescens (strains P26 and B52) in autoclaved (1 2 I "C/ 15 min) skim milk that was inoculated to contain equal populations (- I O5 CFU/mL) of both organisms and incubated at 7 or 13°C for 56 days. Growth of L. monocytogenes was generally enhanced by the presence of P. jluorescens after 7 days of incubation, with the pathogen attaining populations of 1O7 CFU/mL in mixed cultures. However, continued incubation at 7°C led to lower numbers of listeriae in mixed rather than pure cultures, with populations of strain V7 being inhibited approximately eightfold by P. jluorescens B52 following 56 days of storage. Farrag and Marth [ 1051 later showed that inactivation of strain CA was affected by initial levels of P. jluorescens P26, with highest populations being most detrimental to Listeria survival. However, populations of L. monocytogenes strains Scott A and V7 remained unaltered in samples that were initially inoculated to contain P. jluorescens P26 at levels of 103-106CFU/mL. When Farrag and Marth [102] increased the incubation temperature to 13"C, growth of L. monocytogenes was neither enhanced nor inhibited by either Pseudomonas strain during the first 7 days of incubation. However, after 56 days of incubation, final populations of Listeria were as much as 20-fold lower in mixed rather than pure culture. Although these findings and those of Quinto et al. [ 1701 indicate that P. jluorescens was more detrimental to survival of listeriae in skim milk stored at 13 than 7"C, growth and survival of P. jluorescens was not appreciably affected by the presence of L. monocytogenes, with pseudomonads consistently reaching populations of 108--1O9 CFU/mL. In a similar study, Marshall and Schmidt [ 1451 found that growth of L. rnonocytogenes strain Scott A (used in the previous study) in autoclaved skim and whole milk was not enhanced by the presence of Pseudomonas fragi during 8 days of incubation at 10°C. Similarly, growth of P. fragi also was unaffected by L. monocytogenes. Some researchers have speculated that psychrotrophic pseudomonads may be able to utilize some of the nutrients in milk faster than L. monocytogenes; thus suppressing growth of listeriae in milk during refrigerated storage. Marshall and Schmidt [ 1451 investigated this theory by inoculating samples of autoclaved whole milk, skim milk, and reconstituted nonfat dry milk (10% solids) with P. fragi or P. jluorescens (strain T25, P26, or B52); incubating the samples for 3 days at 10°C to obtain --106-107 P. fragi CFU/mL or 104- 1O6 P. jluorescens CFU/mL; inoculating these Pseudomonas cultures with L. monocytogenes; and then incubating the samples for an additional 8 days at 10°C. Throughout this study, addition of listeriae to all milks preincubated with P. jluorescens or P. fragi did not significantly affect growth or survival of either pseudomonad. However, as shown in Figure 12, L. monocytogenes grew faster and attained higher final populations in samples of whole milk that were preincubated with either of the two pseudomonads than in whole milk that was not treated with pseudomonads. L. monocytogenes behaved similarly in both whole and skim milk, with average generation times of approximately 7 and 8 h in milks preincubated with P. jluorescens and P. fragi, respectively (Fig. 13). Although accelerated growth of Listeria was observed in reconstituted nonfat dry milk preincubated with either pseudomonad, generation times for listeriae in either of the two mixed cultures

-

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396

I L. nionocyrogenes

---

+-

L. niotiocylogenes +

P.fragi

------Q.---

L. nronocyrogenes + P . flicorescens

Days

FIGURE 12 Growth of Lisferia monocytogenes at 10°C in whole milk preincubated for 3 days with selected Pseudomonas spp. (Adapted from Ref. 145.) generally did not differ significantly. As was true for whole and skim milk, L. monocytogenes attained populations of 1 X 107to 5 X 107CFU/mL in reconstituted nonfat dry milk, with highest numbers occurring in milk preincubated with P. jluorescens rather than P. fragi. Flavobacterium is another genus of gram-negative psychrotrophic bacteria that is frequently recovered from raw milk, pasteurized milk, and butter. Hence, Farrag and Marth [104] also examined behavior of L. monocytogenes in the presence of flavobacteria in skim milk at 7 and 13°C. Growth of L. monocytogenes strains Scott A, CA, and V7 in autoclaved skim milk was enhanced by the presence of F. lutescens during 14-42 days of a 56-day incubation period at both 7 and 13"C, with these higher populations again being attributed to proteolysis of milk proteins by F. lutescens. However, Flavobacterium sp. ATCC 21429 failed to impact the growth of L. monocytogenes at 7°C and proved to be slightly inhibitory to the same three Listeria strains when samples were held at 13°C. One strain of Bacillus spp. [ 1431 also prevented Listeria growth in raw milk. These results dispel the previous theory and indicate that L. monocytogenes can readily compete with P. fragi, P. jluorescens, and certain Flavobacterium spp. for nutrients in milk and at the same time can outgrow these organisms at refrigeration temperatures

L. monocytogenes in Unfermented Dairy Products 17 16 15 14

13

12 11 10

6

397

-

-

L. monocytogenes

-

L. monocytogenes P . fluorescens

L. monocytogenes P.fragi

-

-

Whole

Skim

Nonfat Milk Solids

Product

FIGURE13 Generation times of Listeria monocytogenes at 10°C in various milks preincubated for 3 days with P. fragi or P. fluorescens spp. (Adapted from Ref. 145.) even if the ratio of L. monocytogenes to pseudomonads or flavobacteria is on the order of 1 : 100,000. Enhanced growth of microorganisms, including L. monocytogenes, in the presence of these psychrotrophs is now known to be related to increased levels of nutrients that occur in milk as a result of proteolytic enzymes produced by these organisms [104,146]. Since many of these enzymes are heat-stable and able to survive pasteurization, raw milk must be handled properly and pasteurized within a reasonable time (i.e., 3-4 days) to prevent conditions that may favor growth of listeriae. As previously noted [156], enhanced growth of listeriae in intensively pasteurized as compared to HTST-pasteurized and raw milk also might be related to this phenomenon.

NONFLUID DAIRY PRODUCTS Although the aforementioned studies demonstrate the ability of L. monocytogenes to grow to potentially hazardous levels in fluid dairy products held at refrigeration temperatures, concern about the behavior of this organism in dairy products extends well beyond fluid milks and cream. As you will recall from Table 5 , nearly 50 recalls have been issued in the United States for Listeria-contaminated ice cream. These recalls, along with FDA reports suggesting that about 3.5% of the ice cream and 8.5% of the ice cream novelties produced in the United States may be contaminated with presumably low levels of L. monocytogenes, have prompted research on the fate of listeriae in frozen dairy products. Additionally, behavior of Listeria during manufacture and storage of nonfat dry milk and butter also has been investigated in the event that these products are inadvertently prepared from skim milk and cream, respectively, that have been contaminated after pasteurization.

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398

Ice Cream Frequently, pasteurized milk that has not been sold in retail stores is returned to dairy factories and reprocessed into chocolate ice cream. Since large commercial refrigeration units often fail to maintain a constant temperature of 4"C, virtually all reclaimed milk has undergone some degree of temperature abuse during the period in which the product was on sale. In addition to possible growth of L. monocytogenes during this 2-week period of "cold enrichment,'' pseudomonads also can grow in milk and produce an environment that is more favorable for growth of Listeria even after pasteurization. The numerous Class I recalls issued since the late 1980s for Listeria-contaminated ice cream prompted Berang et al. [70] to investigate the behavior of L. monocytogenes in inoculated samples of chocolate ice cream (and chocolate milk as discussed earlier) prepared from fresh skim milk and commercial skim milk that was held beyond the expiration date. Although growth of listeriae was certainly not expected in ice cream held at - 18 to -24"C, the pathogen survived equally well in both types of chocolate ice cream. Hence, use of returned milk in chocolate ice cream did not appear to enhance Listeria survival. Long-term survival of L. monocytogenes was also confirmed in a later study [160] in which the pathogen persisted for 14 weeks in ice cream stored at - 18°C with no apparent cell death or injury. In 1996, Dean and Zottola [81] assessed the fate of L. monocytogenes V7 in full-fat ( 10%) and reduced-fat (3%) soft-serve ice cream prepared with and without 14 ppm nisin. Regardless of fat content, L. monocytogenes populations remained constant in ice cream during freezing and 3 months of storage at - 18°C. However, nisin effectively reduced Listeria survival in both full- and reduced-fat ice cream during manufacture, with Listeria populations generally decreasing 2 and 3 orders of magnitude in full- and low-fat ice cream, respectively, following 1 month of frozen storage at - 18°C. Although not currently approved in the United States as an ice cream ingredient, incorporation of nisin into ice cream formulations appears to be an effective, albeit costly, means of inactivating listeriae and reducing the number of Class I Listeria-related recalls that continue to plague the dairy industry. In 1989, Amelang and Doores [2,3] determined the generation times for L. monocytogenes in nine formulations of commercially produced ice cream mix that varied in type and level of fat (cream, butter), sugar (cane sugar, corn sweetener), and milk solids (condensed milk, skim milk, whey powder). To simulate postprocessing contamination, all samples were inoculated to contain 103L. monocytogenes strain Scott A or V7 CFU/ mL and incubated at 4, 2 1, and 35°C. Overall, L. monocytogenes had average generation times of 21.6, 1.08, and 0.79 h in ice cream mixes incubated at 4, 21, and 35"C, respectively, with similar growth rates occurring in mixes containing 10, 14, and 15% fat and held at the same temperature. It is noteworthy that these generation times are markedly shorter than those calculated by Rosenow and Marth [ 1801 for growth of the same strains in whole milk, skim milk, chocolate milk, and whipping cream (see Table 5). Although L. monocytogenes generally behaved similarly in all ice cream mixes incubated at 4 and 2 1"C, differences in generation times were noted at 35°C when the pathogen was cultured in ice cream mixes made with alternative fat and milk solids. At 35"C, growth of listeriae was somewhat enhanced in mixes containing butter rather than cream, skim milk powder, or whey powder rather than condensed skim milk and egg yolk as additional sources of solids. Although the pathogen grew most rapidly in ice cream mix containing a 50 :50 ratio of cream to butter, partial replacement of cane sugar (sucrose: glucose + fructose)

-

L. monocytogenes in Unfermented Dairy Products

399

with corn sweetener (glucose and maltose) or high fructose corn syrup failed to significantly shorten generation times.

Butter In 1988, Olsen et al. [158] examined the fate of L. monocytogenes during manufacture and storage of butter in the event that the product is prepared from contaminated cream. According to their report, pasteurized cream was inoculated to contain 104- 10' L. monocytogenes CFU/g and churned into butter. After removing the buttermilk, washed butter grains were salted to a level of 1.2% and resultant butter was analyzed weekly for listeriae during 10 weeks of storage at - 18, 4-6, and 13°C. During manufacture -95% of the L. monocytogenes population was lost in buttermilk, with the remaining 5% of the population appearing in butter. The pathogen was present at levels of 1.7 X 104to 1.8 X 10' CFU/ g in cream as compared with 1.5 X 103to 1.6 X 104CFU/g in butter, indicating that like Staphylococ-cus aureus [ 1531, L. monocytogenes also favors the water rather than lipid phase during butter making. As shown in Figure 14, Listeria populations increased 1.9 and 2.7 orders of magnitude in butter stored at 4-6 and 13OC, with maximum numbers being observed after 49 and 42 days of storage, respectively. These findings along with similar results by Lanciotti et al. [ 1371 for commercially prepared light butter stored at 4 and 20°C indicate that enough milk solids were trapped in the water phase (containing -6% salt) to support growth of listeriae during storage. Numbers of listeriae then began to decrease; however, the organism was still present at levels >104 CFU/g following 70 days of refrigerated storage. Although freezing the contaminated butter prevented growth

6.00

1 I-/

t

2.001' 0

/ \ /

'

20

4 to 6 O C

*

A

'

40 Days

'

.

a

'

60

"

'

J

80

FIGURE14 Survival of L. monocytogenes in butter manufactured f r o m artificially contaminated cream and stored at 12, 4-6, and -18°C. Each line represents the average of 4 trials. (Adapted f r o m Ref. 158.)

400

Ryser

of L. monocytogenes, the organism was still present at levels of -103 CFU/g after 70 days of storage at -18"C, as was also reported by Slavchev et al. [191]. Thus far L. monocytogenes has not been isolated from pasteurized cream manufactured in the United States; however, given the massive Listeria recall of Texas-produced fluid dairy products, including half-and-half and whipping cream, in May of 1986 (see Table 5 ) , one cannot assume that all pasteurized cream and butter manufactured in the United States and elsewhere will be universally free of listeriae. As you will recall from Chapter 10, one cluster of listeriosis cases in southern California was attributed to consumption of contaminated butter [ 1471. Hence, since at least four Class I recalls have been issued for L. monocytogenes-contaminated butter, and since growth of L. monocytogenes has been demonstrated experimentally in both cream and butter during refrigerated storage, it is necessary to ensure that cream is pasteurized and that recontamination of pasteurized cream is prevented before and during its churning into butter.

Nonfat Dry Milk Dried dairy products, including nonfat dry milk, whey, and casein, also may become contaminated with pathogenic microorganisms both before and after drying. Such concerns have been raised recently in Australia and New Zealand [133]. Although all dry dairy products examined thus far have been Listeria-free, methods used to detect listeriae in these surveys were generally unable to recover cells that may have been injured during the drying process. Two factors, namely, the unusual thermal resistance of L. monocytogenes and the report of a milkborne listeriosis outbreak in Massachusetts during 1983, prompted Doyle et al. [90] to examine behavior of L. monocytogenes during manufacture and storage of nonfat dry milk. Samples of concentrated (30% solids) and unconcentrated (10% solids) skim milk were inoculated to contain 105- 106L. monocytogenes (strain Scott A or V7) CFU/mL and dried to moisture contents of 3.6-6.4% in a gas-fired pilot plant-sized spray dryer with inlet and outlet air temperatures of 165 t 2 and 67 t 2"C, respectively. All samples of nonfat dry milk were stored at 25°C for up to 16 weeks and periodically analyzed for listeriae using both direct plating on McBride Listeria Agar (detects uninjured cells) and cold enrichment in Tryptose Broth (detects injured and uninjured cells). Listeria populations decreased approximately 1.O- 1.5 orders of magnitude during spray drying regardless of whether or not nonfat dry milk was prepared from concentrated or unconcentrated skim milk. Strain V7 was generally hardier than strain Scott A during both spray drying and storage of nonfat dry milk. Twelve to 16 weeks of storage at room temperature were required to decrease populations of strain V7 > 1000-fold in nonfat dry milk, whereas only 6 weeks of storage were necessary to obtain similar decreases in numbers of strain Scott A. Overall, strains Scott A and V7 survived a maximum of 8 and 12 weeks in nonfat dry milk, respectively. Although strain Scott A generally survived equally well in nonfat dry milk prepared from concentrated and unconcentrated skim milk, strain V7 survived 2 weeks longer in nonfat dry milk manufactured from concentrated rather than unconcentrated skim milk. The higher moisture content of nonfat dry milk (i.e., 5.7 and 6.4%) prepared from concentrated skim milk may have enhanced survival of listeriae in this product during extended storage. Overall, populations of L. monocytogenes decreased > 10,000-fold in nonfat dry milk during 16 weeks of storage at room temperature. Hence, if commercially produced nonfat dry milk is ever found to contain L. monocytogenes,

-

L. monocytogenes in Unfermented Dairy Products

401

presumably at very low levels, it may be possible to eliminate this pathogen by holding the product at room temperature for several months.

Abou-Donia, S.A., and A.K. Al-Medhagi. 1992. Detection and survival of Listeria monocytogenes in Egyptian dairy products. J. Dairy Sci. 75 (suppl. 1): 138. 1a. Abou-Eleinin, A.M., E.T. Ryser, and C.W. Donnelly. 1998. Unpublished data. 2. Amelang, J., and S. Doores. 1989. The effect of ingredients in ice cream formulations on the growth of Listeria monocytogenes. Annual Meeting of the Institute of Food Technologists, Chicago, June 25-29, Abstr. 468. 3. Amelang, J., and S. Doores. 1989. The effect of medium, growth phase and temperature on the growth of Listeria monocytogenes in ice cream mix. Annual Meeting of the Institute for Food Technologists, Chicago, June 25-29, Abstr. 469. 4. Andre, P., H. Roose, R. Van Noyen, L. Dejaegher, I. Vyttendaele, and K. De Schrijver. 1990. Neuro-meningeal listeriosis associated with consumption of an ice cream. Med. Mal. Infect. 20:570-572. 5. Anonymous. 1986. Ice cream bars recalled. FDA Enforcement Report, July 16. 6. Anonymous. 1986. Ice cream recalled. FDA Enforcement Report, Oct. 22. 7. Anonymous. 1986. Ice cream recalled. FDA Enforcement Report, Oct. 29. 8. Anonymous. 1986. Ice cream, sherbet and glacee recalled. FDA Enforcement Report, Sept. 3. 9. Anonymous. 1986. Ice milk mix recalled. FDA Enforcement Report, June 25. 10. Anonymous. 1986. Large class I recall made of ice cream because of Listeria. Food Chem. News 28(24):11-12. 11. Anonymous. 1986. Listeria causes class I recalls of ice milk mix, milk. Food Chem. News 28( 16):22. 12. Anonymous. 1986. Milk, chocolate milk, half and half, cultured buttermilk, whipping cream, ice milk, ice milk mix and ice milk shake mix recalled. FDA Enforcement Report, June 25. 13. Anonymous. 1986. Sherbets, non-dairy products, ice milk products, gelati-da products and ice cream recalled. FDA Enforcement Report, Aug. 27. 14. Anonymous. 1987. Chocolate ice cream recalled. FDA Enforcement Report, Sept. 16. 15. Anonymous. 1987. Class I recall made of cheese because of Listeria. Food Chem. News 28(50):52. 16. Anonymous. 1987. FDA launching two-year pathogen surveillance program. Food Chem. News 29(31):10-12. 17. Anonymous. 1987. Ice cream and ice milk recalled. FDA Enforcement Report, Aug. 26. 18. Anonymous. 1987. Ice cream bars recalled. FDA Enforcement Report, Nov. 4. 19. Anonymous. 1987. Ice cream, ice milk and sherbet recalled. FDA Enforcement Report, Feb. 11. 20. Anonymous. 1987. Ice cream, ice milk and sherbet recalled. FDA Enforcement Report, Aug. 19. 21. Anonymous. 1987. Ice cream nuggets recalled. FDA Enforcement Report, Aug. 5. 22. Anonymous. 1987. Ice cream products recalled. FDA Enforcement Report, Sept. 2. 23. Anonymous. 1987. Ice cream products recalled. FDA Enforcement Report, Sept. 16. 24. Anonymous. 1987. Ice cream products recalled. FDA Enforcement Report, Sept. 23. 25. Anonymous. 1987. Ice cream recalled. FDA Enforcement Report, Jan. 28. 26. Anonymous. 1987. Ice cream recalled. FDA Enforcement Report, Feb. 11. 27. Anonymous. 1987. Ice cream recalled. FDA Enforcement Report, May 27. 28. Anonymous. 1987. Ice cream recalled because of Listeria, pottery because of lead. Food Cheni. News 29(21):16-17. 1.

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Anonymous. 1987. Milk industry has spent $66 million on recalls and related expenses, Witte says. Food Chem. News 29( 17):29-30. Anonymous. 1987. More ice cream recalled because of Listeria. Food Chem. News 28(48): 33. Anonymous. 1988. Frozen dessert products recalled. FDA Enforcement Report, July 27. Anonymous. 1988. Ice cream, cheese recalled because of Listeria. Food Chem. News 30(6): 27. Anonymous. 1988. Ice cream pies recalled. FDA Enforcement Report, Dec. 28. Anonymous. 1988. Ice cream products, cheese recalled because of Listeria. Food Chem. News 30(9):47. Anonymous. 1988. Ice cream recalled. FDA Enforcement Report, April 6. Anonymous. 1988. Ice cream recalled. FDA Enforcement Report, Sept. 7. Anonymous. 1988. Ice cream recalled. FDA Enforcement Report, Sept. 14. Anonymous. 1988. Ice cream recalled. FDA Enforcement Report, Nov. 2 . Anonymous. 1988. International Dairy Federation: Group E64-Detection of Listeria rnonocytogenes-sampling plans for Listeria rnonocytogenes in foods, Feb. 9. Brussels. Anonymous. 1988. More cheese, ice cream linked to possible Listeria. Food Chem. News 29( 11):37-38. Anonymous. 1989. Ice cream bars recalled. FDA Enforcement Report, Feb. 15. Anonymous. 1989. Ice cream recalled. FDA Enforcement Report, April 19. Anonymous. 1989. Le contr6le des rbsidus dans les produits laitiers. Bull. 1nf.-Minist. Agric., France I273:22-24. Anonymous. 1990. Frozen yogurt recalled. FDA Enforcement Report, Feb. 7. Anonymous. 1990. Ice cream and frozen yogurt novelties recalled. FDA Enforcement Report, July 10. Anonymous. 1990. Ice cream bars recalled. FDA Enforcement Report, April 25. Anonymous. 1990. Ice cream recalled. FDA Enforcement Report, Nov. 7. Anonymous. 1990. Sherbet, ice milk and ice cream recalled. FDA Enforcement Report, Dec. 5. Anonymous. 1990. USDA, FDA officials report apparent decrease in Listeria isolations. Food Chem. News 32( 1): 12- 15. Anonymous. 1991. Butter recalled. FDA Enforcement Report, Aug. 7. Anonymous. 1991. Ice cream and ice milk recalled. FDA Enforcement Report, June 19. Anonymous. 1992. Butter and butterine recalled. FDA Enforcement Report, July 22. Anonymous. 1992. Ice milk and ice cream recalled. FDA Enforcement Report, Dec. 30. Anonymous. 1993. Ice cream bars recalled. FDA Enforcement Report, Sept. 29. Anonymous. 1994. Butter products recalled. FDA Enforcement Report, Oct. 12. Anonymous. 1994. Ice cream recalled. FDA Enforcement Report., Oct. 5. Anonymous. 1995. Ice cream novelties recalled. FDA Enforcement Report, Dec. 13. Anonymous. 1996. Frozen yogurt recalled. FDA Enforcement Report, Mar.6. Anonymous. 1996. Ice cream and sherbet recalled. FDA Enforcement Report, April 10. Anonymous. 1996. Ice cream, frozen yogurt, sherbet, sorbet and ice cream mix recalled. FDA Enforcement Report, Jan. 3 1. Anonymous. 1996. Ice cream recalled. FDA Enforcement Report, Jan. 3 I . Archer, D.L. 1988. Review of the latest FDA information on the presence of Listeria in foods. WHO Working Group on Foodborne Listeriosis, Geneva, Feb. 15-19. Arias, L., R. Monge, F. Antillon, and E. Glenn. 1994. Occurrence of the bacteria Listeria spp. in raw milk in Costa Rica. Rev. Biol. Trop. 42:711-713. 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. 60:8 1 1-8 16.

30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64.

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77. 78. 79. 80. 81. 82. 83. 84.

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Arnold, G.J., and J. Coble. 1995. Incidence of Listeria species in foods in NSW. Food Australia 47:7 1-75. Bachman, H.P., and U. Spahr. 1995. The fate of potentially pathogenic bacteria in Swiss hard cheese and semihard cheeses made from raw milk. J. Dairy Sci. 78:476-483. Bean, N.H., J.S. Goulding, C. Lao, and J.F. Angulo. 1996. Surveillance of foodborne disease outbreaks-United States, 1988- 1992. M.M.W.R. 45: 1-66. 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. Intern. J. Food Microbiol. 4:249-256. Beckers, H.J., P.H. in’t Veld, P.S.S. Soentoro, and E.H.M. Delfgou-van Asch. 1988. The occurrence of Listeria in food. Foodborne Listeriosis-Proceedings of a Symposium, Wiesbaden, Germany, Sept. 7, pp. 84-97. Berrang, M.E., J.F. Frank, and R.E. Brackett. 1988. Behavior of Listeria monocytogenes in chocolate milk and ice cream mix made from post-expiration date skim milk. J. Food Prot. 5 1 :823 (Abstr.). Brindani, F., and E. Freschi. 1988/1989. Ricerca di Listerirz monocytogenes nel latte di ovicaprini ed in alcuni tipi di formaggio. Annal. Fac. Med. Vet. 8-9:205-219. Busta, F.F., and M.L. Speck. 1968. Antimicrobial effect of cocoa on salmonellae. Appl. Microbiol. 16:424-425. Casarotti V.T., R.G. Claudio, and R. Camargo. 1994. Occurrence of Listeria monocytogenes in raw milk, pasteurized C type milk and minas frescal cheese commercialized in PiracicabaS.P. Arch. Latinoamer. Nutr. 44: 158-163. Cheng, C.C., S.B. Shiau, and H.S. Lin. 1993. Incidence and characterization of Listeria monocytogenes in raw milk and feeds. Taiwan J. Vet. Med. Anim. Husb. 615965. Ciftcioglu, G., M.T. Ulgen, and K. Bostan. 1992. An investigation on the presence of Listeria monocytogenes in ice cream. J. Fac. Vet. Istanbul 18:1-8. Colonna, V., A.M. DiNoto, E.M. Russo Alesi, C. Emanuele, and S. Caracappa. 1994. Observations on the presence of Listeriu monocytogenes in milk products of ovine origin. In Progressi scientifici e technolgici in tema di patologia e di allevamento degli ovini e dei caprini. Societa Italiana di Patologia e di Allevamento degli Ovini e dei Caprini. Atti XI Congress0 Nazionale. Perugia, Italy, June 1-4, p. 439-442. Conner, D.E., V.N. Scott, S.S. Sumner, and D.T. Bernard. 1989. Pathogenicity of foodborne, environmental and clinical isolates of Listeria monocytogenes in mice. J. Food Sci. 54: 15531556. Coskun, S., 0. Onal, M. Keskin, T. Okyay, A. Yuce, and B. Erel. 1993. Investigation of Listeria in raw milk and comparison of culture and ELIS.4 methods. Turkish J. Infect. 7: 329- 332. Da Cruz, I.M.V., M.I. Fernandes, and M.M. Sol. 1990. Incidence of Listeria monocytogenes in Portuguese raw goat’s milk. In: Posters and Brief Communications of the XXIII International Dairy Congress, Montreal, Oct. 8-12. Abst. 77. Davidson, R.J., D.W. Sprung, C.E. Park, and M.K. Rayman. 1989. Occurrence of Listeria monocytogenes, Campylobacter spp., and Yersinia enterocolitica in Manitoba raw milk. Can. Inst. Food Sci. Technol. J. 22:70-74. Dean, J.P., and E.A. Zottola. 1996. Use of nisin in ice cream and effect on the survival of Listeria monocytogenes. J. Food Prot. 59:476-480. Dedie, K. 1958. Weitere experimentelle Untersuchungsbefunde zur Listeriose bei Tieren. In R. Roots and D. Strauch (eds.), Listeriosen, Zbl. Veterinarmed. Beiheft, pp. 99-109. D’Errico, M.M., P. Villari, G.M. Grasso, F. Romano, and I.F. Angelillo. 1990. Isolamento di Listeria spp. da latte e formaggi. Riv. Soc. Ital. Sci. Aliment. 19:47-52. Dijkstra, R.G. 1971. Investigations on the survival times 0 1 Listeria bacteria in suspensions of brain tissue, silage and faeces and in milk. Zbl. Bakteriol. I Abt. Orig. 216:92-95.

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12 Incidence and Behavior of Listeria monocytogenes in Cheese and Other Fermented Dairy Products ELLIOTT. RYSER Michigan State University, East Lansing, Michigan

INTRODUCTION On June 14, 1985, L. monocytogenes emerged from relative obscurity to the front page of many American newspapers because of a large listeriosis outbreak in California that was directly linked to consumption of Mexican-style cheese manufactured in metropolitan Los Angeles. By the time this outbreak subsided in August 1985, as many as 300 cases of listeriosis were reported, including 85 deaths-at least 40 of which were traced to the tainted cheese. In response to this foodborne outbreak of listeriosis, U.S. Food and Drug Administration (FDA) officials added L. monocytogenes to their list of pathogenic organisms that should be of concern to cheesemakers and began surveying various soft domestic cheeses for listeriae. Approximately 6 months later, isolation of L. monocytogenes from several imported Brie cheeses purchased at a supermarket led to the eventual recall of approximately 300,000 tons of Brie cheese imported from France and to a real concern about the incidence of this pathogen in other European cheeses. Recall of this cheese prompted two corrective measures: (a) adoption of a cheese certification program by the United States and France to prevent importation of Listeria-contaminated cheese and (b) initiation of numerous large-scale surveys to determine the extent of Listeria contamination in virtually all types of cheese manufactured in the United States, Canada, and Western Europe. 411

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Throughout 1986 and most of 1987, the impact of Listeria on European cheesemakers was primarily in the form of economic losses from destruction of contaminated product. However, L. rnonocytogenes struck again late in 1987 with the report of a large listeriosis outbreak in Switzerland (see Chap. 10) in which Vacherin Mont d'Or soft-ripened cheese was incriminated as the vehicle of infection. Most recently, tainted Brie cheese prepared from raw milk was responsible for a major listeriosis outbreak in France. These cheeseborne listeriosis outbreaks have prompted worldwide efforts to determine the incidence of Listeria spp. in various cheeses and examine the behavior of L. rnonocytogenes during manufacture and storage of numerous fermented dairy products. The first portion of this chapter summarizes Listeria-related recalls of cheese in the United States and results from surveys dealing with the incidence of listeriae in domestic and imported fermented dairy products. The second half of this chapter addresses the fate of L. rnonocytogenes during manufacture and storage of buttermilk, yogurt, and various cheeses (including whey) and the potential for cheese ingredients, such as rennet, salt brine, and coloring agents, to serve as vehicles of contamination during cheesemaking.

U.S. SURVEILLANCE PROGRAMS AND RECALLS FOR L. MONOCYTOG€N€S IN DOMESTIC AND IMPORTED CHEESE Domestic Cheese The concept of listeriosis as a foodborne illness is not new. As you will recall from Chapter 10, consumption of contaminated raw milk was believed to have caused several cases of listeriosis in post-World War I1 Germany. In 1961, Seeliger [274] also suggested sour milk, cream, and cottage cheese as possible vehicles of infection in this outbreak. Although results from two Yugoslavian studies concerned with behavior of L. rnonocytogenes in various fermented dairy products (i.e., cultured cream, unsalted skim milk cheese, Kachkaval cheese, and yogurt) were published in 1964 [195] and 1981 [281], no surveys dealing with the incidence of listeriae in fermented dairy products were made before contaminated Mexican-style cheese was linked to the California listeriosis outbreak in June of 1985. Public health concerns about presence of L. rnonocytogenes in domestic and imported fermented dairy products as well as other foods, such as meat, poultry, seafood, fruits, and vegetables, can be traced either directly or indirectly to the 1985 listeriosis outbreak in California. Less than 1 month after the first nationwide Class I Listeria-associated recall was issued for 22 varieties (-500,000 lbs.) of Mexican-style cheese contaminated with L. monocytogenes (Table l), the FDA developed a series of programs designed to prevent the recurrence of such an outbreak [280] (Fig. 1). The Domestic Soft Cheese Surveillance Program-the first of the dairy factory initiative programs-was instituted by the FDA in July of 1985 and involved on-site inspection of firms manufacturing soft cheese [ 141. Priority was given to manufacturers of Mexican-style soft cheese, followed by firms producing other ethnic-type soft cheeses, such as Edam, Gouda, Liederkranz, Limburger, Monterey Jack, Muenster, and Port du Salut, made from raw, heat-treated (<71.7"C [16l0F]/15 s) or pasteurized (171.7"C [161"F]/15 s) milk. In addition to determining the firm's compliance with good manufacturing practices (i.e., use of proper pasteurization, cleaning, and sanitizing procedures), FDA inspectors collected and analyzed cheese samples for L. rnonocytogenes using the original FDA pro-

L. monocytogenes in Fermented Dairy Products

4 13

cedure. Cheese samples also were tested for the presence of enteropathogenic strains of E. coli and for phosphatase activity, which if present generally indicates improper pasteurization of cheesemilk and/or subsequent contamination with raw milk. However, suitability of the phosphatase test for cheese has since been questioned. Less than 2 months into this program, FDA officials isolated a pathogenic strain of L. monocytogenes from one sample of domestically produced Liederkranz cheese (see Table 1). The manufacturer subsequently recalled the product nationwide. Following preliminary FDA reports of further Listeria contamination, this recall was extended to include all lots of Brie and Camembert cheese manufactured at the same facility [8,12]. However, final laboratory reports indicated that both Brie and Camembert cheese were contaminated with L. inrzocua, which is nonpathogenic, rather than L. monocytogenes. Although the Domestic Soft Cheese Surveillance Program also was responsible for temporarily closing two soft cheese factories in California that produced phosphatase-positive cheese [9], it must be stressed that L. monocytogenes was never isolated from cheeses produced at either facility. In general, FDA inspections of other soft cheese factories uncovered problems similar to those encountered during inspections of Grade A fluid milk factories: (a) potential bypasses of the pasteurizer, (b) postpasteurization blending of product, and (c) a general lack of education and/or training of plant personnel [223]. Items of particular concern to cheesemakers and that were not generally found during visits to Grade A milk factories included defects in the pasteurization process, discrepancies in pasteurization/production records, and a higher incidence (than in Grade A milk factories) of pathogenic microorganisms (including L. monocytogenes) on environmental surfaces in production and storage areas. Inspections of domestic cheese factories continued throughout 1986, 1987, and 1988 under four separate programs (see Fig. l), with FDA officials reaching nearly half of the 400 soft cheese factories in the United States by April of 1986 and the remaining factories (including follow-up inspections of problem factories) by late 1987 [46]. According to FDA records [loll, L. monocytogenes was confirmed in 12 of 658 (1.82%) domestic cheese samples analyzed during 1986. During these inspection programs, six Class I recalls were issued for various ethnic-type soft and semisoft cheeses containing L. monocytogenes. In response to (a) a 1987 report of a woman who developed listeriosis in San Bernadino, California, after consuming illegally produced Mexican-style cheese and (b) the widespread availability of uninspected, unbranded Mexican-style cheese illegally produced from raw milk in metropolitan Los Angeles [71a], Genigeorgis et al. [175], in conjunction with the California Department of Food and Agriculture, U.S. Department of Agriculture’s Food Safety and Inspection Service (USDA-FSIS), the Immigration and Naturalization Service, and the Los Angeles District Attorney’s Office, surveyed 100 California-produced soft Hispanic-style cheeses that were either seized or purchased undercover between June and November of 1988, Overall, two samples each were positive for L. monocytogenes and L. innocua. These four Listeria-contaminated cheeses had a pH of 6.2-6.5 and were presumably prepared from raw milk as evidenced by a positive alkaline phosphatase test. Given the ability of L. monocytogenes to grow in such cheeses during refrigerated storage and marketing, Hispanic-style cheeses continue to constitute a significant public health threat, with these varieties accounting for 7 of 21 recalls issued through 1996, including one large recall in June 1990 involving approximately 500,000 Ib of product. As previously mentioned, although all products containing L. monocytogenes must be retrieved from the marketplace, formal Class I recalls do not have to be issued for

4 14

Ryser

TABLE 1 Chronological List of Class I Recalls in the United States for Domestic Cheese Contaminated with L. monocytogenes Date recall initiated

Type of cheese

Origin

Distribution

Quantity (lb)

Ref.

Arizona, Arkansas, California, Colorado, Georgia, Guam, Hawaii, Idaho, Illinois, Kansas, Louisiana, Marshal1 Islands, Massachusetts, Nevada, New Jersey, New Mexico, New York, Oklahoma, Oregon, Rhode Island, Samoa, Texas, Utah, Washington state Nationwide, Puerto Rico Arizona, California, Oregon, Texas

-500,000

-10,000 127,607

8, 9, 12 20, 38

Virginia Illinois Kentucky Wisconsin California

Virginia, Washington, DC Illinois, North Carolina, Ohio, Pennsylvania Nationwide California, Washington state Arizona, California, Florida, Texas, Washington state

10,850 1150 -13,800 -1400 Unknown

42, 54 52 51 53, 280 64

1 1/6/90

California

500,000

78

2/1/91 2/14/91

Florida Wisconsin

-1362 >89.000

81 80

Ricotta Jack Cold-pack cheese food

711 1/91 10/28/91 3110192

New York Wisconsin Wisconsin

1109 12,500 Unknown

82 79 83

Queso fresco Limburger

10/14/92 1211 8/92

Washington Wisconsin

Arizona, California, Idaho, Nevada, Oregon, Washington state Southeastern United States Connecticut, Georgia, Illinois, Michigan, New York, Ohio, Pennsylvania, Texas, West Virginia, Wisconsin Florida, New York Iowa, Minnesota, Wisconsin Arizona, California, Colorado, Florida, Georgia, Illinois, Indiana, Maryland, Michigan, Minnesota, New York, Ohio, Pennsylvania, Tennessee, Texas, Vermont, Virginia, Wisconsin Oregon, Washington state Wisconsin

Unknown 1500

85 86

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

6/13/85

California

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

8/ 14/85 3/5/86

Ohio California

911 1/86 4117/87 5/6/87 8/21187 1/29/88

Cheese spread Mozzarella

+

10, 11

L. monocytogenes in Fermented Dairy Products

4 15

Cheese spread

3/4/93

Tennessee

Cream cheese

10119/93

Wisconsin

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

4/15/94 511 1/94 5120194 512 I 194 5/23/94 5/24/94 6/15/94

Wisconsin Massachusetts Texas Texas Texas Wisconsin California

Torte loaf cheese

8111194

Missouri

Swiss cold-pack cheese food Swiss Gorgonzola

811 1 194 I0/28/94 2/2/96

Wisconsin Ohio Wisconsin

Cream cheese with vegetables

10/30/97

Massachusetts

Cream cheese

1 1114197

Massachusetts

Queso fresco Queso fresco

2/4/98 3/23/98

Wisconsin Domestic

Blue cheese Blue cheese salad dressing

411 1/98 511198

Wisconsin Louisiana

a

Later found to contain only L. innocua.

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

11,789

84

3075

88

1429 20 Unknown Unknown Unknown 1220 -5,682

94 89 92 91 91 93 90

301

96

510 2270 4500

95 97 98

7,340

lOOa

Unknown

lOOb

248,938 Unknown

IOOe, lOOf lOOg

Unknown Unknown

lOOc lOOd

California listeriosis outbreak:

DOMESTIC Domestic soft cheese surveillance program - begun July 1985

r

1

2

L. monocytogenes accidentally isolated

d J./

Aged and ripened cheese survey begun January 1986 r

l*

Survey of cheese manufactured

-

IMPORTED

' \

cheese testing program

I-

L. monocytogenes isolated from Italian

1

Cheese under general pathogen surveillance program 1988

- January 1986

-

Continuation of s o f t cheese survey March 1987

1

from French Erie cheese

Romano cheese

- June

1987

I

-

begun April 1986

1

Italian cheese surveillance program begun July 1987 I

I

Survey of import cheese in domestic begun December 1987 status

I

-

FIGURE1 Surveillance programs for Listeria spp. in domestic and imported cheese. (Adapted from Ref. 101.)

-

I

-I

I L

L. monocytogenes in Fermented Dairy Products

417

contaminated products that have not yet reached retail stores. Since such situations typically lead to nonpublished ''internal recalls" issued by the manufacturer, far more cheese was likely destroyed during this 11-year period than has actually been reported. Several such informal recalls involved a part-skim milk cheese manufactured in California [57] as well as ricotta, Parmesan, and mozzarella cheese of uncertain origin [233]. Following a report by Ryser and Marth [259] that L. rnonocytogenes can survive more than 1 year in Cheddar cheese (i.e., well beyond the mandatory 60-day aging period for Cheddar cheese manufactured from raw milk), the FDA modified its Domestic Cheese Program in August of 1987 to include cheese prepared from unpasteurized milk [46]. Between April and October of 1987, 181 samples of domestic aged (held a minimum of 60 days at 1:1.7"C [35"F]) natural cheese manufactured from raw milk, as well as similar imported cheeses in domestic status, were collected from retail stores by FDA field personnel and analyzed for L. rnonocytogenes (Table 2). These efforts uncovered one positive sample-a sharp Cheddar cheese manufactured in Wisconsin, which was subsequently recalled from the market in July of 1987 (see Table 1). Late in 1987, the FDA announced plans for a 2-year pathogen surveillance program [43] which was designed to examine domestic and imported cheese as well as other highrisk foods (i.e., milk, vegetables, and seafood) for the presence of L. rnonocytogenes and other selected pathogens, including Vibrio cholerae, V. parahaernolyticus, Escherichia coli, enteropathogenic E. coli, Staphylococcus aureus, Salmonella spp., Yersinia enterocolitica, Clarnpylobacterjejuni, and C. coli. Under this program, samples of soft-ripened and raw milk cheese as well as imported hard and artificial blended cheese were examined for all of the aforementioned organisms except Vibrio spp. Domestic cheeses were collected at the wholesale level, whereas samples of imported cheese were obtained from retail stores. Although this program prompted only one Listeria -related recall of domestic cheese during 1989 and 1990, five separate recalls of Anari and Halloumi cheese imported

TABLE 2 Incidence of L. monocytogenes in

"Domestic" Cheese Manufactured from Raw Milk-FDA 1987a

Type of cheese Blue Brick Cheddar Colby Edam Goat Gouda Monterey Jack Swiss Other Total

Number of samples analy zed

(%>

18 5 71 8 4 6 1 9 42 17

0 0 l b (1.4) 0 0 0 0 0 0 0

181

1 (0.55)

~

Includes imported cheese in domestic status. 2 samples with L. innocua. Source: Adapted from Ref. 101. a

Number of positive samples

4 18

Ryser

from Cyprus were reported during this same 2-year period along with one additional recall of Italian soft-ripened/semisoft cheese. However, since additional cheese-related recalls after 1990 have been limited to three imported cheeses, the present FDA inspection program in combination with increased vigilance on the part of cheesemakers appears to be highly effective in limiting consumer exposure to both domestic and imported Listeriacontaminated cheese. Several other fermented dairy products also were examined for L. monocytogenes in conjunction with the FDA Dairy Initiative Program [ l o l l (see Chap. 11). In 1986, 10 samples of cottage cheese were found to be free of listeriae. Other than cheese and cheese food, 1% fat cultured buttermilk [33,35] and frozen yogurt [7 1 ] are the only other domestically produced, fermented dairy products known to have been contaminated with L. monocytogenes. The first of these products was included in a 1986 Class I recall involving approximately 1 million gallons of dairy products (fluid milk, chocolate milk, half-andhalf, whipping cream, ice milk, ice milk mix, ice milk shake mix, ice cream, and ice cream mix), all of which presumably contained L. monocytogenes. Three years later, officials from the Wisconsin Department of Agriculture, Trade and Consumer Protection issued a statewide recall for one particular brand of frozen yogurt after routine testing revealed the presence of L. monocytogenes in one sample of mandarin orange frozen yogurt [73]. Both of these products were retrieved from the marketplace without incident.

Imported Cheese France International concern over the potential health hazard of consuming Listeria-contaminated cheese also is rooted in the California listeriosis outbreak of 1985. This outbreak and an earlier link between consumption of French Brie and/or Camembert cheese and several outbreaks of foodborne illness in the United States and Europe caused by enterotoxigenic and/or enteropathogenic E. coli [2 17,234,2961prompted a meeting in September of 1985 between FDA officials and representatives of the French Embassy/French Delegation on Food Safety and Food Distribution to discuss the FDA’s plans for inspecting imported soft cheese [ 131. Late in September, representatives from the Codex Committee on Food Hygiene and the International Dairy Federation agreed with FDA officials that a “Code of Hygienic Practices” should be developed for manufacturing fresh and soft cheese [7]. Use of raw milk (a known source of L. monocytogenes) to improve organoleptic properties of certain cheeses was cited as a particular area of concern. Although a basic certification program for soft cheese produced in France was in operation for some time, agreement on a general Code of Hygienic Practices for manufacture of soft cheese was not reached during the remainder of 1985. In January of 1986, as part of a research effort to enhance recovery of listeriae from cheese, FDA officials inadvertently isolated a pathogenic strain of L. monocytogenes from two uninoculated control samples of French Brie cheese purchased at a local supermarket [ 19,1011. Ironically, both cheeses were prepared from pasteurized milk in a cheese factory certified by the French government under the existing soft-ripened cheese agreement. In response to these findings, the first in a series of six nationwide recalls was issued in February of 1986 for Listeria-contaminated French Brie cheese (Table 3). The following week, the French firm that manufactured the tainted cheese agreed to cease all production [19]. Shipment of additional cheese that was previously certified as “Listeria free” by an

L. monocytogenes in Fermented Dairy Products

4 19

independent French laboratory (certification by the French Ministry of Agriculture began January 1, 1986) was also stopped pending identification of the contamination source. In addition, all suspect lots of French Brie cheese en route to the United States were detained on entry and tested for Listeria by the FDA before being released. Shortly thereafter, sampling was extended to include virtually all lots of French Brie cheese produced by this manufacturer. The French cheese industry was dealt its most serious blow in March of 1986, when approximately 660 million pounds of Brie cheese produced by five different manufacturers were recalled in the United States (Table 3). This recall, which involved nearly 60% of all Brie cheese marketed in the United States, immediately raised the possibility of blocklisting French firms that produced contaminated cheese [2 I ]. Consequently, FDA officials immediately began testing all shipments of French soft-ripened cheese as well as 20% of French soft/semisoft cheese and 20% of all other imported soft cheeses for Listeria, E. coli, and phosphatase (see Fig. I ) [24]. Recognizing the danger of contracting listeriosis from consuming contaminated French soft-ripened cheese, FDA officials drafted the following proposal to detain, test, and certify all French soft-ripened cheeses exported to the LJnited States [72]: FDA intends to detain any entry that is found to be Listeria-positive, regardless of the species of ListcJriafound. French soft-ripened cheese without a certificate indicating negative results for the Listeria analysis or with a positive analysis for Listeria .will be detained. . . . In addition, all French soft-ripened cheeses are to be sampled and analyzed for the presence of Listeria. [Under the previous agreement, cheeses were shipped with a certificate of analysis which only indicated the level of E. coli and the absence of phosphatase.] The analysis may be carried out by a private laboratory after the cheese has arrived in the United States, or alternatively the analysis may be conducted so that the availability of the results will coincide with the arrival of the shipment in the United States. The timing of the analysis is important to ensure that the nature of the cheese tested, particularly the pH, is identical to that examined by the FDA, if we decide to perform an audit on the entry. Thus, importers of French softripened cheese should provide FDA with certificates indicating the dates testing was initiated and completed. Cheese shipments will not be released without a certificate indicating negative results for Listeria analysis. . . .

In this proposal, FDA officials stressed that other methods used to detect Listeria in cheese should conform to the 7-day FDA method described in Chapter 7 and also suggested that 1 / 16-in-thick slices from the cheese surface (sample with highest pH) be analyzed for listeriae rather than cross-sectional plugs of cheese. Following FDA threats to halt importation of soft-ripened cheese, the French Ministry of Agriculture agreed to begin lot-by-lot testing in April of 1986, as outlined in the March FDA proposal [25]. However, French authorities stressed that such a program would not be practical on a long-term basis and hoped that the FDA would accept an expansion of’the existing factory/product certification program to include Listeria testing in the near future. Beginning in May 1986, FDA officials announced that all shipments of French soft-ripened cheese lacking certification of analysis for Listeria would be detained [23]. Inspections during the next 2 months uncovered L. rnonncytogenes in two French cheeses-a noncertified Brie and a 6-lb certified lot of Muenster [23]-both of which were presumably recalled internally. Continued problems with Listeriu-contaminated French soft-ripened cheeses prompted FDA officials to revise the imported cheese surveillance program in August of 1986 [IS]. These changes allowed immediate detention of French cheeses that were: (a)

R yser

420

TABLE 3 Chronological List of Class I Recalls in the United States for Imported Cheese Contaminated with L. rnonocytogenes Type of cheese

Date recall initiated

Country of manufacture

Brie Brie Brie

2112/86 2114/86 2/14/86

France France France

Brie Brie Brie Brie Brie Brie

2/14/86 2/14/86 212 1186 2/24/86 3/14/86 411/86

France France France France France France

Soft-ripened: Tourre de 1’Aubier and Fromage des Burons Soft-ripened: Tourre de 1’Aubier

6/23/86

France

8/13/86

France

Semisoft: Morbier Rippoz Soft-unripened, full fat Semisoft

8118/86 4/16/87 1/27/88

France France Italy

Distribution

Quantity

Ref.

Bermuda, Nationwide Georgia, New Jersey Colorado, Connecticut, Florida, Louisiana, Maryland, Massachusetts, New Jersey, New York, Ohio, Washington, DC Florida, New York, Washington, DC Nationwide Oregon, Washington state Illinois, Minnesota, New Jersey Nationwide Colorado, Connecticut, Georgia, New Jersey, New York, North Carolina, Texas, Washington, DC New York, Ohio, Pennsylvania

57,000 2-6-lb wheels 40 cases 100 cases

17, 19, 26, 29 16 16

California, Illinois, Maine, Massachusetts, New Jersey, New York, Oregon Illinois, Massachusetts, Michigan New Jersey, New York, Texas Nationwide

10 cases Unknown Unknown 909 cases -660 million lb -230 Ib Unknown

1056 Ib -1600 lb 15 wheels 410cartons

16 16, 37 15, 37 17, 39 36, 40 27, 36

28,31 32 28, 30, 34 47, 48 59

421

L. monocytogenes in Fermented Dairy Products Semiseft: L’P,mu!ette Dmish Esrem Semisoft: L’Amulette Danish Esrom Semisoft: L’Amulette Danish Esrom Blue

Anari Anari Anari Halloumi Italian soft ripened and semisoft Fontina

Limburger jarisberg

2/ 11/88 21 12/88 21 18/88

EefiF-Ek Denmark Denmark

4/6/88

Denmark

5/26/89 7/27/89 8/9/89 9/15/89 7/27/90

Cyprus Cyprus Cyprus Cyprus Italy

4/6/93

Sweden

2/29/96

Germany

6i7i96

Norway

CdifGrni2 East, Midwest, North, South Florida, New Jersey, New York, Massachusetts California, Florida, Illinois, Massachusetts, Michigan, Minnesota, New Jersey, New York, North Carolina, Oregon, Pennsylvania, Texas New York Illinois, Texas New York Florida, New Jersey, New York California, Connecticut, New Jersey, New York, Pennsylvania California, Connecticut, Maryland, Massachusetts, Minnesota, New Hampshire, New York, North Carolina, Pennsylvania, Rhode Island, Washington state Florida, Indiana, Maine, Massachusetts, New Jersey, New York, Ohio, Utah, Virginia Alaska, Cdifoniia, Guam, Hawaii, Idaho, Montana, Nevada, Oregon, Utah, Washington state

Unknown -11,500 lb -1,150 lb

56, 57, 60 56, 57, 61 56-58, 62

-5,000 Ib

55, 56

50 cases 79 cases 80 cases 14,400 Ib Unknown

68 69 70 75 76

85,080 lb

87

813 lb 30,727 lb

100 99

R yser

422

manufactured at a non-government-certified factory, (b) unaccompanied by a Listeriafree government certificate, (c) positive for phosphatase, or (d) manufactured by one of several firms that were block-listed for Listeria. Although these changes made importation of French cheeses more difficult, sampling of cheeses that were manufactured at certified factories and accompanied by Listeria-free certificates was decreased to the 20% level. Between June and August of 1986, four additional Class I recalls were issued for French semisoft/soft-ripened cheese contaminated with L. monocytogenes (see Table 3). Two of three firms involved in these recalls were previously block-listed by the FDA [ 181. An additional Class I recall issued for semisoft Morbier Rippoz cheese (see Table 3) was accompanied by the following press release [34]: Although Listeria is a rare cause of human illness, it can be life-threatening to pregnant women and their fetuses, frail elderly persons or other persons with weakened immune systems. In healthy adults, it is a transient illness with such mild-to-moderate flu-like symptoms as fever, headaches, and/or gastrointestinal tract distress.

The language used in such press releases also has received considerable attention. These messages to the public must be firm enough to accomplish the goals of the recall but not so alarming as to create an undue panic. After considerable consultation, the governments of France and the United States reached agreement on a French certification program for soft cheese [49,101]. Under this program, which began February 15, 1987, cheeses were tested before shipping using methods that were mutually acceptable by both governments. French cheeses manufactured at certified factories would be sampled at the 5% level, whereas other French cheeses (and cheeses manufactured in other countries without certification programs) would be analyzed at the 20% level. In the event of a Listeria-positive shipment, personnel at the French cheese factory would be required to investigate the potential source of contamination and analyze every lot of cheese for listeriae in at least the next 20 consecutive shipments destined for the United States. Although a positive finding would not automatically result in suspension of the certified status for a cheese factory under this program, FDA officials reserved the right to initiate detentions and/or recalls if a product was found to contain L. monocytogenes. After this certification program was accepted, only one additional recall involving a French soft-ripened full-fat cheese has been reported (see Table 3).

Other Western European Countries Despite the adverse publicity that the French cheese industry received throughout 1986 and 1987, it must be recognized that the problem of Listeria-contaminated cheese was not limited to France. Between October and December of 1986, FDA inspectors isolated Listeria spp. from 4 of 74 (5.4%) cheeses imported from Italy, two cheeses of which also contained high levels of phosphatase [45]. After finding similar percentages of positive samples during January, February, and March of 1987, FDA officials told representatives of the Italian government either to submit a draft for a certification program (or recommend an alternate solution) or face a ban on importation of potentially hazardous cheeses into the United States. As of April 30, 1987, only 13 of all Italian cheese samples analyzed complied with current FDA safety standards: free of Listeria, phosphatase and enteropathogenic strains of E. coli. Additionally, 144 cheese samples examined as part of an import alert were suspected of containing L. monocytogenes [44]. After isolating listeriae from Italian Pecorino Romano cheese prepared from goat’s milk (see Fig. 1) in June of 1987, [280], the previous import alert was extended to include

L. monocytogenes in Fermented Dairy Products

423

both soft and hard varieties of Italian cheese [50]. (This was the first instance in which L. monocytogenes was isolated from hard cheese.) Subsequently, the FDA ordered intensified sampling of soft and hard cheese for the next 2 months [44]. Late in 1987, FDA officials also increased the number of cheeses sampled from Austria, Denmark, Germany, Italy, and Switzerland as part of the agency’s ongoing imported cheese surveillance program (see Fig. 1) [57]. Although this action prompted the recall of several Danish cheeses in early 1988 (see Table 3), no additional Class I recalls were issued during the remainder of 1988 for imported cheese contaminated with L. monocytogenes. Heightened concern over the presence of this pathogen in European cheeses (which sterns from the 1987 cheeseborne outbreak of listeriosis in Switzerland) and subsequent initiation of corrective action are probably both responsible for the lack of Class I recalls issued during the remainder of 1988 and early 1989. However, during the latter half of 1989, FDA officials issued (a) four separate Class I recalls for Listeria-contaminated soft cheeses manufactured in Cyprus (see Table 3) and (b) an import alert for contaminated soft and hard cheeses produced by two Italian firm [ 1651. The overall situation regarding presence of L. monocytogenes in imported cheese has greatly improved since 1986 [77] with only four additional recalls of imported cheese issued since 1990. However, sporadic detection of listeriae in imported cheeses suggests that limited surveillance of such products is still necessary to safeguard public health.

SURVEYS AND MONITORING PROGRAMS FOR LISTERIA SPP. IN CHEESE PRODUCED OUTSIDE THE UNITED STATES In response to the 1985 cheeseborne listeriosis outbreak in California, scientists worldwide began analyzing many types of cheese for Listeria spp. Given the possible ramifications of selling Listeria-contaminated cheese and the fact that large quantities of European specialty cheeses are exported to the United States and Canada, high priority was given to determining the incidence of listeriae in Western European cheeses. Since 1986, over 50 surveys have dealt with the incidence of Listeria spp. in various cheeses. However, since sampling designs (i.e., number and size of sample, site of sample collection [cheese factory or retail store], age of sample, portion of cheese analyzed [surface, interior, or both]) and methods for detecting and identifying listeriae vary widely among these surveys, many of the Western European studies and those conducted elsewhere need to be interpreted with some caution.

Canada Reports from federal monitoring programs in both Canada and the United States [57,196] indicate that the incidence of listeriae in Canadian cheese (and nonfermented dairy products as described in Chap. 11) is relatively low. In the only Canadian survey thus far reported, Farber et al. [ 1661 examined 182 samples of soft and semisoft cheese for listeriae using the original FDA method. The cheeses analyzed in this survey were produced at 61 different cheese factories, most of which were located in the provinces of Ontario and Quebec. Although all cheeses examined were Listeria-free, 19 of 79 samples (24%) were positive for phosphatase, which suggests that these cheeses may have been prepared, at least in part, from raw milk. The only additional information concerning Canadian cheese is the unconfirmed isolation of L. monocytogenes from Cheddar and Colby cheeses [207],

Ryser

424

both of which were manufactured from raw milk and held a minimum of 60 days at 2 1.7"C (235°F) as required by the Canadian government. Although the incidence of L. monocytogenes in Canadian-produced cheese appears to be low, the pathogen has been detected in cheese exported to Canada from several Western European countries, including Denmark, France, Switzerland, and Germany [66,196]. In conjunction with the Canadian survey just discussed, Farber et al. [ 1661 also examined 187 samples of Western European soft and semisoft cheese (98 different brands from 12 different countries) that were regularly exported to Canada. Three soft and semisoft cheeses produced by the same manufacturer in France were positive for Listeria spp. (Table 4). Two cheeses contained L. monocytogenes alone, whereas the third contained both L. monocytogenes and L. innocua. In keeping with the previously described 1988 policy regarding foods that have been directly linked to major listeriosis outbreaks, Canadian officials immediately recalled the contaminated cheese. Although some of the tainted cheese was likely consumed before the recall, no cases of listeriosis linked to consumption of this cheese were reported. Despite being labeled as manufactured from pasteurized milk, the three French cheeses from which listeriae were isolated yielded positive results with the phosphatase test as did other cheeses imported from Denmark, Finland, and Switzerland. Such findings, along with unpublished reports of phosphatase in pasteurized dairy products, have raised serious questions as to the validity of the phosphatase test. Results from one study [244] demonstrated that certain heat-labile, microbially produced alkaline phosphatases can mimic the natural phosphatase found in milk and produce false-positive results in the Scharer test. Hence, the ability of the phosphatase test to determine whether or not a dairy product such as cheese was made from pasteurized milk (or from pasteurized milk contaminated with raw milk) needs to be reexamined.

TABLE 4 Incidence of Listeria spp. in Soft/Semisoft European Cheeses Exported to Canada Between October 1985 and March 1987

Country of origin Austria Denmark Finland France Germany Greece Italy Netherlands Norway Portugal Sweden Switzerland Total

Number of positive samples (%)

Number of samples anal yzed

L. monocytogenes

L. innocua

Other Listeria spp.

2 43 1 104 16 2 3 2 7 2 2 3

0 0 0 3 (2.9) 0 0 0 0 0 0 0 0

0 0 0 1 (1.0) 0 0 0 0 0 0 0 0

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

187

3 (1.6)

1 (0.5)

0

Source: Adapted from Ref. 166.

L. monocytogenes in Fermented Dairy Products

425

France Beginning in early 1986, sporadic Listeria-contamination problems have been associated with French soft cheeses exported to the United States and Canada as well as England [ 1961, Germany [74], the Netherlands [ 1 141, Norway [ 196,3081, Sweden [ 1961, and Australia [ 1911. Hence, in an effort to bolster public confidence in the safety of cheeses produced in France, the French government, in cooperation with the Veterinary Service for Food Hygience in France, conducted a series of systematic surveys to determine the incidence of Listeria spp. in French cheeses destined for domestic and foreign markets (Table 5) [ 1 19,181 1. Overall, I .34% of predominantly soft, 30-day-old French cheeses examined during 1986 and 1987 contained detectable levels of both L. monocytogenes and other Listeria spp., with few differences observed between cheeses destined for domestic or foreign consumption. It also is noteworthy that comparable levels of contamination were seen in soft cheeses prepared from raw and pasteurized milk. These findings agree with those of most other surveys which suggest that soft cheeses are most likely to become contaminated with L. monocytogenes during the latter stages of manufacture and ripening. Although L. monocytogenes also was recovered from 10.3% of soft/semisoft French cheeses marketed in Sweden from 1989 to 1993 [210], most surveys have suggested contamination rates of < 10% with I03 of 2275 (4.5%) French cheeses surveyed (Table 6) reportedly harboring L. monocytogenes. However, contamination rates of 46.9 and 87.0% have been reported for soft surface-ripened cheeses prepared from raw milk. In the latter survey [279], L. monocytogenes populations of 106 CFU/g were detected on the cheese surface, with serotype 1/2 predominating. In another French survey [ 1SO], workers at the Veterinary Service for Food Hygiene recovered I,. monocytogenes as well as L. innocua and other Listeria spp. from 0.3 to 3.5% of cottage, soft-ripened, and semihard cheeses examined (Table 6). However, unlike the aforementioned survey, comparable contamination rates were observed for soft-ripened cheese prepared from raw and pasteurized milk. Additional efforts in France have focused on characterizing listeriae isolates from cheese and other milk products. Listeria spp. recovered from French dairy products during 1986 included L. monocytogenes (370 strains), L. innocua (134 strains), L. seeligeri (17

TABLE 5 Incidence of Listeria spp. in French Cheese Destined for Domestic (France) and Foreign Markets during 1986 and/or 1987

Market DomesticJ Foreignb

Type of cheese

Number of samples analyzed

Soft Other Soft (pasteurized milk) Soft (raw milk)

Total Results from January to November 1987. Results from January 1986 to September 1987. Source: Adapted from Ref. 119. a

Number of positive samples (%) ~

L. monocytogenes

Other Listeria spp.

192 I35 736 355

2 (1.0) 0 1 1 (1.5) 6 (1.7)

3 (1.6) I (0.7) 1 0 (1.4) 5 (1.4)

1418

19 (1.34)

19 (1.34)

426

Ryser

TABLE 6 Incidence of Listeria spp. in Cheeses Manufactured Outside the United States

Country Europe Belgium

Czechoslovakia

Denmark France

Germany

Type of cheese Soft Unspecified Unspecified Unspecified Soft ripened Sheep's milk Hard Unspecified Soft/semisoft Unspecified Soft ripened (raw milk) Soft, surface-ripened (raw milk Soft (raw milk) Soft (heat-treated milk) Soft ripened (pasteurized milk) Soft (pasteurized milk) Soft semisoft Semihard Blue Cottage Unspecified Soft Soft Soft Soft (raw milk) Soft (unripened) Soft (mold-ripened)

Number of samples analyzed

886 929 262 37 77 10 33 24 46 25 330 23 32 5 873 32 174 289 126 149 242 712 248 166 22 8 117

L. monocytogenes

62 (6.9) 214 (23) 35 (13.4) 0 6 (7.8) 0 0 2 (8.3) 0 8 (32.0) 3 (0.9) 20 (87.0) 15 (46.9) 0 12 (1.4) 3 (9.4) 18 (10.3) 10 (3.5) 0 2 (1.3) 20 (8.3) 33 (4.6) 3 (1.2) 7 (4.2) 2 (9.1) 0 4 (3.4)

L. innocua ND ND ND 0 ND ND ND ND ND ND 6 (1.8) ND 13 (40.6) 2 (40.0) 5 (0.6) 5 (15.6) ND 1 (0.3) 0

0

ND 58 (S.1) 16 (6.4) ND 2 (9.1) 0 5 (4.3)

Other Listeria SPP. ND ND 68 (25.9) 0 ND ND ND ND ND 8 (32.0) 1 (0.3) ND 1 (3.1)" 0 3 (0.3) 0 ND 1 (0.3) 0 0 61 (25.2) 4 (0.6)" 0 ND 0 0 0

Ref.

210 151

300 119 229 229 229 229 210 119 180 228 164 164 180 164 210 180 180 180 257 287 272 290 164 302 302

L. monocytogenes in Fermented Dairy Products

Ireland Italy

Soft (smear-ripened) Soft/semisoft Soft/semisoft Semisoft Semisoft Semisoft (unripened) Semisoft (mold-ripened) Semisoft (smear-ripened) Semihard Semihard Hard Acid Acid Fresh Processed Unspecified Soft Soft Semisoft Moulded Mold-ripened Hard Hard Curd Processed Processed Soft Soft/semisoft Soft Soft Soft Soft Soft

427

41 256 31 268 45 89 12 7 237 108 42 61 48 149 21 89 25 15 25 10 10 1s

10 15 20 15 40 17 1284 400 54 29 21

3 23 1 9 4 2 7 6 2 2 1

(7.3) (9.0) (3.2) (3.4) 0 (4.5) 0 (28.6) (3.0) (5.6) (4.8)b (3.3) (2.1)

0 0

8 (9.0) 0 0 0 2 (20.0) 0 1 (6.7) 0 0 0 0 1 (2.5) 0 65 (5.1) 8 (2.0) 2 (3.7) 0 2 (1.6)

5 (12.2) ND ND 7 (2.6) 1 (2.2) 1 (1.1) 0 0 14 (5.9) ND 0 22 (36.1) ND 0 0 17 (19.0) ND ND ND ND ND ND ND 0 ND 0 ND ND 140 (10.9) ND 4 (7.4) ND 2 (1.6)

0 7 (2.7) ND 8 (?.O)a 0 3 (3.4)a 0 0 0 ND 1 (2.4)a 1 (1.6)a ND

0 0 0

ND 4 (26.7)' ND ND ND 4 (26.7)' ND 0 ND 0 ND ND 218 (17.0)d ND 0 ND 0

302 119 210 287 272 302 302 302 287 290 289 289 290 289 289 273 253 194 253 253 253 194 253 194 253 194 196 141 137 134 146 174 222

Ryser

428

TABLE 6 Continued

Country

Type of cheese Soft Soft Soft Soft unripened Soft unripened Soft ripened Soft surface-ripened (mold) Soft surface-ripened Fresh Fresh Fresh Soft/semisoft Soft/semisoft Semisoft Ripened Hard Hard Hard Goat’s milk Goat’s milk Sheep’s milk Unspecified Unspecified Unspecified Unspecified Unspecified Unspecified Asiago

Number of samples analyzed

30 12 8 69 18 136 16 90 239 38 17 64 36 118 50 99 40 10 24 21 40 1846 373 115 75 62 52 12

L. monocytogenes 0 1 (8.3) 0 6 (8.7) 0 9 (6.6) 0 1 (1.1) 0 2 (5.3) 0 0 1 (2.8) 0 1 (2.0) 0 0 0 0 1 (4.8) 0 24 (1.3) 22 (5.9) 6 (5.2) 0 3 (4.8) 4 (7.7) 0

L. innocua 1 (3.3) ND 1 (12.5) ND ND ND ND 10 (11.1) 0 2 (5.3) ND ND ND 5 (4.2) 0 0 0 ND ND ND ND ND 50 (13.4) 3 (2.6) ND ND 0 ND

Other Listeria SPP0 ND 0 ND ND ND ND 1 (1.1)C 0 0 ND ND ND 0 0 0 0 ND ND ND ND ND 70 (18.8)b 0 ND ND 0 ND

Ref.

293 243 256 250 136 250 146 293 137 146 174 136 210 137 256 137 146 136 124 136 124 109 138 246 236 127 119 124

L. monocytogenes in Fermented Dairy Products

Netherlands

Norway Spain

Crescenza Gorgonzola Gorgonzola Gorgonzola Gorgonzola Gorgonzola Mozzarella Mozzarella Mozzarella Mozzarella Mozzarella Mozzarella Mozzarella Mozzarella Pecorino Talleggio Talleggio Taleggio Soft (raw milk) Soft (pasteurized milk) Soft (domestic) Soft (imported) Fresh Soft Semihardlhard Blue (raw milk) Unspecified Unspecified (raw milk) Unspecified (raw milk) Unspecified (pasteurized milk) Queso fresco / cottage

429

212 67 58 44 40 25 94 74 50 30 29 24 20 14 8 45 38 12 938 484 850 90 23 14 20 11 100 49 42 21 91

-

0 (9.0) (5.2) (9.1) (5.0) (8.0) (16.0) 0 0 0 4 (13.8) 0 2 (10.0) 0 0 0 0 0 43 (4.6) 10 (2.0) 0 10 (11.0) ND ND 0 0 0 1 (2.0) 10 (23.8) 0 7 (7.7)

6 3 4 2 2 15

ND 28 (41.8) 21 (36.2) ND 4 (10.0) ND 10 (10.6) 2 (2.7) 0 0 2 (6.9) ND ND ND ND 7 (15.6) ND ND ND ND ND ND ND ND 0 ND ND 0 ND ND 4 (4.4)

ND 1 (l.l)a 0 ND 0 ND 1 (l.l)a 2 (2.7)e 0 0 1 (3.4)' ND ND ND ND 2 (4.4) ND ND ND ND NDg ND 1 (4.3)h 1 (4.3)g 0 ND ND 1 (2.0)a ND ND 2 (2.2)a

124 246 242 243 146 174 246 146 103 164 242 124 243 174 124 242 243 124 294 294 254 254 220 220 220 21 1 196 247 130 130 120

Ryser

430

TABLE 6 Continued

Country Sweden Switzerland

Turkey

Yugoslavia United Kingdom England England/ W ales

Type of cheese Soft/semisoft soft Soft (mold-ripened) Soft (smear-ripened) Semisoft Semisoft (mold-ripened) Semisoft (smear-ripened) Semisoft (smear-ripened) Hard Unspecified Kashor White Unspecified Unspecified (raw milk) White-brined Soft Soft/semisoft Soft Soft Soft Soft Soft ripened Soft unripened Hard Ewe’s milk Goat’s milk

Number of samples analyzed

L. monocytogenes

L. innocua

Other Listeria SPP.

Ref.

27 604 54 18 205 26 1 343 69 88 17 30 30 224 40 170

0 40 (6.6) 0 4 (22.2) 4 (1.9) 7 (2.7) 33 (9.6) 6 (8.7) 0 0 0 0 4 (1.8) 2 (6.1) 9 (5.3)

ND 38 (6.3) 0 3 (16.7) 0 ND ND 13 (18.8) 0 ND ND ND 7 (3.1) ND ND

ND 0 ND ND 0 ND ND ND 0 1 (5.9) ND ND 0 ND ND

210 123 41 41 123 123 123 41 123 119 186 186 190 186 151

25 1 12 1437 25 1 222 131 769 366 66 141 476

1 (0.4) 0 16 (1.1) 10 (4.0) 23 (10.4) 0 63 (8.2) 4 (1.1) 1 (1.5) 1 (0.7) 22 (4.6)

9 (3.6) ND ND ND ND ND ND ND ND ND ND

0 ND ND ND ND ND ND ND ND ND ND

213 210 177 213 24 1 118 188 188 188 188 188

L. monocytogenes in Fermented Dairy Products

Northern Ireland Scotland Elsewhere Australia

Brazil Costa Rica Egypt Japan

Jordan Morocco United Arab Emirates

431

Soft Soft Unspecified

33 27 305

Soft Soft Unspecified Unspecified Minas frescal Soft Damietta Kareish Domestic fresh Domestic soft/semisoft Domestic semi-hard/hard Imported fresh Imported soft/semisoft Imported semi-hard/hard Imported other White brined Domestic fresh Imported mold-ripened Domestic Imported

437 28 338 126 20 20 50 100 92 94 105 94 418 403 55 67 20 45 53 196

ND, not determined. a L. seeligeri. Isolated from sheep's milk cheese. non-L. monocytogenes. 185 L. welshimeri, 18 L. ivanovii, 15 L. murrayi. L. grayi. 63 L. welshimeri, 6 L. ivanovii, 1 L. murrayi. g Lisreria sp. L. welshimeri.

0 3 (11.1) 3 (1.O)C

15 1 6 1 9 1 1

16

2

(3.4) (0.2) (1.8) (0.8) 0 (45.0) (2.0) (1.0) 0 0 0 0 (3.8) 0 0 0 0 0 0 (1.0)

0 ND 0

ND 0 ND 1 (0.8) 0 ND 1 (2.0) 3 (3.0) ND ND ND ND ND ND ND ND ND ND 0 2 (1.0)

1 (3.3)a ND 1 (3.3)a

191 258 110

24 (5.5)' 0 ND 0 0 ND 0 1 (l.O)h ND ND ND ND ND ND ND ND ND ND 0 0

102 102 298 187 129 23 1 167 167 232 232 232 232 232 232 232 161 159 159 184 184

432

Ryser

strains), and L. ivanovii (1 strain), with 299 of 370 (80%) and 48 of 370 (13%) L. monocytogenes strains belonging to serovars I /2 and 4b, respectively. Additional surveys conducted in France [ 1 19,1811 and Belgium [ 1031 from 1985 to 1990 indicated that a disproportionately large number of L. monocytogenes strains isolated from cheese and other dairy products were serovar 1/2. This situation appears to be reversed in the United States, with isolates of serovar 4b typically outnumbering those of serovar 1/2. Phage typing has become a useful means of characterizing particular L. monocytogenes strains isolated from dairy products and of trackmg the probable source of foodborne listeriosis outbreaks. Although only 33 3 % of all L. monocytogenes strains isolated from French dairy products during 1986 and 1987 were typeable using the available set of phages, some phage types were unique to particular regions within France [ 1191. In some instances, excellent correlations were observed between specific phage types and certain cheese varieties, with some phage types even being specific to a particular dairy. Such findings have lead to better control of the listeriosis problem within the dairy industry. The inadvertent isolation of L. monocytogenes from French soft-ripened cheese by FDA officials in January of 1986 prompted several additional surveys of French cheese exported to other Western European countries. Working in The Netherlands, Beckers et al. [ 114,1151 examined 69 samples of French soft cheese (i.e., Brie and Camembert) for L. monocytogenes using both direct plating and cold enrichment. The pathogen was recovered from 7 of 69 (10.1%) cheeses at levels ranging between 103and 106 CFU/g. Cold enrichment uncovered three additional cheeses with L. monocytogenes for a total of 10 positive samples. Although all 10 Listeria-positive cheeses were prepared from raw milk, comparable rates of contamination have frequently been reported for cheese manufactured from raw and pasteurized milk [ 119,1801.

Germany As mentioned in Chapter 10, Germany experienced a major outbreak of listeriosis shortly after World War 11. This outbreak, which may have resulted from consuming contaminated raw milk, led to an increased interest in listeriosis research, and this in turn prompted Prof. H. P. R. Seeliger to publish his time-honored monograph Listeriosis in 1961. During the last 40 years, the late Prof. Seeliger emerged as one of the world’s leading authorities on listeriosis. In addition, he operated a listeriosis research center at the Institut fur Hygiene und Mikrobiologie der Universitat Wurzburg to which Listeria isolates could be sent for biochemical and serological confirmation. Hence, it is not surprising to learn that the incidence of listeriae in cheese has received considerable attention in Germany. Although spared from the heavy economic losses experienced by the United States and France, Germany and most other European countries have not escaped the Listeria problem completely unscathed. Despite rigorous testing, Listeria-laden German blueveined cheese was recalled from France [196], with a similar recall being issued for sour milk cheese exported to Canada [66,196] and The Netherlands [196]. Consequently, a series of Listeria-monitoring programs were introduced for German soft, semisoft, semihard, and hard cheeses as well as cultures, cheese byproducts, and the general environment within cheese factories. Since 1990, German officials have been enforcing a policy similar to that adopted in Canada in which only contaminated foods previously associated with foodborne listeriosis outbreaks are recalled from the marketplace. Results from various surveys made since 1986 (see Table 6) indicate that 0-9.1 %

L. monocytogenes in Fermented Dairy Products

433

(average of 3.9%) and 0-28.6% (average of 3.6%) of the soft and semisoft cheeses marketed in Germany contained L. monocytogenes, respectively, with the highest incidence of listeriae generally occurring in smear-ripened varieties. With few exceptions, L. innocua was isolated more frequently from soft and semisoft cheese than was L. monocytogenes. Although somewhat similar average percentages were reported for the incidence of L. monocytogenes in semihard (3.8%) and hard cheese (4.8%), the two hard cheeses that contained L. monocytogenes were reportedly manufactured from ewe’s rather than cow’s milk. Overall, it appears that the Listeria contamination rate for hard cheeses prepared from cow’s milk may still be relatively low, as also was observed in Switzerland (see Table 6). Hence, these results from Germany generally agree with those from other surveys in that L. monocytogenes was found more frequently in high-rather than low-moisture cheese. Of the three remaining categories of German cheese shown in Table 6, only acid curd cheese was positive for listeriae. The apparent absence of Listeria spp. from samples of fresh (i.e., cottage) and processed cheese is not entirely unexpected, since procedures used to manufacture these cheeses include relatively severe heat treatments. Even if a few listeriae survived the manufacturing process, most, if not all, of the survivors would have been sublethally injured during exposure to heat and/or acid and would therefore be unable to grow in most selective enrichment broths that are commonly used for examining cheese. In the only other study thus far reported, Weber et al. [302] examined various German cheeses, including 11 types manufactured from ewe’s and goat’s milk, for listeriae (Table 7). Although all cheeses prepared from ewe’s or goat’s milk were free of L. monocytogenes, L. innocua was detected in one sample of fresh goat’s milk cheese. Despite the ability of lactating sheep and goats to shed L. monocytogenes in their milk, as further evidenced by isolation of L. monocytogene:?from approximately 4 of 480 (0.8%) samples of raw goat’s milk in England [188], relatively few additional studies have dealt with the incidence of listeriae in ewe’s and goat’s milk cheese. Nevertheless, in addition to the aforementioned survey of German hard cheese produced from ewe’s milk [287l, Tham [291] also reported isolating L. monocytogenes from one sample of 8week-old goat cheese marketed in Sweden.

TABLE 7 Incidence of Listeria spp. in Domestic and Imported Cheese Analyzed in Germany Between October 1987 and June 1988 ~

Type of cheese/milka Fresh / Cow Fresh/Goat Soft/Cow Soft/Goat Soft/Ewe Semisoft/Cow Semisoft/Ewe Semihard/Cow Hard/Cow

Number of samples analyzed

21 1 307 1 3 144 6 22 4

Milk from which cheese was manufactured. Source: Adapted from Ref. 302. a

Number of positive samples (%)

L. monocytogenes

L. innocua

2 (9.5) 0 8 (2.6)

0 1 (100.0) 11 (3.6) 0 0 8 (5.6) 0 11 (50.0) 0

0 0 19 (13.2) 0 0 0

434

Ryser

The incidence of L. monocytogenes in the remaining cheeses prepared from cow’s milk was similar to those values obtained in other studies (see Table 6), with the pathogen being detected in 2.6 and 13.2% of the soft and semisoft (presumably mold- and smearripened varieties) cheeses examined, respectively. The unusually high incidence of L. munocytogenes in fresh curd cheese (9.5%) is probably the result of contamination during later stages of manufacture and/or packaging as well as the small number of samples examined. As was true for surveys of French soft-ripened cheese discussed earlier in this chapter, most L. monocytogenes isolates were serovar 1/2 (22 strains) as also reported by Schonberg et al. [273], with the remaining seven isolates being classified as 4ab or 4b. However, since most clinical L. monocytogenes isolates from Germany are serovar 4b, it appears that some questions remain concerning the ability of present isolation methods to recover L. monocytogenes serovar 4b as compared with other serovars, from various foods, including cheese.

Italy Public health concerns raised in the United States following the isolation of L. monocytogenes from imported cheese prompted over 15 surveys of cheeses manufactured in Italy (see Table 6). Overall, L. monocytogenes was recovered from 196 of 6382 (3.1%) Italian cheeses surveyed, with this pathogen being most prevalent in Gorgonzola (7.3%), followed by mozzarella (6.3%) and various soft cheeses (4.2%). In another study, Cantoni et al. [128] reportedly isolated L. monocytogenes from 14 of 375 (3.7%), 14 of 216 (6.5%), and 5 of 95 (5.3%) samples of Gorgonzola (blue-veined), Tallegio (soft, surface-ripened), and other Italian cheeses, respectively. Although a follow-up study demonstrated L. monucytugenes at levels of <100 to 12,000 CFU/g in Gorgonzola and Taleggio cheese, respectively, the pathogen was never recovered from 1150 samples of soft, semisoft, semihard, or hard cheese or from 72 samples of pasta filata-type cheese such as provolone and mozzarella. When present, however, L. monocytogenes serovar 1 typically predominated [242] as has been reported for other European cheeses. Between January 1987 and September 1988, Massa et al. [222] also examined 54 soft rindless (i.e., Mascarpone, mozzarella, Crescenza) and 67 soft thin-rind (i.e., Italico, Caciotta) cheeses produced by both large and small northern Italian factories for listeriae and E. coli. Listeria rnonocytogenes was detected in only 2 of 47 (4.2%) thin-rind cheeses manufactured by one small factory, with core samples from these two positive cheeses being negative for the pathogen. Although all other thin-rind and rindless soft cheeses were free of L. monocytogenes, two mozzarella cheeses contained detectable levels of L. innucua. According to these investigators, E. coli populations in these cheeses ranged from <10 to 8 X 105 CFU/g, with the two L. rnonocytogenes-positive cheeses containing 2 104E. coli CFU/g. However, since 14 similar Listeria-free cheeses also contained >103 E. coli CFU/g, E. coli is clearly a poor indicator organism for possible presence of listeriae.

Switzerland Although several major Western European countries have experienced various degrees of economic loss from Listeria-contaminated cheese, thus far only Switzerland, France, and the United States have been forced to deal with major outbreaks of cheeseborne listeriosis. Well before the 1987 listeriosis outbreak in Switzerland linked to consumption of Vacherin Mont d’Or soft-ripened cheese, Swiss officials began examining various cheeses for lister-

L. monocytogenes in Fermented Dairy Products

435

iae. Although these surveys apparently were prompted by the 1985 listeriosis outbreak in California, an unusually high incidence of listeriosis in certain areas of Switzerland which could not yet be explained may have provided added incentive to initiate these surveys. A two-stage Listeria-monitoring program was later established for cheese and other dairy products with random testing of 10-g samples obtained at both the factory and retail level [ 1961. According to the Federal Bureau of Health, such samples must be completely free of L. monocytogenes before the product is deemed acceptable. Working in Switzerland, Breer [ 1231 examined 799 domestic and imported cheese samples for listeriae during the winter of 1985/1986. Various Listeria spp. were detected in 19.2% of the soft surface-ripened cheeses, all of which were traced to 10 Swiss and a few foreign manufacturers. During follow-up investigations of these 10 cheese factories in Switzerland, Listeria spp. were isolated from surfaces of various cheeses and also from curing and smearing brines, waste-water sinks, and surfaces of wooden boards used in cheese ripening. In addition, identical serovars of L. monocytogenes (1 /2b and/or 4b) and/ or L. innocua were isolated repeatedly from the same cheese factories. These findings demonstrate that ample opportunity existed for cheese to become contaminated with listeriae during the later stages of manufacture and ripening. Subsequently, Breer [ 1231 reported that 4.9 and 4.7% of all cheeses sold in Switzerland contained L. monocytogenes or L. innocua, respectively (see Table 6). Of equal importance is the fact that both Listeria spp. were isolated more frequently from soft (6.6, 6.3%) than semisoft cheese (1.9,0%) and that neither organism was detected in 88 samples of hard cheese. During 1986, Breer [122] also found that 12.9 and 10.0% of soft surface-ripened cheeses manufactured in Switzerland were contaminated with L. monocytogenes and L. innocua, respectively (Table 8). The incidence of both Listeria spp. was generally twice as high in smear- rather than mold-ripened cheese. As in previous studies, the rate of Listeria contamination was typically independent of the type of milk (raw or pasteurized) from which smear-ripened cheeses were manufactured. These Swiss studies, along with several of the aforementioned German surveys, indicate a greater likelihood of isolating L. monocytogenes and L. innocua from high rather than low-moisture cheese, with special emphasis on mold- and smear-ripened varieties.

TABLE8 Incidence of Listeria spp. in Soft Mold- and Smear-Ripened Cheeses Manufactured in Switzerland During 1986 from Pasteurized and Raw Milk

Type of cheese

Number of positive samples (%)

Number of samples analyzed

L. monocytogenes

L. innocua

22 9

2 (9.1) 0

1 (11.1)

17 22

3 (17.6) 4 (18.2)

4 (23.5)

70

9 (12.9)

7 (10.0)

Mold-ripened Raw milk Pasteurized milk Smear-ripened Raw milk Pasteurized milk Total Source: Adapted from Ref. 122.

0

2 (9.1)

436

Ryser

In support of this observation, Bannerman and Bille [ 11I] found that 110 of 449 (24.5%) rinds from soft cheese produced in Switzerland were contaminated with Listeria spp., including a high percentage of samples with L. monocytogenes. During an additional survey made between October 1986 and September 1987 [41], L. monocytogenes was detected in 4 of 18 (22.2%) and 6 of 67 (8.7%) smear-ripened soft (i.e., Limburger, Romadur, Muenster, Reblochon) and semisoft (i.e., St. Paulin, Tilsiter, Mutschli, Raclette) cheeses, respectively, with many cheeses also containing L. innocua. From the apparent widespread distribution of Listeria within some cheese factory environments, it follows that cheeses prepared from raw and pasteurized milk are equally likely to contain listeriae. Additional information concerning the incidence and control of listeriae in dairy factories and other food processing facilities is given in Chapter 17.

Other European Countries As already implied, recent Listeria problems that have affected the European cheese industry are not limited to France, Germany, Italy and Switzerland. Other European nations, including Austria, Belgium, Denmark, England, Finland, Greece, Hungary, The Netherlands, Spain, and Sweden, also expressed concern in a March 1989 poll conducted by the International Dairy Federation [ 1961. According to the survey, L. monocytogenes was isolated from domestic cheese sold in Austria (soft cheese), Belgium (rind-type cheese), Denmark (various soft cheeses), England (various cheeses), Ireland (soft farm-house cheese), The Netherlands (young farm-house Gouda), and Sweden (goat’s milk cheese), with this pathogen later being identified in cheeses from Czechoslovakia (soft-ripened) [229], Greece (Feta) [ 1511, Ireland (soft) [196], Turkey (soft) 1186,1901 and Yugoslavia (white brined) [ 15 11 (see Table 6). Denmark, England, and Sweden have also experienced problems with soft or semisoft cheeses imported from Denmark, France, Germany and/ or Italy (Table 9). In contrast, Norway [ 1 10,3051 and Spain [ 1961 have been primarily affected by imported soft and/or blue-veined cheeses. Thus, in addition to France, Italy, Switzerland, and Germany, Austria, Belgium, Denmark [67], England, Finland, Greece, Hungary, The Netherlands, and Sweden also have developed programs to actively monitor the incidence of listeriae in domestic/imported cheese (especially soft surface-ripened varieties) and/or manufacturing environments within cheese factories. As of March 1989 [196], Austria, Denmark, France, Germany, Greece, Hungary, Italy, Sweden and Switzerland had regulations regarding the sale of cheese and other foods contaminated with L. monocytogenes, with most European countries now attempting to prevent distribution and sale of cheese (and in some instances other ready-to-eat foods) containing 2100 L. monocytogenes CFU/g. Since these observations along with the 1987 listeriosis outbreak in Switzerland involving Vacherin Mont d’Or cheese both support the widespread notion that L. monocytogenes poses a significant health threat to certain segments of the population, the European Economic Community Consumers Association published a list of soft and semisoft cheeses manufactured in France and Switzerland that should be avoided by susceptible individuals-pregnant women, immunocompromised adults, and the elderly. This highly controversial list of cheeses (and brands when applicable) included Brie (La Renommee), Muenster (Ermitage), Crkme de Bleu (Diapason), Lys Bleu, Camembert (I Signy), Tilsit, Fourme de Bresse, Bleu de Bresse, Reblochon, Pont L’Eveque, Gruykre, and Vacherin Mont d’Or. In February of 1988, a case of cheeseborne listeriosis was reported in England in which a 40-year-old woman contracted meningitis shortly after consuming Anari-type soft

L. monocytogenes in Fermented Dairy Products

437

TABLE 9 Incidence of L. monocytogenes in Cheeses Marketed in Sweden from 1989 t o 1993 Type of cheese Country of origin Austria Denmark England France Germany Greece Italy Netherlands Norway Romania Spain Sweden Total

White mold

Green/Blue mold

__

0/1"

Heat-treated

o/ 1

0/3

0/8

1/ 122 (0.8)

4/26 (15.4)

0/3 1

7/302

-

__

0/5

0/25 -

o/ 1

-

o/ 1

-

0/3 ~~

Number of positive sampleshumber of samples analyzed (%). Source: Adapted from Ref. 210. a

Other

0/13

0/27 0/12 0123 1/19 (5.3)

15/119 (12.6) 0/8

~~~

Smear-ripened

0/46 0/12 5/144 (3.5) 1/31 (3.2) o/ 1 1/36 (2.8) 0/2 o/ 1 o/ 1 o/ 1 0126

0/19

15/154 (9.7)

Type of cheese milk

-

-

3/18 (16.7) o/ 1

0/14 0/3

1/2 (50.0) 0/2 -

0/4

-

-

o/ 1

-

o/ 1

-

Raw -

13/30 (43.3) -

o/ 1

13/31 (41.9)

438

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goat’s milk cheese that contained L. monocytogenes at levels >107CFU/g (see Chap. 10). During follow-up investigations at the factory [224], the same L. monocytogenes strain also was isolated from 8 of 11 and 4 of 8 factory and/or retail samples of Halloumi and Cheddar cheese, respectively, as well as single samples of Gjestost and soft chive cheese. In addition, L. innocua also was recovered from several samples of Halloumi and Cheddar cheese. As in the previously described studies by Pini and Gilbert [241] and Massa et al. [222], no clear relationship was observed between the presence of L. monocytogenes/L. innocua and coliformslE. coli. According to several additional surveys, some Costa Rican [2311 and Turkish cheeses [ 1481 contained 104L. monocytogenes and 2 102coliform CFUI g. Hence coliforms appear to be relatively poor indicators of Listeria contamination. The Public Health Laboratory Service in London coordinated a large-scale survey in which various dairy products marketed in England and Wales were sent to 46 laboratories throughout the country for Listeria testing. Results from this comprehensive survey (see Table 6) indicated that 8.2, 1.1, 1.5 and 4.1% of the soft-ripened, soft-unripened, hard, and goat’s milk cheese manufactured in England and Wales contained L. monocytogenes; 75, 42 and 7 isolates classified as serovar 1/2, 4b and 4, respectively. Among the soft ripened varieties, 13 cheeses harbored > 103 L. monocytogenes CFU/g with 3 samples exceeding 105 CFU/g. Of these 13 cheeses, 7 were prepared from raw milk, with only one of the cheeses being manufactured in the United Kingdom. In contrast, only 2 of 33 cheeses prepared from ewe’s or goat’s milk contained >500 L. monocytogenes CFU/g. Overall, the incidence of this pathogen was similar in imported (7.4%) and UK-produced cheese. These incidence rates and serovar distribution patterns for L. monocytogenes in soft-ripened and unripened cheese are generally similar to those observed in most other European studies [209,2101. As just suggested, numerous surveys for incidence of listeriae in cheese also have been completed in many of these aforementioned countries which, with the exception of Denmark and France, have not experienced major economic problems associated with Listeria-contaminated cheese. Following the 1986 report of an English woman who contracted listeriosis after consuming French soft cheese [ 1121, two English researchers [241] examined 45 domestic soft cheeses as well as 177 soft cheeses imported from France, Italy, Cyprus, Germany, Denmark, and Lebanon for Listeria spp. and E. coli. (Table 10). Overall, L. monocytogenes was isolated from 2 of 45 (4.4%) English cheeses and 21 of 177 (11.9%) soft cheeses imported from France, Italy, and Cyprus. Populations of L. monocytogenes in contaminated cheese ranged from <102 to 105 CFU/g, with 9 of 12 French cheeses containing 2 104CFU/g. Despite differences in media and methods used in various surveys, the contamination rate of 14.1% for soft French cheeses calculated in this study was close to the 14.5% previously observed for French soft cheese exported to The Netherlands. As was true for previous surveys of French dairy products, all strains of L. monocytogenes (except one nontypable strain) were of serovar 1/2 or 4b, with the former predominating. Listeria innocua, the only other Listeria sp. detected during this survey, was isolated from 9 of 85 (10.6%), 7 of 44 (15.9%), 2 of 45 (4.4%), and 1 of 6 (16.7%) soft cheeses produced in France, Italy, England, and Denmark, respectively, with 6 of 222 (2.7%) cheeses containing both Listeria spp. Although E. coli populations exceeded 10 CFU/g in 73 of 222 (32.9%) cheeses examined, no correlation was again observed between the presence of L. monocytogenes or L. innocua and contamination with E. coli. In fact, E. coli was detected at >10 CFU/g in only 10 of 23 (43.5%) cheeses that contained the pathogen. In this study, 10 of 23 (43.5%) cheeses contaminated with L. monocytogenes were prepared from pasteurized milk, whereas 2 and 11 of the remaining positive cheeses were manufactured from raw milk and milk of undetermined processing,

L. monocytogenes in Fermented Dairy Products

439

TABLE 10 incidence of L. rnonocytogenes and E. coli in Soft Cheese Sampled in England During 1987

L. monocytogenes

Country of origin England France Italy Cyprus West Germany Denmark Lebanon Total

Number of samples analyzed

Number of positive samples

85 45 44 20 17 6 5 222

Level/g

Number of samples with >I0 E. coli CFU/g (%)

12 (14.1) 2 (4.4) 7 (15.9) 2 (10.0) 0 0 0

<102-105 <:102 < 102-1 0 4 <:102 ND ND ND

32 (37.6) 14 (31.1) 12 (27.3) 3 (15.0) 9 (52.9) 2 (33.3) 1 (20.0)

23 (10.4)

ND- 105

73 (32.9)

ND, not detected. Source: Adapted from Ref. 241.

respectively. Thus, as in previous studies, the type of milk (i.e., raw or pasteurized) from which cheese is made appears to be a poor indicator of possible Listeria contamination. Problems regarding the occasional presence of listeriae in soft cheese also have surfaced in the Scandinavian countries, with L. monocytogenes being recovered from 0.3% of Norwegian cheeses [ 1511 and also identified in Danish Esrom and Blue Costello cheese that was exported to Norway [196,308], Sweden [196], and the United States. Although four Class I recalls were issued for Danish Esrom and Blue cheese in the United States (see Table 3), both of these cheeses (-20% of which were contaminated) were on sale for up to 2 months in Norway before being removed from the market, apparently without incident [3O5]. Danish officials also took steps to prevent unsold cheese from reaching consumers and have since developed a Listeria surveillance program [67] similar to that instituted in the United States with routine testing of various cheeses as well as cheesemaking facilities. Additional concern over the microbiological safety of various cheeses also led to isolation of L. monocytogenes serovar 1/2b from two presumably French soft-ripened cheeses (one prepared from raw milk and one from pasteurized milk) that were exported to Norway and Sweden [292]. Surface and interior samples from the raw milk cheese contained 7.5 X 105and 1.0 X 102L. monocytogenes CFU/g, respectively, whereas corresponding samples from the pasteurized milk cheese contained 4.0 X 106 and 1.O X 106 L. monocytogenes CFU/g. The reasons for nonuniform distribution of listeriae in softripened cheese will be explored in the second half of this chapter. Although cheese prepared from raw milk contained 3 X 106to 7 X 106colifomr CFU/g, coliform tests indicated that the remaining cheese manufactured from pasteurized milk was fit for consumption. These findings reinforce the fact that coliform-free cheese may not necessarily be free of L. monocytogenes.

Other Countries Reports of Listeria-contaminated cheese in countries beyond North America and Europe also are beginning to surface (see Table 6). In 1987, L. monocytogenes was recovered

440

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from ricotta cheese manufactured in Melbourne, Australia [298]. This event, along with identification of L. monocytogenes in the same imported brands of Danish blue and French brie cheese that were recalled in the United States [ 1911 prompted Venables [298] to determine the incidence of listeriae in Camembert, blue vein, ricotta, cottage, pasta filata, high-moisture, low-acid, and other cheese varieties manufactured in and around Melbourne. Overall, L. monocytogenes was recovered from 6 of 338 (13%)cheeses produced by five different manufacturers, with the pathogen identified as being present in pasta filata (three samples), ricotta (two samples), and shredded (one sample) cheese. One cheese also contained L. seeligeri. Simultaneous identification of L. monocytogenes in environmental samples from all factories producing Listeria-positive cheese strongly suggests that these cheeses were contaminated during manufacture and/or ripening. In keeping with U.S. policies, attempts were made to remove tainted cheese from the marketplace. Furthermore, after thoroughly cleaning and sanitizing the factory, government officials required that Listeria-free cheese be produced for 12 consecutive days before being released to the public. Recent discovery of nonpathogenic listeriae in raw milk from neighboring New Zealand also has prompted authorities in that country to institute a similar environmental monitoring program for all cheesemakers who export their products. Information regarding the presence of listeriae in dairy products produced elsewhere is still reasonably scant, with results from a March 1989 IDF Survey [ 1961 indicating that L. monocytogenes had not yet been isolated from any dairy products manufactured in South Africa, Israel or the former Soviet Union. Most cheese-related surveys from other countries have yielded negative results, with detection of L. monocytogenes being limited to certain high-moisture domestic cheeses produced in Costa Rica [23 I], Egypt [ 1671, and Venezuela [ 1471 along with imported (presumably European) soft cheeses marketed in Japan [232] and the United Arab Emirates 11841. Given the enormous volume of dairy products exported to other countries and the fact that L. monocytogenes has been isolated from the natural environment of all seven continents except Antarctica, it appears that developing countries are unlikely to remain completely untouched by the problems associated with Listeria-contaminated foods. Consequently, interest in the incidence of listeriae in dairy products and other ready-to-eat foods will likely continue in the years ahead.

BEHAVIOR OF L. MONOCYTOGENES IN FERMENTED MILKS Before the well-known 1985 outbreak of cheeseborne listeriosis occurred in California, very little information was available about the behavior of L. monocytogenes in fermented milks and cheese. In fact, at the time of this outbreak, a search of the scientific literature uncovered only four such studies, which were reported from Bulgaria [ 195,2951 and Yugoslavia [278,281] between 1965 and 1979. Hence, in addition to prompting numerous surveys for Listeria spp. in cheese, discovery of cheese as an important vehicle in foodborne listeriosis has led to well over 100 publications addressing the fate of L. monocytogenes in fermented dairy products during manufacture and storage. As described elsewhere in this book, cows, sheep, and goats can shed L. monocytogenes naturally in their milk during lactation. According to results from recent environmental surveys of dairy processing facilities, ample opportunity exists for this pathogen to enter pasteurized milk as a postpasteurization contaminant before the fermentation process

L. monocytogenes in Fermented Dairy Products

44 I

begins as well as afterward as a contaminant of the finished product. Thus far most studies have dealt with behavior of L. monocytogenes in fermented dairy products inoculated with the pathogen either before or after fermentation, with relatively few studies addressing the fate of listeriae in fermented dairy products manufactured from naturally contaminated raw milk. Although the extent to which L. monocytogenes survives in cultured dairy products is partly dictated by whether or not the pathogen enters the product before or after fermentation, viability of Listeria in fermented dairy products, particularly cheese, depends on the type of product in which the pathogen is found as well as the degree of acid tolerance possessed by the contaminating strain [ 1721. Hence, to better understand the complex interactions between the various factors that affect viability of listeriae in cheese (i.e., amount, activity and type of starter culture, a,, pH, salt content, temperature during manufacture and storage), it is appropriate to begin this section by first discussing the behavior of L. monocytogenes in milk fermented with niesophilic arid thermophilic lactic starter cultures. Commingled with this information will be data concerning the fate of this foodborne pathogen during manufacture and storage of cultured buttermilk, cream, and yogurt. The viability of L. monocytogenes in coagulants (e.g., calf rennet, microbial rennet, and bovine-pepsin rennet extract), coloring agents (e.g., annatto), and starter distillates (e.g., natural flavor compounds derived from cultured milk) used in cheesemaking also will be considered before our discussion of natural cheeses and cold-pack cheese food. Two additional areas of concern to cheesemakers, namely, the fate of L. monocytogenes in whey and salt brine solutions, will be examined at the end of this chapter.

Starter Cultures, Cultured Milks, and Cream Fermented or cultured buttermilk, cream, and yogurt were among the first dairy products to be mass produced commercially using pure bacterial starter cultures. Today, two mesophilic (optimal growth at 30°C) lactic acid bacteria starter cultures, namely, Lactococcus lactis ssp. luctis and Lactococcus lactis ssp. cremoris, (formerly Streptococcus lactis and Streptococcus cremoris, respectively), are commonly used either alone or in combination to manufacture cultured buttermilk and cream, whereas a mixture of two thermophilic (optimal growth at or above 37°C) lactic acid bacteria, namely, Streptococcus salivarius ssp. thermophilus and Lactobacillus delbriickii ssp. bulgaricus (formerly Streptococcus thermophilus and Lactobacillus bulgaricus, respectively), is used to produce yogurt. These same mesophilic and thermophilic starter cultures also are used to produce over 400 varieties of cheese that were recognized by the United States Department of Agriculture [297] in 1978 with over 1200 cheese varieties now being in existance worldwide. The variety of cheese to be produced depends, in part, on which of the lactic acid bacteria are used, either alone or in combination, as the starter culture.

Mesophilic Starter CuItu res Viability 01' L. monocytogenes in the presence of mesophilic lactic acid bacteria was first examined by Schaack and Marth [269]. Samples of autoclaved skim milk were inoculated to contain approximately 10' L. monocytogenes CFU/mL along with 0.1,0.5, 1.O, or 5.0% of a S. cremoris or S. lactis milk culture and then fermented at 21 or 30°C for 15 h to simulate preparation of a conventional bulk starter culture for cheesemaking. As shown in Figure 2, L. monocytogenes grew to some extent in all samples during fermentation regardless of the species of lactic acid bacteriurn, inoculum level, or incuba-

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442

-1

mm]

-E

2

=0.10/0

= 1.0%

21°C

30°C

1.50

c1

M

tl,

1.25

0

-g,

.d Y

1 .o

0

a

'c

0.75

; I .-

0.50

M

c cd

v

0.25

3ooc

2 I "C

S. _ crerrioris __

-S. lactis

FIGURE2 Changes in populations of L. monocytogenes in skim milk following a 15hour fermentation at 21 and 30°C with 0.1, 0.5, 1.0, or 5.0% (v/v) added S. cremoris or S. lactis. (Adapted from Ref. 269.) tion temperature. Maximum Listeria populations after 15 h of incubation at 21 "C were -1.0-2.3 orders of magnitude lower in skim milk containing 0.1-5.0% of either starter culture than in control samples without starter culture. Increasing the incubation temperature to 30°C led to final Listeria populations that were -2.5-4.0 orders of magnitude lower than in controls. As expected, growth of L. monocytogenes increased as the starter culture inoculum level decreased from 5.0 to 0.1%. Although acid production by the starter culture played a major role in limiting multiplication of Listeria, with the pathogen never growing at pH (5.0, two situations were reported in which the organism was almost completely inhibited at pH 5.6-6.0-well above the minimum pH value generally required for growth. Although still in use today, conventionally prepared bulk starter cultures have one major disadvantage in that excessive acid development from an inherent lack of buffering capacity frequently leads to some cell damage and partial loss of starter culture activity. Consequently, in industrially prepared bulk starter cultures, the final pH is now normally held constant at 5.1-5.2 by either adding (manual or automated) a neutralizer during the fermentation process (Le., external pH control) or by using a specially prepared growth medium containing chemical buffers which solubilize as the pH decreases (i.e., internal pH control). Given the popularity of internal pH-controlled media for starter culture preparation, researchers at the University of Wisconsin investigated the ability of L. monocytogenes strains Scott A, V7, and CA to compete with S. lactis [303] and S. cremoris [304] in one commercially available starter culture medium having internal pH control. In both studies,

L. monocytogenes in Fermented Dairy Products

443

-

the starter culture medium was inoculated to contain 103 L. monocytogenes CFU/mL together with either a 0.25 or 1.0% inoculum of S. lactis or S. cremoris and inoculated at 21 or 30°C for 30 h. Growth of the pathogen was only partially inhibited by S. lactis and S. cremoris when compared with starter-free controls, with the greatest inhibition occurring at the higher inoculum level and higher temperature. However, Listeria populations of 10'- 106and 104-10' CFU/mL developed in samples fermented with S. lactis and S. cremoris, respectively, when these cultures were ready for use (pH 5.5) after 15 to 18 h of incubation. Since neither conventional bulk starter technology nor internal pH-controlled media will completely inhibit this pathogen, manufacturers of cheese and other fermented dairy products should not discount the starter culture as a possible source for Listeria but rather should adopt rigorous sanitation standards and Hazard Analysis Critical Control Point (HACCP) programs to minimize Listeria contamination and potential product loss. Ultrafiltered milk, a type of concentrated milk sometimes used for commercial manufacture of certain cheeses including mozzarella, ricotta, cottage, and Cheddar, also possesses a higher buffering capacity than that of unfiltered milk because of higher concentrations of proteins and insoluble salts. Consequently, listeriae may have greater opportunity to grow in ultrafiltered as opposed to unfiltered milk during fermentation. El-Gazzar et al. I1581 examined this question by inoculating samples of unfiltered skim milk as well as retentate (concentrated twofold and fivefold by volume) and permeate from unfiltered skim milk to contain 103-10' L. monocytogenes CFU/mL together with 107-108 L. lactis subsp. cremoris CFU/mL. In contrast to the aforementioned studies involving skim milk and internal pH-controlled bulk starter media, Listeria failed to grow in ultrafiltered milk containing starter culture, with populations remaining constant in unfiltered slum milk and decreasing up to 10- and 100-fold in 2-fold retentate and permeate, respectively, after 36 h of incubation at 30°C. Increased inactivation of L. monoc-ytogenes in the permeate as compared with retentate and unfiltered skim milk is again related to the lower buffering capacity of the permeate which results from ultrafiltration. When these samples were refrigerated at 4"C, L. monocytogenes persisted 4-6 weeks in slum milk (pH 4.2), 3-5 weeks in retentate (pH 4.6), and 1 week in permeate (pH 4.1). Thus, fermentation of ultrafiltered milk at 30°C will not guarantee complete inactivation of Listeria even after the finished product is moved to refrigerated storage.

Cultured Buttermilk Cultured buttermilk is essentially pasteurized skim milk that has undergone a 12- to 15hour fermentation at 20°C with S. cremoris or S. lactis (0.5% initial inoculum) and certain flavor-enhancing lactic acid bacteria such as Leuconostoc cremoris or L. dextranicum. After fermentation, the final product is packaged and refrigerated until consumed. As part of a follow-up study [270], all 15-h-old fermented milk samples from the aforementioned study by Schaack and Marth [269] were stored at 4°C and examined for L. monocytogenes. Survival of the pathogen ranged from an average of 5 weeks in skim milk fermented at 30°C with a 5.0% inoculum of S. cremoris to 12.5 weeks in skim milk fermented at 21°C with a 0.1% inoculum of S. cremoris (Table 11). Similarly, Listeria viability averaged 2.5- 13.0 weeks in skim milk previously fermented at 30 and 2 1"C with 5.0 and 0.1% S. lactis, respectively. Slower inactivation of the organism in skim milks fermented at 21 rather than 30°C may be related to the rate of acid production during fermentation, since pH values for fermented milks ranged between 4.3-6.0 and 4.2-4.6 immediately after 15 h of incubation at 2 1 and 3OoC,respectively. The fact that L. monocytogenes can survive 10.5 weeks in this refrigerated product (fermented 15 h at 21°C with

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TABLE 11 Weeks of Survival of L. monocytogenes in Skim Milks Fermented with S. cremoris or S. lactis at 21 and 30°C for 15 h and then Stored at 4°C S. cremo ris

S. lactis

Inoculum (%)

21°C

30°C

21°C

30°C

0.1 0.5 1.o 5.0

12.5a 10.5 8.0 9.0

6.5 6.0 4.5 5 .O

13.0 ND 8.5 6.0

2.0 ND 3.0 2.5

ND, Not determined. a Average of two trials. Source: Adapted from Ref. 270.

0.5% S. cremoris) emphasizes the importance of maintaining sanitary conditions during manufacture of cultured buttermilk. In addition to entering pasteurized skim milk as a postpasteurization contaminant, L. monocytogenes also can be introduced into cultured buttermilk as a postfermentation contaminant. Choi et al. [ 1331 studied the second of these scenarios. Samples of commercially produced, cultured buttermilk (pH 4.2 1) were inoculated separately with each of four strains to contain an average of 3.5 X 103L. monocytogenes CFU/mL and stored at 4°C. Under these conditions, the pathogen survived an average of 22.8 days (Table 12), with populations of three of four Listeria strains decreasing more than 100-fold during the first 8- 12 days of refrigerated storage. It is important to realize that although L. monocytogenes was inactivated faster when added directly to cultured buttermilk than when skim milk was fermented into a “buttermilk-like” product in the previous study [270], the pathogen still survived throughout the normal shelf life of the product. Furthermore, results from an earlier study [ 1821 demonstrating that E. coli and Enterubacter aerogenes

TABLE 12 Survival of L. monocytogenes in Inoculated Samples of Commercially Produced Buttermilk and Yogurt Stored at 4-5°C

Product

Initial

Final

Survival (dayS >

3.55

4.21

4.38

22.8

4.26 4.36

4.02 4.03

4.08 4.09

21.2 24.7

4.2 1 4.70 2.10 7.10

4.03 4.03 4.10 4.10

4.10 4.10 4.10 4.10

24.7 22.3 3 9

Buttermilk Plain yogurt Brand Da Brand Yb Vanilla-flavored yogurt Brand D Brand Y Plain low fat yogurt Plain low fat yogurt

‘’ Custard-style.

PH

L. monocytogenes inoculum (log,, CFU/mL)

Fluid-style. Source: Adapted from Refs. 133, 279.

L. monocytogenes in Fermented Dairy Products

445

are inactivated faster than L. monocytogenes in cultured buttermilk imply that coliformfree buttermilk may not necessarily be free of listeriae. These findings again emphasize the importance of good sanitation in producing Listeria-free buttermilk.

Cultured Cream Unlike cultured buttermilk, far less is known about the viability of L. monocytogenes in cultured cream. In the only study reported thus far, Stajner et al. [281] manufactured cultured cream from naturally contaminated raw cow’s milk containing approximately 5 X 1O5 L. monocytogenes CFU/mL. According to these Yugoslavian authors, viable listeriae were detected in the finished product throughout 7 days of storage at 3-5°C.

Thermophilic Starter Cu Itures Practically speaking, thermophilic fermentations used to produce yogurt and certain cheeses (e.g., Swiss, Parmesan, mozzarella, and Romano) are not normally continued beyond 4-6 h. The only two exceptions are in production of Bulgarian buttermilk and acidophilus milk, which require thermophilic fermentations of 10- 1 2 and 18-24 h, respectively. Therefore, primary emphasis will be placed on behavior of Listeria during the first 6 h of fermentation. In addition to determining the fate of L. monocytogenes in the presence of mesophilic starter cultures [269], Schaack and Marth [268] also investigated the ability of this organism to grow during fermentation of skim milk with thermophilic lactic acid bacteria. As in the previous study, samples of autoclaved skim milk were inoculated to contain -103 L. monocytogenes CFU/mL. After adding 0.1, 1 .O, or 5.0% (Streptococcus thermophilus, Lactobacillus bulgaricus, or a mixture of the two species, all samples were examined for numbers of listeriae during 15 h of incubation at 37 and 42°C. Limited growth of L. monocytogenes occurred in all samples, with the organism generally attaining maximum populations after 6 h of incubation at either temperature (Fig. 3). At this point, Listeria populations were generally I .O-1.5 orders of magnitude lower in fermented than in unfermented control samples, as was also reported by Lukasova, [212], indicating that growth of the pathogen was markedly suppressed by the thermophilic starter culture, particularly when used at inoculuni levels of 5%. In addition, greater inhibition of listeriae was consistently observed in rnilks fermented at 42 rather than 37°C. Listeria monocytogenes behaved similarly in milks fermented with S. thermophilus, L. bulgaricus, and a mixture of both starter cultures during the initial 6 h of incubation; however, viability of the pathogen in milks fermented beyond 6 h varied with the species of lactic acid bacterium used in the fermentation. Although populations of listeriae remained relatively unchanged in all milks fermented 6-15 h with S. thermophilus (final pH of 4.55-4.90), the pathogen frequently survived only 9-15 h in milks fermented with L. bulgaricus alone. Rapid inactivation of the pathogen coincided with pH values 1 4 . 0 which developed in milks fermented 9-15 h with L. bulgaricus. The combination of L. bulgaricus and S. thermophilus was more inhibitory to Listeria than was S. thermophilus alone but less inhibitory than was L. bulgaricus alone. Although populations of listeriae failed to decrease in milks fermented at 37°C with the mixed starter culture, some inactivation was noted in all corresponding samples incubated at 42°C. As was true when L. bulgaricus was used alone, inactivation of listeriae by the mixed starter culture again was most pronounced in samples having pH values 54.0. Following 15 h of incubation, all fermented milks in this study were stored at 4°C

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446

-E

1.75

h

5 LL

1.50

U

2

1.25

c! c

.g

-

1.0

*I 1

a a

0.75

3

0.50

0

B;:[

.”

to 0)

2

0.25

...

6 0

... ... ...

37oc

37°C

42°C

42°C

L. bulrraricu$

S, thermophilug

37°C

42OC

L. bulearicus arid -

S. IhermoohiluS

FIGURE3 Changes in populations of L. monocytogenes in skim milk following a 6-

hour fermentation at 37 and 42°C with 0.1, 1.0, and 5.0% (v/v) added Lactobacillus bulgaricus, Streptococcus thermopilus, or L. bulgaricus + S. thermopilus. (Adapted from Ref. 268.)

and monitored for listeriae by Schaack and Marth [270]. Using S. thermophilus alone, L. monocytogenes survived 21-32 and 5-15 weeks in milks fermented at 37 and 42OC, respectively (Table 13). Failure of these milks to attain pH values 14.0 after fermentation with S. thermophilus helps explain the unusually long survival of listeriae. As expected, L. bulgaricus was most detrimental to listeriae with the pathogen surviving beyond 15 h only in samples fermented with the lowest inoculum. Using a 0.1 % L. bulgaricus inoculum the pathogen was eliminated from milks fermented at 37 and 42°C following 7 and 3 days

TABLE 13 Weeks of Survivalaof L. monocytogenes

in Skim Milks Fermented with S. thermophilus or S. thermophilus + L. bulgaricus (LEST) at 37 and 42°C for 15 h and then Stored at 4°C

LBST

S. thermophilus Inoculum (%)

37°C

42°C

37°C

42°C

0.1 1.o 5 .O

28.5 32.0 21.0

15.0 8.5 5 .O

7.5 1.5 1.o

1.5 12 h 15 h

Average of two trials. Source: Adapted from Ref. 268. a

L. monocytogenes in Fermented Dairy Products

447

of refrigerated storage, respectively. Milks cultured with a combination of S. thermophilus and L. bulgaricus yielded results that were between both extremes observed when the two starter cultures were used separately (Table 13). Once again, L. monocytogenes survived longer in refrigerated milks fermented at 37 than 42"C, with slower inactivation in the former being attributed to slightly higher pH values. These findings are similar to those of Zuniga-Estrada et al. [309], who found that Listeria persisted for only 8 h when milk containing 1O3 L. monocytogenes and 1O6 S. salivarius subsp. thermophilusll. delbrueckii subsp. bulgaricus CFU/mL was fermented at 42°C to pH 4.4.

Yogurt As previously mentioned, L. monocytogenes can enter yogurt either before the fermentation as a milk contaminant or afterward as a contaminant of the finished product. Schaack and Marth [268,270] examined the behavior of Listeria under the first of these conditions by inoculating yogurt mix to contain -103-104 L. monocytogenes (strains V7, OH, Scott A, or CA) CFU/mL and 2% of a commercial starter culture containing S. thermophilus, L. bulgaricus, and Lactobacillus acidophilus. Yogurt mix was fermented at 45°C for 5 h and then stored at 4°C. Populations of all four Listeria strains increased an average of 2.5- to 10.0-fold in yogurt mix during the fermentation. After finished yogurt at pH 4.75 was refrigerated at 4"C, numbers of listeriae decreased, with strains V7, OH, Scott A, and CA surviving 7-12, 7-12,4-12, and 1-5 days, respectively. The pH of yogurts in which listeriae were last detected ranged from 3.88 to 4.1 1. However, several later studies have shown that Listeria survival is more variable than previously observed, with L. monocytogenes persisting 2-7 days [221], 4 days [4], 3-5 days, 18-24 days [203], and 14-25 days [251] in yogurts of pH 4.2, 4.0-4.3, 4.1-4.7, 3.8-3.9, and 4.5-5.0, respectively, during refrigerated storage, with growth of the pathogen also general1y not being observed during fermentation. Furthermore, in one French study [ 1401 involving highly acidic yogurt (pH 3.5) prepared from yogurt mix inoculated to contain 102-107 L. monocytogenes CFW/ mL, the pathogen was eliminated after only 1-2 days of storage at 4°C. Although acid development and differences in acid tolerance/injury during manufacture [ 1721 play major roles in determining the fate of listeriae in yogurt, other factors including various starter culture metabolites and antilisterial compounds (bacteriocins) produced by certain strains of L. acidophilus [248] also may contribute to the rapid demise of Listeria in yogurt, provided this lactic acid bacterium is in the product, with addition of 100 mgl-' lysozyme to yogurt mix before fermentation also reportedly decreasing Listeria survival in the finished product [251]. Although nearly all yogurt in the United States is now prepared from cow's milk, this product is sometimes manufactured from ewe's and/or goat's milk, particularly in Europe and the Middle East. In a 1964 Bulgarian study [ 1951,L. monocytogenes viability in yogurt prepared from naturally contaminated ewe's milk was markedly influenced by the storage temperature, with the pathogen surviving <24-48 h and >6 days in yogurt held at 18-22 and 10°C, respectively. Faster demise of listeriae at the higher rather than lower storage temperature was attributed to increased acid production by S. thermophilus and L. bulgaricus. When Abdel-Gawad et al. [personal communication] recently prepared yogurt from cow's, ewe's and goat's milk inoculated to contain 103-104L. monocytogenes CFU/ml, 97-99.9% of the population was sublethally injured within 24 h of manufacture with no cells repairing and/or surviving in any yogurt samples (pH 4.0N4.3) beyond 5 days of cold storage. Many dairy industry personnel believe that contamination of yogurt is more likely

448

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to occur after rather than before fermentation. Choi et al. [ 1331 simulated postfermentation contamination of yogurt by inoculating two commercial brands of plain and vanilla-flavored custard-and fluid-style yogurt to contain 104- 1O5 L. monocytogenes CFU/mL. The pathogen survived an average of 2 1.2-24.7 days in yogurt held at 4°C (Table 12), with most listeriae being inactivated during the first 8-12 days of refrigerated storage. Khattab et al. [203] reported similar findings with listeriae persisting 18-24 days in experimentally produced Egyptian yogurt (pH 3.8-3.9) inoculated to contain 106L. monocytogenes CFU/mL. After inoculating various formulations of experimentally produced yogurt to contain 106L. monocytogenes CFU/mL, Griffith and Deibel [ I891 also detected the pathogen in yogurt samples having a pH value of 4.3 following 28 days of storage at 4°C. Although the type of yogurt had no apparent effect on Listeria survival in these studies, the point at which yogurt became contaminated greatly influenced survival of listeriae, with Schaack and Marth [270] showing that the pathogen survived only 1- 12 days when added to yogurt mix before fermentation. Siragusa and Johnson [279] conducted a similar study in which three different brands of commercial, unflavored, low-fat yogurt were inoculated to contain approximately 1O2 and 107L. monocytogenes CFU/g and then stored at 5°C. Listeriae survived <3 days in yogurt inoculated with low levels of the pathogen even though pH values of yogurt were similar to those in the study by Choi et al. [133] (Table 12). Using the high inoculum, viable listeriae were found for only 9 days, with populations decreasing approximately 100-fold each after 3 and 6 days of refrigerated storage. Ayre et al. [ 1061 reported similar results using an L. monocytogenes inoculum level of 107CFU/g. In contrast, Ribeiro and Carminati [251] examined the effect of yogurt pH on Listeria survival by inoculating experimentally produced (pH 4.5), commercial fruit (pH 4.05) and plain yogurt (pH 3.76) to contain -104 L. monocytogenes CFU/mL. Overall, the pathogen survived 2-3 days, 3-4 days, and 12-14 days in refrigerated yogurts having pH values of 3.76, 4.05 and 4.5, respectively, thereby verifying the impact of pH on Listeria survival. However, Griffith and Deibel [ 1891 reported that L. monocytogenes populations decreased approximately four orders of magnitude in artificially acidified (pH 4.2) rather than fermented yogurt during the first 6 days of storage at 4°C. Hence, decreased tenacity of L. monocytogenes in inoculated yogurt samples in these studies as compared with the work by Choi et al. [ 1331 again demonstrates that factors other than pH also are contributing to the demise of Listeria. Many yogurts and cultured buttermilks marketed today have pH values of approximately 4.0 and 4.3, respectively. Since large populations of L. monocytogenes were inactivated faster in yogurt than in buttermilk during refrigerated storage, one would expect a lower incidence of listeriae in commercial yogurt. Evidence from FDA surveys discussed earlier in this chapter supports this view, since thus far the pathogen has been detected in commercial buttermilk but not yogurt, with 100 retail and farm-produced samples also negative for L. monocytogenes in the United Kingdom [202]. However, as was true for buttermilk, E. coli and Enterobacter aerogenes are also inactivated faster than L. monocytogenes in yogurt during refrigerated storage [ 1821. Hence, coliform-free yogurt may not necessarily be free of Listeria. This is important to remember when results of coliform tests on these products are interpreted. It is evident from this discussion that good sanitation practices are of utmost importance in producing Listeria-free yogurt, buttermilk, and other fermented milk products. Since yogurt is occasionally used as an ingredient in other foods, Sikes [277] investigated the fate of L. monocytogenes in low-moisture (1.9% water), medium-acid (pH 4.9)

-

-

-

L. monocytogenes in Fermented Dairy Products

449

yogurt-based dairy bars supplied to the U.S. military. These bars, which contained -34% heavy cream, 27.5% yogurt, 27.5% cream cheese, and I I % of other ingredients (i.e., sugar, sunflower oil, whey), were inoculated to contain approximately 1 X 10' L. monocytogenes strain Scott A CFU/g and then periodically examined for numbers of listeriae during extended storage at 25°C. Results indicated that Listeria populations decreased only approximately 100-fold after 40 days of incubation, thus demonstrating the ability of this organism to persist in low-moisture, medium-acid foods. As will soon be discussed, similar behavior has been reported for L. monocytogenes in semihard cheeses, such as Cheddar, Colby, and Gouda, which also have pH values near 5.0.

Traditional Fermented Milk Products In certain African and Middle Eastern countries, traditionally fermented ethnic milk products such as ergo (Ethiopia) and labneh (United Arab Emirates) comprise a significant portion of the daily diet. Although produced from commercially pasteurized milk under generally adequate hygienic conditions, these products are also frequently prepared at home (particularly in rural areas) from raw milk which is allowed to undergo a natural souring (i.e., fermentation) at ambient temperature without the addition of starter cultures. Hence, the likelihood for the presence of Listeria and other bacterial pathogens in raw milk combined with an often questionable fermentation and a short product shelf life makes the microbiological safety of such home-fermented milks particularly suspect. Working in Zimbabwe, Dalu and Feresu [ 1421 examined the fate of L. monocytogenes in commercially fermented milk prepared from both raw and pasteurized milk. Commercial fermented milk was prepared from pasteurized milk which was inoculated to contain 104L. monocytogenes CFU/mL together with an active mesophilic starter culture and then packaged and fermented 24 h at ambient temperature, whereas both traditionally fermented milks were prepared by allowing raw milk to sour naturally in earthenware pots during 24 h of incubation at ambient temperature. Overall, Listeria populations increased 5 10-fold in all three milks during fermentation, with the final product attaining a pH of 4.3--4.6. After 5 days of storage at 4 and 20°C, numbers of listeriae generally decreased about 10- and 100-fold, respectively. Consequently, survival of L. monocytogenes beyond the normal 3-7 day shelf life of these traditionally fermented milks poses a legitimate public health concern. In 1993, Ashenafi [ 1041 prepared ergo, a traditional Ethiopian fermented milk, from boiled milk that was inoculated to contain 103L. monocytogenes CFU/mL along with Lactobacillus and Streptococcus starter organisms from a previous batch of product. After the first 12 h of a 24-h fermentation at 25OC, numbers of listeriae generally increased 10to 100-fold i n ergo (pH 4 . 5 - 5 3 , with the highest populations being observed in samples fermented in unsmoked as compared with olive wood-smoked glass containers. During the next 12 h, the pathogen was slowly inactivated as the pH of the product decreased to 1 4 . 0 . Continued ambient storage led to complete demise of Listeria in ergo fermented in wood-smoked and unsmoked containers within 36-48 and 48-60 h, respectively. Consequently, traditional preparation of ergo in rural areas using wood-smoked fermenting vessels appears to be beneficial in minimizing Listeria survival in this product which is typically prepared from raw milk and consumed immediately after the 24-h fermentation period. Labneh, a Middle Eastern yogurt-like product, is produced using a normal yogurt starter culture of L. delbreuckii subsp. bulgaricuslS. salivarius subsp. thermophilus. Fol-

450

Ryser

lowing approximately 5 h of incubation at 42"C, the product is salted (1% NaCl), poured into muslin bags, and hung in a cooler for 48 h. Gentle mixing to obtain a smooth consistency follows, after which the finished product (pH 3.8) is packaged for sale. These manufacturing steps which afford many opportunities for contamination prompted Gohil et al. [ 1851 to assess the fate of L. rnonocytogenes in labneh as a postfermentation contaminant. When commercially produced labneh was inoculated to contain l O4 L. rnonocytogenes CFU/g and stored at 4"C, the pathogen survived 2 and 7 days in product of pH 3.8 and 4.5, respectively, with the addition of 1% NaCl not appreciably altering Listeria survival. Raising the holding temperature to 10, 20, or 30°C led to more rapid demise of listeriae, with samples generally being free of L. rnonocytogenes after 2-3 days of storage.

BEHAVIOR OF L. MONOCYTOG€N€S IN CHEESE Considerable work has been done at the University of Wisconsin-Madison and elsewhere to define the behavior of L. rnonocytogenes during manufacture and ripening of various types of cheese. Most of these studies describe what might happen if cheese is prepared from contaminated milk. Since North American and European surveys have indicated that soft/semisoft cheeses ripened with mold or bacteria are most frequently contaminated with L. rnonocytogenes, research dealing with such varieties will be discussed first, followed by data on ripened cheeses of progressively lower moisture content, goat's milk cheese, unripened cheese, whey cheese, and cold-pack cheese food. Whereas this chapter concludes with a discussion of L. rnonocytogenes behavior in whey and brine solutions, we will begin this section by first examining the viability of listeriae in coagulants, coloring agents, and starter distillates, all three of which are commonly used in cheesemaking.

Coagulants To produce cheese curd, milk must first be coagulated or clotted, which can be done either by acidification or addition of a coagulating enzyme. In the first method, an active lactic starter culture is used to lower the pH of the milk to 4.6-4.7 (isoelectric point of casein), at which point the casein micelles in the milk precipitate and form a coagulum. Alternatively, coagulation is occasionally accomplished by adding food-grade acids directly to milk. Coagulation of milk by either means of acidification is primarily confined to the manufacture of cottage cheese and a few ethnic varieties of fresh cheese. The second method, in which a coagulating enzyme is added to destabilize the casein micelles and clot milk at a near-neutral pH, is used to manufacture virtually all other types of cheese. Coagulants presently used in cheesemaking include calf rennet extract, chymosin, bovine pepsin-rennet extract, and microbial rennet. Traditionally, calf rennet is extracted from the lining of the abomasum (fourth stomach) of suckling calves and contains two enzymes-pepsin and chymosin (the latter is most important for coagulation of milk). Shortage of calf rennet following World War I1 led to the use of bovine pepsinrennet, an extract obtained from the abomasum of somewhat older calves, that can be substituted for calf rennet. Increased production costs of both calf rennet and bovine pepsin-rennet have in turn prompted development of several rennets of microbial origin. Thus far enzyme preparations obtained from molds belonging to the genus Mucor (particularly M. rniehei) have proven to be the most satisfactory substitutes for animal rennet. Since two of four coagulants used in cheesemaking are of animal origin, and since these animals sometimes carry L. rnonocytogenes, this pathogen might occasionally appear

L. monocytogenes in Fermented Dairy Products

451

in both crude enzyme preparations and finished coagulant at the time of shipping. Although microbial rennet should be free of Listeria spp. when manufactured, the presence of listeriae within the rennet manufacturing facility or the cheese factory environment could contaminate any of these products if mishandled. Given the recovery of an apparent sodium benzoate-resistant strain of L. monocytogenes from commercially produced calf rennet extract [ 1571, the possible presence of L. monocytogenes in coagulants should be of concern to cheesemakers, with the International Dairy Federation also contemplating the addition of rennet to its list of cheesemaking ingredients to be examined for Listeria spp. [288]. During 1988 and 1989, El-Gazzar and Marth published results from three studies examining the viability of listeriae in calf [ 1531, bovine pepsin [ 1541, and microbial rennet [ 1561. In each of these studies, commercially produced, Listeria-free rennet was inoculated to contain approximately l 03,l 04,l 05,or 1O6 L. monocytogenes CFU/mL and analyzed for listeriae during 56-70 days of storage at 7°C using both direct plating and cold enrichment. All samples of calf and bovine pepsin-rennet inoculated with the two lowest levels of Listeria were free of the pathogen after 14-28 days of storage at 7°C (Table 14). Even though 42-56 days of storage were required to eliminate the pathogen from samples containing initial inocula of approximately 1O5 and 1O6 L. rnonocytogenes CFU/mL, the reader is reminded that all four inoculum levels used in these studies were many times greater than levels that might occur naturally in commercially produced coagulants. Hence, barring contamination in the cheese factory, these findings suggest that calf and bovine pepsin-rennet are normally held long enough in distribution channels to ensure cheesemakers that both coagulants are Listeria-free. Inactivation of L. monocytogenes in calf rennet and pepsin-rennet probably results from the combined effects of 5% propylene glycol, 2% sodium propionate, 0.1% (or more) sodium benzoate, 14-21 % salt, and a relatively low pH of 5.6. Results from several studies assessing the viability of L. monocytogenes in the presence of benzoic acid and sodium propionate are discussed in Chapter 6. Unlike calf and bovine pepsin-rennet, more than 70 days of storage were required to eliminate L. monocytogenes at even the lowest inoculuni level from microbial rennet (see Table 14). Enhanced survival of listeriae in microbial rennet may be related to the nature of the coagulant itself as well as the presence of fewer preservatives. Although L. monocytogenes is unlikely to enter microbial rennet during manufacture, the relatively high incidence of listeriae in cheese factories may lead to inadvertent contamination of the coagulant during cheesemaking. Considering the tenacity of L. monocytogenes in microbial rennet and the long shelf life of this product, it may be prudent for cheesemakers periodically to verify that the microbial rennet they are using is indeed Listeria-free.

Coloring Agents and Starter Distillates Depending on local preference, various yellow-orange colorants such as annatto (an extract from annatto seed [Bixia orellana]) and turmeric (an extract from the turmeric root [Curcuma longa]) can be added to milk at the beginning of cheesemaking, with annatto most commonly being used in the manufacture of Cheddar, Colby, Muenster, and brick cheese. Although freshly prepared colorants are unlikely to contain microbial pathogens, inadvertent exposure of these coloring agents to L. monocytogenes in the cheese factory could lead to production of a contaminated product. Thus, in addition to the aforementioned coagulants, El-Gazzar and Marth [ 1551 also investigated the fate of listeriae in five commercially available annatto/turmeric extracts that were inoculated to contain approxi-

Ryser

452

TABLE 14 Survival of L. monocytogenes Strain CA in Three Milk Coagulants Stored a t 7°C ~~

~

~

Number/mL after days of storage

0

Product Calf rennet extract

Bovine pepsin-rennet extract

Microbial rennet

a

2.5 1.1 3.0 2.0 9.5 2.0 7.5 1.0 6.0 7.0 2.0 1.0

X

7 103

x 10'

10' 106 X 103 x 10' X X

x 105 x 106 x 103 X X

103 10'

x 106

(+) Positive result by cold enrichment. ( - ) Not detected after 6 weeks of cold enrichment.

Source: Adapted from Refs. 153, 154, 156.

1.5 X 3.5 x 6.0 X 6.0 X

1.0 x 2.0 x 1.5 x

1.7 1.7

X X

1.5 x

102( + ) a 102(+) 104(+) 104(+) 10 30 103 104 103 103 10' 105

14 <10 ( - ) b <10 (-)

1.2 x 103 (+I 4.5 x 107(+) <10 (-) <10 (-) 1.0 x 10' 1.0 x to2 7.6 X 10' 1.0 x 103 2.3 X 10' 4.0 X 10'

28

42

<10 (-) <10 (-) 30 (+I 2.0 x 102(+) <10 (-)

<10 (-)

<10 (-)

<10 (+) <10 (+) 2.8 X 10' 2.2 x 102 2.5 X 10' 3.6 X 10'

56

70

-

-

<10 (-) <10 (-) <10 (-> <10 (-) 1.2 x 10' 1.4 X 102 1.6 X 10' 7.0 X 10'

-

<10 (-) <10 (-)

<10 (-) <10 (-) <10 (-) <10 (+) <10 (+) 3.3 x 102 4.4 x 10' 2.3 X 10' 9.3 x 10'

-

-

8.5 9.0

40 90 X X

103 10'

L. monocytogenes in Fermented Dairy Products

453

mately 103--1O7 L. monocytogenes strain CA/mL and stored at 22°C. Regardless of the initial inoculum level, populations of listeriae immediately decreased 2 4 orders of magnitude in all colorants, with the pathogen being completely inactivated immediately after addition to three of five extracts. The almost instantaneous death of Listeria in these three colorants was attributed to the presence of propylene glycol and a pH of 13.3 (one extract). Although Listeria populations of 5800 CFU/mL were observed in the two remaining colorants immediately after inoculation, with the highest level of listeriae, these samples were free of the pathogen following 7 days of ambient storage. Overall, these findings indicate that the length of time that these colorants spend at ambient temperatures during distribution and before use at the cheese factory is more than adequate to inactivate small numbers of listeriae that might enter as chance contaminants. Small levels of starter distillates, that is, mixtures of natural flavor compounds such as diacetyl obtained by distilling specially cultured milks, are frequently used to enhance the flavor of cottage and processed cheese as well as ice cream, margarine, butter, yogurt, snack foods, and certain types of candy. Hence, in connection with the study just described, El-Gazzar and Marth [ 1551 also examined the fate of listeriae in a commercially available starter distillate that was inoculated to contain 102- I O6 L. monocytogenes strain CA, Scott A, or V7 CFU/mL and held at 7°C. Overall, strain CA decreased to nondetectable levels in all samples after 2-7 days of storage depending on the initial inoculuni, whereas 728 days of incubation were required to eliminate strains Scott A and V7 from similar samples. Therefore, barring inadvertent contamination in the cheese factory, the time involved in shipping and distributing these starter distillates should be more than sufficient to eliminate any inadvertent Listeria contaminants.

Mold-Ripened Cheeses Mold-ripened cheeses can be divided into two categories: (a) white mold cheeses, which are surface-ripened by either Penicillium camemberti, Penicillium caseicolum, or Penicillium candidum (i.e., Brie and Camembert), and (b) blue-mold or blue-veined cheeses in which ripening results from growth of Penicillium roqueforti or P. glaucum throughout the cheese (i.e., Roquefort, blue, Gorgonzola). The relatively high moisture content of these surface-ripened cheeses, along with a nearly neutral pH in fully ripened cheese, allows rapid growth of L. monocytogenes as well as other foodborne pathogens that would normally be inhibited in more acidic cheeses. Since mold-ripened cheeses also are highly susceptible to surface contamination during ripening, it is not surprising that Brie and Camembert were among the first varieties of cheese in which L. monocytogenes was detected and the behavior of the organism studied.

Camembert Cheese Pasteurized milk was inoculated to contain approximately 500 L. monocytogenes strains Scott A, V7, CA, or OH CFU/mL and manufactured into Camembert cheese by Ryser and Marth [ 2601. Following 10 days of storage at I5"C/95% relative humidity (RH) to permit proper growth of P. camemberti on the cheese surface, all cheeses were wrapped in foil and ripened at 6°C. Wedge (pie-shaped), surface, and interior samples of cheese were diluted in Tryptose Broth and analyzed for listeriae at appropriate intervals using both direct plating and cold enrichment. Populations of L. monocytogenes increased 5- to 10-fold during the first 24 h after manufacture; however, this increase probably did not result from growth of the organism

-

454

Ryser

during cheesemaking. Numerous studies have shown that bacterial populations typically increase 5- to 10-fold during curd formation as a direct result of entrapment of organisms in curd particles, with the exact level of increase dependent on the moisture content of the cheese. In all likelihood, L. monocytogenes was similarly concentrated during formation of Camembert cheese curd. Entrapment of L. monocytogenes in the curd is further supported by the fact that, in this study, only 1.3% of the original Listeria inoculum in the milk was lost in the whey. Yousef et al. [308] later demonstrated that the failure to observe L. monocytogenes population increases of approximately 5- to 10-fold after formation of Camembert as well as Cheddar and cottage cheese curd was probably related to the method of sample preparation. Their improved procedure in which curd samples were homogenized in warm (45°C) Tryptose Broth containing 2% trisodium citrate was subsequently used to examine behavior of L. monocytogenes during manufacture and storage of brick [264], Colby [306], feta [238], blue [237], and Parmesan cheese [307]. During the initial 17 days of cheese ripening, the first 10 days of which occurred at 15"C, populations of three of four L. monocytogenes strains decreased 10- to > 1000fold, with lowest numbers generally being observed in surface samples (Fig. 4). On further ripening at 6"C, all four Listeria strains grew (particularly between 25 and 30 days of storage) and attained maximum populations of 106- 108CFU/g in wedge and surface samples from fully ripened cheese; however, maximum listeriae populations were generally

-

8.0

8.0

s!

3

/

.-c

E

;3 35.0

"

0

"5

10

15

20

75

30

35

40

ULLld 45

50

55

4

60

.

65

5

Days

FIGURE4 Behavior of L. monocytogenes strain CA (solid symbols) and pH (open symbols) during ripening of Camembert cheese. Solid symbols at <1.0 log,, strain CA/g indicate results for cold enrichment. Numbers indicate the week at which strain CA was found, whereas an "x" signifies that the pathogen was not detected after 8 weeks of cold enrichment. (Adapted from Ref. 260.)

L. monocytogenes in Fermented Dairy Products

455

10- to 100-fold lower in interior samples from the same cheeses. Although growth of L. monocytogenes clearly paralleled the increase in pH of the cheese during ripening, with growth usually commencing after the cheese attained a pH value of 5.75-6.25, decreased viability of three of four Listeria strains in surface samples having pH values of 6.256.50 suggests that factors other than pH, including the presence of potentially inhibitory surface bacteria and yeasts, may also be involved in controlling growth of this pathogen in Camembert cheese. Results from a subsequent study by Ryser and Marth [ 2621 showed greater growth of L. monoc-ytogenesin filter-sterilized Camembert cheese whey previously cultured with P. camemberti than in uncultured whey adjusted to pH values of 5.60-6.80 and thus suggest that P. camemberti is not involved in reducing Listeria populations on the surface of Camembert cheese. In support of these findings, Geisen et al. [ 1731 also failed to observe any antilisterial activity among several strains of P. camemberti that were tested against L. monocytogenes in vitro. However, in the work by Ryser and Marth [260], possible antilisterial activity from yeasts and non-lactic acid bacteria (i.e., micrococci, coryneforms) that are naturally present on the cheese surface during initial stages of ripening was not precluded. To simulate contamination of cheese in the ripening room, Ryser and Marth [260] also inoculated surfaces of 10-day-old wheels of Listeria-free Camembert cheese to contain 2-40 L. monocytogenes (four strains tested separately) CFU/20 cm2.All cheeses were then ripened at 6°C for 60 days, during which time 10-g surface samples were analyzed for listeriae. Three of four L. monocytogenes strains grew on the surface of the cheese and attained maximum populations of 10'- 105CFU/g (Fig. 5 ) . Although the remaining Listeria strain failed to grow on the cheese surface after 60 days of storage, the pathogen was routinely detected throughout the ripening period using cold enrichment. These findings, along with the unfortunate recall of over 300,000 tons of French Brie cheese, stress the importance of manufacturing surface-ripened soft cheeses from high-quality, Listeriafree milk and observing good sanitary practices in the ripening room. Nonetheless, even under ideal manufacturing and ripening conditions, such cheeses may still inadvertently become contaminated with listeriae. Since Vacherin Mont d'Or and Brie de Meaux (a raw milk cheese) were directly involved in two major outbreaks of listeriosis in Europe (see Chap. lO), scientists in both Europe and North America have been exploring various means of eliminating this pathogen from such cheeses that are surface ripened with mold and/or bacteria. Not surprisingly, Banks [ 1 101 reported that L. monocytogenes grew rapidly in Camembert cheese prepared from raw milk, reaching a population of 106 CFU/g in fully ripened cheeses. Although heat treating this Listeria-contaminated milk at subpasteurization temperatures (i.e., 62.8 or 65.6"C) led to markedly lower Listeria populations in the cheese immediately after manufacture, the pathogen was not completely inactivated, with some growth being reported during 7 weeks of cheese ripening. In another report [ 1451, addition of lactoperoxidase system components to the surface of soft bacterial smear-ripened French cheese containing 102--1 O6 L. monocytogenes CFU/g led to complete inactivation of the pathogen following 4 days of storage at 15°C. In 1989, Hughey et al. [ 1931 showed that lysozyme was only bacteriostatic to L. monocytogenes in Camembert cheese. Incorporating 2- 10% carrot juice into homogenized Brie cheese was also effective in minimizing growth of L. monocytogenes in refrigerated samples [ 1 171. Although several additional reports also attest to the usefulness of x-ray [ 12I] and gamma irradiation [ 1621 in eliminating high

-

Ryser

456

./

/

Days

FIGURE5 Growth and survival of L. rnonocytogenes on the surface of Camembert cheese. Half-solid and solid symbols at < I log,o Listeria/g indicate that the organism was detected in one of t w o or two of t w o samples, respectively, using cold enrichment. Numbers indicate the week at which L. rnonocytogenes was found. (Adapted from Ref. 260.) populations of L. monocytogenes from Camembert and other soft surface-ripened cheeses, such treatments do not appear to be very practical, since ripening of the cheese will be adversely affected. Although initial attempts by Asperger et al. [ 1051 failed, results from several subsequent studies yielded more promising findings concerning the use of nisin and nisin-producing starter cultures to eliminate chance Listeria contaminants from soft surface-ripened cheese. When Maisner-Patin et al. [216] used a nisin-producing strain of L. lactis subsp. lactis to manufacture Camembert cheese from milk inoculated to contain 101, 103,or 105 L. monocytogenes strain V7 CFU/mL, numbers of listeriae decreased dramatically 6-9 h into cheesemaking because of the presence of -700 IU nisin/g of curd. Richard [252] also reported that inactivation of L. monocytogenes can be further enhanced by directly adding as little as 25 IU nisin/mL to the milk at the start of cheesemaking. Inhibition of Listeria continued during the first 2 weeks of ripening, however, regrowth of survivors was then reported in the presence of 250-300 IU nisin/g, first on the cheese surface and later in the interior, with the rate and extent of regrowth again paralleling the increase in cheese pH during ripening. Addition of other inhibitory organisms, including Enterococcus faecalis and Luctobacillus paracasei, to milk failed to arrest L. monocytogenes growth in Camembert cheese during extended ripening. However, a difference of 2.4 log CFU/g between numbers of L. monocytogenes in cheeses made with and without nisin-producing starter cultures was maintained throughout 6 weeks of ripening.

L. monocytogenes in Fermented Dairy Products

457

Nisin was most effective when the milk for cheesemaking contained 10' L. monocytogenes CFU/mL, with the pathogen being absent in 25-g samples even after 6 weeks of ripening. These findings agree with those of Sulzer and Busse [285], who used a different nisinproducing strain of L. lactis subsp. lactis. Although the nisin system has limits to preventing regrowth of Listeria during cheese ripening, use of nisin-producing starter cultures appears to be a relatively simple and effective means of minimizing Listeria growth during Camembert cheese manufacture provided that the cheese milk is of good hygienic quality and contains < 103L. monocytogenes CFU/mL. At least five additional studies have addressed the fate of L. rnonocytogenes during manufacture and ripening of Camembert cheese [ 108,225,282,284,2891.Regardless of the method of inoculation (i.e., milk, surface, brine), the basic conclusions from these reports were similar to those reached by Ryser and Marth [260] years earlier in that (a) L. monocytogenes failed to grow during cheesemaking, (b) low numbers of listeriae were recovered during the period of rapid growth of Penicillium candidum on the cheese surface, (c) populations of listeriae in cheese increased rapidly after 21 to 28 days of ripening, and (d) maximum Listeria populations of >106 CFU/g were detected in surface slices from fully ripened cheese. However, Terplan et al. [289] found that interior samples from 4to 56-day-old Camembert cheeses ripened at 5°C consistently contained < 100 L. monocytogenes CFU/g and never attained pH values >5.6 even after 56 days of ripening. Hence, under these conditions, growth of the pathogen was probably suppressed or severely retarded. However, some cells may have been sublethally injured during continuous exposure to this acidic environment, which in turn would have probably decreased the number of Listeria colonies observed on selective plating media. Two of these studies also addressed the influence of cheese ripening temperature on Listeria growth [ 108,2841. As expected, growth of L. monocytogenes was enhanced as the cheese storage temperature was increased from 3 to 15°C in response to more rapid ripening of the cheese and a concomitant increase in pH. Current evidence suggests that this pathogen behaves similarly in naturally contaminated, conimercially produced soft and semisoft mold-ripened cheese. While conducting a survey of soft/semisoft cheese sold in Canada, Farber et al. [ 1661 discovered eight 4month-old French cheeses, presumably of the BrieKamembert variety, that contained 104-105L. monocytogenes CFU/g. Following 1 year of continuous storage at 4"C, Listeria populations remained constant in one cheese and decreased only 10- to 100-fold in the seven remaining cheeses. In view of these results, it is easy to understand why this pathogen has been most frequently detected in soft/sernisoft cheeses that have been surface ripened by molds.

-

Blue Cheese Blue-veined cheeses such as blue, Roquefort, and Gorgonzola that are ripened internally and sometimes externally with P. roqueforti or P. glaucum also have been examined for their ability to support growth and survival of listeriae. Papageorgiou and Marth [237] used the modified Iowa method to manufacture blue cheese from pasteurized milk inoculated to contain approximately 1000 L. rnonocytogenes (strains Scott A or CA) CFU/mL. All cheeses were ripened 84 days at 9-12°C (90-98% RH) and then held an additional 36 days at 4°C. Numbers of listeriae increased by an average of 1.50 log,, CFU/g during the first 24 h of manufacture, with increases of 0.62 and 0.71 log,, CFU/g being attributed to entrapment of the organism within curd particles and growth, respectively. Growth of L.

458

Ryser

monocytogenes occurred primarily during the first 9 h of manufacture and ceased when the pH of the cheese dropped below 5.0. As expected, somewhat less growth occurred in two lots of cheese with particularly rapid acid production. Unlike the behavior of L. monocytogenes in Camembert cheese, the pathogen not only failed to grow during ripening of blue cheese, but decreased in numbers by two to nearly three orders of magnitude during the first 56 days of storage at 5°C (Fig. 6). These decreases, which occurred despite favorable pH values that developed during ripening from growth of P. roqueforti, were most likely caused by formation of free fatty acids [27 11, with listeriostatic/listeriocidal levels of caproic, caprylic [204], lauric, and other medium-chain fatty acids [205] produced as by-products of P. roqueforti growth during ripening of blue-veined cheeses. However, at least eight different P. roqueforti strains can also produce listeriocins in laboratory media [ 1731. Nonetheless, combined effects of a relatively high pH and low storage temperature were probably responsible for both Listeria strains surviving at least 120 days in all lots of blue cheese. Although additional tests

f

0

+T r i a l

I

__t_ Trial 2 ._f_ l r i a l 3

20

40

60

80

100

120

140

Days 7

6

?i

-*

5

Trial 1 Trial 2 Trial 3

4 0

20

40

60

80

100

120

140

Days

FIGURE6 Changes in population of L. monocytogenes strain Scott A and pH during ripening of blue cheese. (Adapted from Ref. 237.)

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showed that strain Scott A was evenly distributed throughout blocks of 120-day-old blue cheese, strain CA was far less tolerant to environmental conditions on the cheese surface and was detected in such samples only after cold enrichment. The lengthy survival of L. rnonocytogeizes in blue cheese, coupled with the recall of Danish blue cheese and isolation of L. rnonocytogenes from Italian Gorgonzola cheese, all stress the importance of preparing blue-mold cheeses from properly pasteurized milk under good hygienic conditions to prevent a possible public health problem involving Listeria.

Bacterial Surface-Ripened Cheeses This group of cheeses consists of soft and semisoft varieties that are ripened under conditions which induce a progression of microbial growth on the cheese surface. Examples of such cheeses include brick from the United States, Pont 1'EvEque and Saint Paulin from France, Tilsiter from Germany, Trappist from Yugaslavia, Havarti from Denmark, Be1 Paese from Italy, and Limburger from Belgium. Differences between these varieties result from the shape of the cheese as well as the amount and/or type of surface growth. Microorganisms are not normally added as pure cultures but rather develop naturally on the cheese surface, since ripening conditions promote growth of organisrns that are normally present in the ripening room. Proper aging of these surface- or smear-ripened cheeses results from the sequential growth of halotolerant yeast, lactic acid-metabolizing bacteria (Micrococcus spp.), and Brevibacteriurn linens, the last-named organism being essential for proper flavor development. As in mold-ripened cheeses, a pH gradient also develops during aging of bacterial surface-ripened cheeses, which in turn creates a more favorable environment for growth of contaminating microorganisms, including Listeria.

Brick Cheese Using the washed curd procedure, Ryser and Marth [264] prepared brick cheese from pasteurized milk inoculated to contain 102- 1O3 L. rnonocytogenes (strain OH, Scott A, V7, or CA) CFU/mL. Following manufacture, cheeses were smeared with a culture of B. linens and ripened at 15"C/95% RH for 2, 3, and 4 weeks to simulate production of mild, aged, and Limburger-like brick cheese, respectively. Since a natural pH gradient develops as brick cheese ripens, three types of cheese samples-surface, interior, and slice (surface and interior)-were analyzed for numbers of listeriae during 20-22 weeks of additional storage at 10°C. Populations of strains OH, Scott A, CA, and V7 increased approximately 64.6-, 37.2-, 7.4-, and 6.8-f0ld, respectively, on completion of brining approximately 32 h after the start of cheesemaking. Since a population increase of approximately 10-fold can be attributed to entrapment of listeriae within curd particles, with relatively few organisms appearing in the whey, growth of L. rnonocytogenes during the latter stages of cheesemaking before brining was confined to strains OH and Scott A. Numbers of listeriae remained relatively stable at 103-104CFU/g of cheese during brining; however, a few organisms were leached from the cheese into the 22% NaCl brine solution. Information on behavior of Listeria in salt brine solutions appears at the end of this chapter. Strains OH (isolated from Liederkranz, a bacterial surface-ripened cheese formerly produced in Ohio) and Scott A grew rapidly during the initial 2 weeks of smear development required to manufacture mild brick cheese and generally attained maximum populations of approximately 6.20 and 6.60, 6.90 and 7.0, and 5.10 and 5.60 log,,,CFU/g in 4-

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week-old slice (pH 6.0-6.5), surface (pH 6.5-6.9), and interior (pH 5.6-6.2) samples, respectively. During the remaining 20 weeks of ripening at 1O"C, numbers of strains OH and Scott A generally decreased only 1- to 7-fold in mild brick cheese. Both strains also behaved similarly in aged and Limburger-like cheese during smear development and extended storage at 10°C. In contrast, strains CA and V7 failed to grow appreciably during or after smear development, despite favorable pH values of 6.8-7.4 in fully ripened cheese. Although strains CA and V7 were detected only sporadically in 4- to 26-week-old samples of mild, aged, and Limburger-like cheese at levels ranging between 2.7 and 4.6 log,, CFU/g, both strains were routinely recovered from 24- to 26-week-old slice, surface, and interior samples after cold enrichment. Hence, all four L. monocytogenes strains survived beyond the normal shelf life of brick cheese. Subsequent experiments [26 11 dealing with possible antilisterial effects of several sulfur compounds (i.e., methyl sulfide, dimethyl disulfide, and methyl trisulfide) produced during ripening of brick cheese failed to explain the inability of strains CA and V7 to grow in mild, aged, and Limburger-like brick cheese. Additional possibilities include (a) inhibition of strains CA and V7 by smear-ripening organisms such as Geotrichum candidum [2 151, Lactobacillus plantarum [ 1631, Brevibacterium linens [2 181, enterococci [ 178,2661, coryneform bacteria [266], and/or certain staphylococci [266], all of which can reportedly produce bacteriocin-like substances active against listeriae or (b) heightened sensitivity of these L. monocytogenes strains to the inhibitory effects of certain listeriocidal fatty acids (i.e., linoleic) and monocglycerides [301] produced during cheese ripening. In conjunction with the previously mentioned European study involving Camembert cheese, Terplan et al. [289] also assessed the behavior of Listeria during manufacture and ripening of red smear-ripened ("brick-like") cheese. When this cheese was produced from pasteurized milk inoculated to contain 95 L. monocytogenes CFU/mL, numbers of listeriae increased 10-fold after the coagulum was cut as a result of entrapment within the curd; however, no growth of the pathogen was detected during the remainder of cheese manufacture. In fact, unlike the study by Ryser and Marth [264], Listeria populations decreased 10-fold by the time the cheese (pH 4.9) was ready for brining, with the cheese containing only 9 L. rnonocytogenes CFU/g after brining. Following 8 days of smear development at 16S°C/93% RH, all cheeses were ripened at 5°C for an additional 62 days. Listeria populations close to the cheese surface increased from 2.5 X 10' CFU/g immediately after smear development to 1.5 X 104 CFU/g in 14-day-old cheese, during which time the pH increased from 4.9 to 5.1. Continued ripening of brick-like cheese at 5°C led to development of stable L. monocytogenes populations of 2.5 X 105CFU/g in 1-cm thick surface slices of 42-day-old cheese. However, unlike the study by Ryser and Marth [264], the pathogen was never detected in interior cheese samples that were more than 4 days old despite pH values of 5.7 in interior samples of 56-day-old cheese. Although results of Ryser and Marth [264] suggest that L. monocytogenes should at least have been isolated occasionally from interior samples of brick-like cheese, the FDA procedure used in this study was unable to detect listeriae in these samples, possibly because of acid injury which may have occurred during exposure to pH values 5 5.2 for as long as 6 months.

Ti lsiter Cheese In 1995, Bachmann and Spahr [ 1071 manufactured Tilsiter cheese (a semifirm, slightly yellow, smear-ripened variety similar to brick cheese) from milk inoculated to contain 104 L. monocytogenes CFU/mL. Overall, their findings were similar to those observed

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for brick cheese containing strains L. rnonocytogenes CA and V7 [264], with Listeria populations varying between 103 and 104 CFU/g in Tilsiter cheese during 90 days of ripening at 10- 13°C.

Trappist Cheese The ability of L. monocytogenes to survive the Trappist cheesemaking process and persist during 90 days of ripening was investigated by Kovincic et al. [206]. Cheeses were prepared from pasteurized milk inoculated to contain I 02- 1O5 L. rnonocytogenes CFU/mL and a 1% starter culture inoculum of L. lactis subsp. Zactis and L. Zactis subsp. crernoris. After rennet coagulation, the curd was cooked at 39°C for 45 min, hooped, drained, and pressed for 10-12 h. Thereafter, the cheese was brine salted (18% NaC1,4 h), dried (5 days at 16-18”Q waxed, and aged at 10°C for up to 90 days. Populations of L. rnonocytogenes increased -10-fold in the finished cheese during the first 30 days of ripening, stabilized over the next 30 days, and then gradually decreased to levels approaching the original inoculum after 90 days of storage. Similar results were also obtained when L. rnonocytogenes was added to the curd/whey mixture rather than the pasteurized milk during cheesemaking. The limited growth and extended survival of L. rnonocytogenes in Trappist cheese (pH 4.9, 30% moisture, 1.4% NaCI) is generally similar to what has been observed for several common varieties of semisoft/hard cheeses to be discussed shortly.

Soft Italian Cheeses Most of the Italian soft cheeses are classified as pasta filata or “plastic curd” cheeses and include such varieties as mozzarella, Provolone, Caciocavallo, and Scamorze with mozzarella clearly being the most economically important. Traditionally, fresh mozzarella has been produced from the high-fat milk of the water buffalo, whereas mozzarella cheese for use on pizza is manufactured from cow’s milk and aged several weeks to develop proper elasticity and meltability. Production of these pasta filata-type cheeses is similar in that the resulting curd is always stretched in hot (160-190°F) water and then kneaded and molded into a characteristically shaped mass of curd which is hardened in cold water, brine salted, and ripened for various times.

Mozza re1la The severe heat treatment that the cheese curd receives and the reported thermal tolerance of listeriae led Buazzi et al. [125] to assess the fate of L. rnonocytogenes during manufacture of mozzarella cheese. When this cheese was prepared from pasteurized milk inoculated to contain 104- 105L. rnonocytogenes strain OH, CA, or V7 CFU/mL, Listeria populations of 104-105 CFU/mL were reported in the curd after cutting, cooking, and cheddaring. However, immersing and stretching the curd 3 to 4 min in hot water (77°C) led to the complete demise of the pathogen. Given that the temperature of the curd was maintained at 65°C for at least 2 min and that L. monocytogenes has a reported D-value of 28.1 s at 65°C [214], mozzarella cheese should be Listeria-free even if small numbers of the pathogen are present in the curd before stretching. These findings are generally similar to those reported during manufacture of traditional mozzarella cheese from buffalo milk [299], with L. rnonocytogenes populations decreasing at least 100-fold during brief stretching of the curd at 90-95°C. Although a few survivors remained after curd stretching and molding, all cheeses prepared from milk inoculated to contain 1O3 and I O5 L. rnonocy-

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togenes CFU/mL were free of this pathogen after 24 and 48 h of refrigerated storage, respectively. The heat treatment given to mozzarella cheese curd is clearly sufficient to inactivate small numbers of listeriae that might be present. However, ample opportunity exists for postprocessing contamination as evidenced by recent surveys and a Class I recall that was issued in early 1991 for over 89,000 lb of mozzarella cheese harboring L. monocytogenes. Stecchini et al. [283] addressed the issue of postprocessing contamination by inoculating the surface and packaging fluid of mozzarella cheese with L. monocytogenes and then storing the product at 5°C for up to 21 days. Under these conditions, numbers of listeriae increased about 10,000-fold during 21 days of storage, with inclusion of a crude heattreated bacteriocin preparation from L. lactis subsp. lactis yielding final populations only 10-fold lower as compared with untreated controls. Thus, manufacturers of mozzarella cheese must adhere to good manufacturing and sanitary practices to prevent contamination and growth of L. monocytogenes to potentially hazardous levels in this product during storage.

Semisoft and Hard Cheeses By definition, hard cheeses are those which contain 5 40% moisture. Cheeses in this category include such varieties as Edam and Gouda (which can also be classified as semisoft cheeses) as well as Colby, Cheddar, Swiss, Emmentaler, Gruykre, Romano, and Parmesan, the last two of which are very hard grating cheeses. Transformation of chalky, acidtasting curd into a ductile, full-flavored cheese is accomplished during ripening through the action of milk enzymes, rennet, and various microorganisms in the cheese, including the starter culture. The biochemical changes which occur during cheese ripening are complex and involve hydrolysis of fats and proteins with subsequent decarboxylation, deamination, and/or dehydrogenation as well as production of carbonyls, nitrogenous compounds, fatty acids, and sulfur compounds, all of which contribute to the overall flavor of the final product. The amount of aging needed to obtain a fully ripened cheese is directly related to moisture content, with a minimum of 2 and 10 months of ripening being required for Edam (-40% moisture) and Parmesan cheese (-30% moisture), respectively. The current popularity of many of these cheeses, along with the ability of L. monocytogenes to survive in acidic environments during refrigerated storage, has prompted a series of studies examining the behavior of L. monocytogenes during manufacture and storage of at least seven semisoft and hard cheese varieties.

Gouda and Maasdam Cheeses Beginning with semisoft/hard cheeses, Northolt et al. [235] prepared Gouda and Maasdam cheese in The Netherlands from pasteurized milk inoculated to contain approximately 500 L. monocytogenes CFU/mL. Gouda cheese was manufactured according to standard procedures, with the exception that one lot was prepared using 0.3 rather than 0.6% starter culture to obtain a cheese with an unusually high moisture content of -45%. Maasdam cheese was prepared using a culture of propionic acid bacteria in combination with 0.6% mesophilic lactic starter. After brine salting, Gouda cheese was ripened 6 weeks at 13OC, whereas Maasdam cheese was ripened 2 weeks at 13"C, 2 weeks at 18OC, and then stored at 4°C for an additional 2 weeks. As in previous studies, entrapment of listeriae within curd particles during cheesemaking resulted in population increases of approximately 10-fold as compared with the

L. monocytogenes in Fermented Dairy Products

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original level in pasteurized milk. However, some Listeria growth was noted during manufacture, with populations increasing an additional fourfold in normal Gouda and Maasdam cheese before brining. Six hours after manufacture, slightly higher Listeria populations were detected in Gouda cheese of high rather than normal moisture. Although numbers of listeriae in interior samples from both Gouda and Maasdam cheese remained relatively constant at 104CFU/g during the first 2 weeks of ripening, the pathogen was not detected in cheese samples taken at or near the surface. After 6 weeks of ripening, L. monocytogenes reappeared in surface samples from all cheeses at levels between 102and 104CFU/g. In contrast, numbers of listeriae in interior samples from 6-week-old cheese were only fourto eightfold lower than populations in the same cheeses immediately after brining. Although L. monocytogenes survived best in high-moisture Gouda cheese which had a pH of 6.0, the selective plating medium used in this study, TrypaBavine Nalidixic Acid Serum Agar, proved to be less than optimal for recovery of stressed or acid-injured listeriae that probably were present in fully ripened Gouda and Maasdam cheese having pH values of 5.48 and 5.44, respectively. I-

Colby Cheese Yousef and Marth [306] prepared Colby cheese from pasteurized milk inoculated to contain 102-103L. monocytogenes (strain V7 or CA) CFU/mL. Following manufacture, all blocks of cheese were held at 4°C for 140 days. During cheesemaking, most Listeria cells were trapped in curd, with an average of only 2.4% of the original inoculum escaping in whey. Populations of L. monocytogenes in cheese increased an average of 1.27 orders of magnitude after pressing-about 29 h after the start of manufacture (Fig. 7). Since an increase of no more than one order of magnitude can be attributed to entrapment of listeriae within curd particles after cutting, these findings suggest that slight growth of the organism did occur, particularly during

" 1

3 Time

FIGURE7 Behavior of L. rnonocytogenes during manufacture and ripening of Colby cheese. (Adapted from Ref. 306.)

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the later stages of cheesemaking and pressing. Numbers of both Listeria strains remained relatively constant in cheese during the first 40 days of ripening, after which populations decreased almost linearly (Fig. 7). Viability of L. rnonocytogenes was strongly influenced by moisture content with strain V7 decreasing more than twice as fast in cheese containing 38.5% (D-value of 54 days) rather than 42.3% (D-value of 124 days) moisture, which is well above the maximum allowable moisture content of 40.0% for Colby cheese. Behavior of L. rnonocytogenes in cheese of normal moisture content also was strain dependent, with strain CA being less stable than strain V7. However, strains V7 and CA were still detected in 140-day-old Colby cheese by direct plating, with cold enrichment results from a follow-up study [308] indicating that both strains were still viable in 5- to 8-month-old Colby cheese stored at 4°C. According to the FDA, Colby and other selected cheeses can be manufactured from raw or heat-treated (subpasteurization) milk provided that the finished cheese is held a minimum of 60 days at or above 1.7"C (35°F) before sale in an attempt to eliminate pathogenic microorganisms. These results (and those for Cheddar cheese to follow) have recently prompted the FDA to reconsider the adequacy of this aging requirement for cheeses prepared from raw milk.

Cheddar Cheese Normal stirred-curd Cheddar cheese, which has a moisture content only slightly less than Colby cheese (i.e., 36-38%), was manufactured by Ryser and Marth [259] from pasteurized whole milk inoculated to contain approximately 5 X 102L. rnonocytogenes (strain Scott A, V7, or CA) CFU/mL. The resulting 10-lb blocks of cheese were ripened at 6 and 13°C and assayed for numbers of listeriae at appropriate intervals. All curd samples examined during manufacture contained approximately 5 X 102 L. rnonocytugenes CFU/g, which suggests that the organism was only minimally concentrated in the curd and failed to grow during cheesemaking. However, since only 6.4% of the initial Listeria inoculum was recovered in the whey, the expected 10-fold increase from entrapment of the organism in curd particles probably went unnoticed because of inadequate sample preparation methods which have since been improved in our laboratory [308]. Numbers of listeriae increased slightly in cheese during pressing, with all three strains attaining maximum populations of 3.50-3.75 log,, CFU/g after 14-35 days of ripening at 13 (Fig. 8) and 6°C (Fig. 9). This population increase, which was approximately 10-fold higher than that of the original inoculum in milk, probably resulted because of enhanced recovery of the pathogen from older cheese which was easier to homogenize rather than from actual growth, as shown by Yousef et al. [308]. After 35 days of storage at either temperature, Listeria populations in cheese began to decrease, with all cheeses maintaining pH values of 5.04-5.09 throughout ripening. Strains Scott A, V7, and CA survived 70-224, 126-196, and 70-126 days in cheese ripened at 13"C, respectively, whereas the same strains remained viable for 70-154, 126-434, and 70-154 days in cheese aged at 6°C. Thus, except for strain V7, which was still present in one block of 434-day-old cheese at a level of 30 CFU/g, the remaining two strains survived equally well in cheeses ripened at either temperature. These findings suggest different acid tolerances among the L. rnonocytogenes strains tested, as has also been reported by Gahan et al. [172]. Additional experiments with strains V7 and CA demonstrated that L. rnonocytogenes was uniformly distributed in Cheddar cheese during at least the first 98 days of ripening at 6°C. Working in England, Banks [ 1101 also reported that L. rnonocytogenes persisted 8-9 months and up to 7 months in Cheddar cheese prepared from Listeriacontaminated raw and subpasteurized (i.e., 62.8 or 65.6"C) milk, respectively. Hence,

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L. monocytogenes in Fermented Dairy Products 4.0

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FIGURE 8 Survival of L. monocytogenes strain V7 in Cheddar cheese ripened at 13°C. Open symbols at < I log,,, Listeria/g indicate that the organism was not detected after 8 weeks of cold enrichment, whereas half-solid or solid symbols at < I log,, Listeria/ g indicate that the pathogen was detected in one of two or two of two samples, respectively, using cold enrichment. Numbers indicate the week at which L. monocytogenes was found. (Adapted from Ref. 259.) these data provide some of the strongest evidence for the inadequacy of the 60 day/? 1.7"C minimum holding period for cheeses manufactured from raw milk. Several subsequent studies examined the effect of Cheddar cheese compositional changes on Listeria survival during cheese ripening. Mehta and Tatini [226] assessed the behavior of L. monocytogenes strains Scott A and V7 in stirred-curd Cheddar cheese containing 1.3 or 2.5% NaCl or an equal molar mixture of NaCl and KCl. Lowering the level of NaCl enhanced destruction of Listeria, with 1.3% NaC1, 2.5% Na/KCl, and 2.5% NaCl decreasing populations 4.3-, 2.3- and 0.4-orders of magnitude in cheese, respectively, after 10 weeks of aging at 7°C. Thus, in addition to being healthier, low-sodium Cheddar cheese appears to be safer in regard to listeriae. When these same investigators [227] prepared stirred-curd Cheddar cheese from whole milk and reduced fat milk (1.55 or 2.0% milkfat), L. monocytogenes strains Scott A and V7 persisted in the finished cheese during 20 months of storage at 7OC, with no survival differences being observed between full fat (28.9% fat) and reduced fat (20.7%) cheese. However, Listeria survival in Cheddar cheese is strongly influenced by milk fat composition and release of free fatty acids during cheese ripening. Schaffer et al. [271] increased the levels of both long-chain (c18, c18.2) and unsaturated fatty acids in milk by feeding cows a diet of extruded soybeans or sunflower seeds and then using this milk to manufacture stirred-curd Cheddar cheese containing L. monocytogenes strains Scott A and V7 as previously described. During manufacture, numbers of listeriae increased 1.O- 1.5 orders of magnitude as previously reported

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FIGURE 9 Survival of L. monocytogenes strain V7 in Cheddar cheese ripened at 6°C. See Figure 8 for explanation of symbols. (Adapted from Ref. 259.) by Ryser and Marth [259]. More important, after 120 days of ripening at 7°C L. monocytogenes populations were five- to eight orders of magnitude lower in cheeses prepared from modified fat milk as compared with unmodified milk. Although some inconsistences were noted in performance of sunflower-and soybean-modified milk, inactivation of L. monocytogenes always occurred most rapidly in cheeses containing the highest levels of free fatty acids, with oleic, linoleic, lauric, and myristic acids shown to be major contributors to Listeria destruction during cheese ripening.

Swiss Cheese Unlike the aforementioned cheeses, manufacture and ripening of Swiss cheese involves several decidedly different steps, including cooking of the curd at 50-53°C and ripening the finished cheese at an elevated temperature for "eye" development. These observations prompted Buazzi et al.[ 1261 to examine the fate of L. monocytogenes during manufacture and ripening of Swiss cheese. When rindless Swiss cheese was prepared from pasteurized milk inoculated to contain 104-105 L. monocytogenes strain V7, CA, or OH CFU/mL, the pathogen was generally unable to grow during cheesemaking, with populations increasing 43% during the early stages of cooking owing to physical concentration and curd shrinkage. Thereafter, about 57% of the population in the curd was inactivated after 3040 min of cooking at 50°C. After pressing, the curd contained 50% fewer listeriae, with this population decreasing most sharply after 30 h of brining at 7°C. Storing the finished cheese (pH 5.2-5.4) 10 days at 7°C reduced the Listeria population to very low numbers. Complete inactivation of the pathogen occurred after 66-80 days of ripening at 24"C,

L. monocytogenes in Fermented Dairy Products

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with production of propionate by eye-forming bacteria likely contributing to the death of listeriae. Two studies conducted in Switzerland [ 107,20I ] demonstrated that the environments within Emmenthaler and Gruykre cheese (i.e., other varieties of Swiss cheese) also are not conducive to Listeria survival, with the pathogen no longer being present in 24hour-old cheeses (pH 5.2-5.4) prepared from raw milk inoculated to contain 104L. monocytogenes CFU/mL.

Parmesan Cheese Parmesan cheese, a hard cheese with a low moisture content, was prepared by Yousef and Marth [ 3071 from pasteurized milk inoculated to contain 104- 1O5 L. monocytogenes (strains V7 or CA) CFU/mL. Unlike the cheeses discussed previously, a lipolytic enzyme (lipase) is often added to cheesemilk to produce the characteristic flavor of fully ripened Parmesan cheese. In addition, the coagulum is cut into very small particles which are cooked at -52°C (125°F) for 45 min until the pH decreases to 6.1. This step serves to expel whey, thus producing a dry, rice-like curd which can be pressed to form a very dense, low-moisture cheese. Following manufacture, the cheese produced in this study was brine salted (22% NaC1) for 7 days at 13OC, dried 4-6 weeks in a humidity-controlled chamber at I3"C, vacuum-packaged, and ripened at 13°C for a minimum of an additional 9 months. During the first 2 h of cheesemaking, populations of both Listeria strains increased approximately 6- to 10-fold, largely from entrapment of the organism within curd particles (Fig. 10). Although Listeria counts remained relatively stable during cooking of the curd at 52°C ( I 25°F) for 45 min, populations decreased appreciably during pressing of the curd. During brining, drying, and ripening at 13°C numbers of both Listeria strains decreased almost linearly, with estimated D-values ranging between 8 and 36 days. Using direct plating, strains V7 and CA were no longer detected in cheese after 21-1 12 and 14-63 days of ripening at 13"C, respectively. Despite large differences in survival of L. monocytogenes between different batches of cheese, both Listeria strains decreased at a faster rate in Parmesan than in Gouda, Maasdam, Cheddar, and Colby cheese during ripening. Decreased viability of the pathogen in Parmesan cheese is probably related to a combination of factors, including (a) action of lipase added to the milk, (b) heat treatment that the curd receives during cheesemaking, and (c) lower moisture content (and water activity) of the fully ripened cheese. To decrease the moisture content and develop proper flavor, the present regulation in the United States requires that Parmesan cheese be aged a minimum of 1 0 months regardless of whether or not the cheese is prepared from raw or pasteurized milk, According to the results of this study, such an aging process should be sufficient to produce Listeria-free Parmesan cheese.

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Hard Italian-Type Cheese Working in Italy, Comi and Valenti [ 1351 inoculated the surface and interior of three freshly prepared hard Italian-type cheeses (a, 0.95-0.98, pH 5.2-5.5) to contain 104- 105 L. monocj~togenesCFU/g. As was true for Parmesan cheese, the pathogen failed to grow in this relatively hard cheese, with Listeria populations decreasing 10- to 100-fold in both surface and interior samples from all three cheeses during the first 28 days of ripening at 4°C. Although the pH of surface and interior samples increased to 5.4-5.5 and 5.8-6.0, respectively, after 35 days ripening, Listeria populations continued to decrease, with the pathogen only being detected by cold enrichment (i.e., populations < 10' CFU/g) in sam-

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FIGURE10 Survival of Listeria rnonocytogenes strain V7 during manufacture and ripening of Parmesan cheese. (Adapted from Ref. 306.)

ples from 35-day-old cheeses. Compositional analysis of these cheeses suggested that the amount of moisture lost after 35 days of ripening (a, 0.95-0.96) may have offset the benefit for Listeria growth caused by the increase in pH.

Hispanic Cheeses Traditional Hispanic-type cheeses comprise a wide range of white cheeses produced in Mexico and in Central and South America. Some of the most popular varieties, including Queso Blanco, Queso Fresco, and Queso de Puna, are high-moisture fresh cheeses consumed shortly after manufacture, whereas others, such as Queso Anejo, Queso de Bola, Queso de Crema, Queso de 10s Ibores, and Queso de Prensa, are lower in moisture and undergo various degrees of aging. Although 30 min of heating at 80-85°C is more than adequate to inactivate L. monocytogenes during manufacture of Queso Blanco cheese [ 1791, typical production practices for Hispanic-type cheeses involve extensive curd manipulations, including hand stirring, salting, and molding, any of which can easily lead to

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product contamination. Queso Blanco and Queso Fresco cheese pose a particular threat to the industry given the involvement of these cheeses in the 1985 listeriosis outbreak in California.

Queso Blanco Cheese In 1995, Glass et al. [179] reported on the behavior of L. monocytogenes in starter culturefree Queso Blanco cheese containing citric, malic, or acetic acid as acidulants and ALTA (a commercial bacteriocin preparation resembling pediocin AcH) as an antilisterial agent. After the finished product (pH 5.2) was inoculated to contain 106L. monocytogenes CFU/ g, populations increased about 10-fold in cheeses containing citric or malic acid during 42 days of storage at 4”C, whereas numbers of listeriae decreased slightly in cheeses prepared with acetic acid. These findings are consistent with those of other investigators who used various laboratory media acidified with malic, citric, or acetic acid (see Chap. 6). Addition of 0.6% ALTA to these cheeses yielded slightly lower Listeria counts as compared with cheeses without ALTA. Using an L. monocytogenes inoculum level of 102 CFU/g, these workers concluded that acetic acid was significantly more effective than malic or citric acid in reducing numbers of L. monocytogenes in Queso Blanco cheese and that addition of ALTA provided added protection against this pathogen. Similar benefits also were reported when Queso Blanco cheese was prepared using a nisin-producing starter culture, with L. monocytogenes populations being about 1000-fold lower in such cheeses (pH 5.3) after 21 days of storage at 4 or 12°C than in nisin-free controls [144]. Although direct addition of Nisaplin (1000 AU/mL) to the cheese milk yielded Listeria populations 100-fold lower in 1-day-old cheeses than in Nisaplin-free controls, the pathogen recovered to control levels within 21 days at 12°C. Hence, incorporating a nisinproducing starter culture was superior to direct addition of Nisaplin for minimizing survival of L. monocytogenes in Queso Blanco cheese during storage.

Queso de 10s lbores Cheese The fate of L. monocytogenes in Queso de 10s Ibores cheese (a hard, ripened cheese of pH -5) also was indirectly determined by Mas and Gonzalez-Crespo [219] using commercially available cheeses of various ages. Overall, detecting Listeria spp. in 5 of 10, 2 of 10, and 1 of 10 cheeses that had been aged for 7, 30, and 60 days, respectively, suggests that this hard, low-moisture cheese will not support long-term survival of listeriae.

Pickled Cheeses The terms pickled and white-brined are often used to describe a group of soft/semisoft, white curd cheeses to which large quantities of salt are added as a preservative. Cheeses belonging to this group are principally manufactured in countries bordering the Mediterranean Sea and include such varieties as feta (Greece), Turkish white-brined cheese (Turkey), Teleme (Bulgaria), Domiati (Egypt), and Kareish (Egypt). Some of these cheeses are frequently prepared from ewe’s, goat’s, or buffalo’s milk. Depending on the cheese variety, salt either can be added directly to the milk or curd, or the finished cheese can be stored in salt brine, salted whey, salted skim milk, or dry salt. The extreme tolerance of L. monocytogenes to high concentrations of salt, along with the organism’s ability to grow at refrigeration temperature, has made these cheeses of particular interest to food microbiologists working with Listeria.

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Feta and Turkish White-Brined Cheese In 1989, Papageorgiou and Marth [238] described the fate of L. monocytogenes during manufacture, ripening, and storage of feta cheese. During the course of this work, there was an unconfirmed report of a woman in New York who delivered a stillborn infant in December of 1987 after consuming feta cheese contaminated with L. monocytogenes. Hence this study, which will now be discussed, took on added importance. According to these authors, cow's milk was inoculated to contain approximately 5 X 103L. monocytogenes (strain Scott A or CA) CFU/mL. After warming milk to 35"C, a 1% commercial starter culture of S. thermophilus/L. bulgaricus was added. Forty min after addition of rennet, the coagulum was cut and the resulting curd was transferred to metal hoops. Following 6 h of draining, cheeses were removed from the hoops and placed in a 12% salt brine solution for 24 h. The following day, all cheeses were transferred to 6% salt brine at 22°C for 4 days until the cheese attained a pH of 4.3-4.4. Finally, cheese in the same 6% brine solution was moved to storage at 4°C. Cells of L. monocytogenes were entrapped in the curd during cheesemaking, with populations nearly 10-fold greater in curd than in inoculated milk. Only about 3.2% of the original inoculum was lost in the whey. During whey drainage and the first 1-2 days of ripening at 22"C, numbers of listeriae increased - 1.5 log,,, CFU/g, with both strains attaining maximum populations of approximately 1 X 106 CFU/g (Fig. 11). Although growth of both Listeria strains generally ceased at pH values between 4.6 and 5.0, numbers of listeriae remained virtually constant in cheese during 2-5 days of storage at 22°C in

- 7

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FIGURE11 Fate of L. rnonocytogenes strain Scott A during manufacture and early brining of feta cheese. (Adapted from Ref. 238.)

L. monocytogenes in Fermented Dairy Products

471

6% brine solution. Both salt brines in which feta cheese was ripened and/or stored were positive for listeriae (these details appear in our discussion of brine solutions at the end of this chapter). Although all feta cheeses older than 5 days maintained a pH of 4.3, both L. monocytogenes strains survived >90 days in finished cheese stored at 4°C (Fig. 12). However, differences between the two Listeria strains were noted, with populations of strains Scott A and CA decreasing 1.28 and 3.07 log,, CFU/g in 90-day-old as compared with 2-day-old feta cheese, respectively. Sarumehmetoglu and Kaymaz [267] reported that L. monocytogenes behaved similarly in Turkish white-brined cheese prepared from artificially contaminated raw milk, with numbers of listeriae generally decreasing < 100fold in the finished cheese during 90 days of refrigerated storage. Although feta and Turkish white-brined cheese can be prepared from raw milk, ripening such cheese at or above 1.7"C for 60 days will not in any way guarantee that the final product is Listeria-free with long-term survival of this pathogen highly probable. Hence, it would appear prudent to manufacture these cheeses only from pasteurized milk under good hygienic conditions to decrease the chance of a public health problem involving Listeria.

Domiati Cheese Domiati cheese, a popular fresh white-brined cheese most commonly consumed in Egypt and other parts of the Middle East, was prepared by Tawfik [286] using a 1 : 1 mixture of pasteurized cow: buffalo milk to which 7.5% NaCl, 0.5% Lactobacillus casei, and 106 L. monocytogenes were added. Listeria populations increased approximately 10-fold in the finished cheese as a result of entrapment within the curd and then decreased over time, with the pathogen surviving 4-8 weeks when the cheese was stored in salted whey at 20-25°C. Similar findings were reported by Ahmed et al. [ 5 ] , with L. monocytogenes strain V7 being inactivated in Domiati cheeses (pH 4.5-5.5) containing 5 and 10% NaCl

&-

Trial 1 Trial 2 Trial 3

2 1 0

8

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20

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FIGURE12 Survival of L. rnonocytogenes strain Scott A during storage of feta cheese. (Adapted from Ref. 238.)

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during 4 weeks of ripening at 30°C. According to Abou-Donia and Al-Medhagi [3], L. monocytogenes also persisted no more than 8 weeks in naturally contaminated retail samples of Domiati cheese. A decrease in cheese pH from 6.1 to 3.8 and an increase in salt content from 6.8 to 11.4% are clearly responsible for limiting growth and survival of L. monocytogenes in this product.

Bulgarian White-Pickled Cheese As early as 1964, Ikonomov and Todorov [ 1951 reported manufacturing white-pickled cheese from ewe's milk containing 102-103 L. monocytogenes CFU/mL. A mixture of 0.1% S. Zactis and Lactobacillus casei served as the starter culture. Although L. monocytogenes persisted 15-30 days in white-brined cheese ripened at 18-22OC, the pathogen survived twice as long when the same cheese was ripened at 12-15°C. In addition to storage temperature, Listeria viability also was partly dependent on the amount of acid produced in the cheese during ripening, with pH values of approximately 4.3 and 4.6 being reported as lethal to listeriae in cheese ripened at 12-15 and 18-22OC, respectively.

Sudanese White-Pickled Cheese Working in the United States, Abdalla et al. [l] prepared a traditional Sudanese whitepickled cheese from Lactococcus starter and Lactococcus starter-free pasteurized milk that was inoculated to contain 105L. monocytogenes Scott A CFU/mL. During cheesemaking, Listeria populations increased approximately 10-fold to 106 CFU/g in the cheese, with growth and acid production by the starter culture being prevented by addition of 8% NaCl to the milk during cheesemaking. Consequently, the finished product had a pH of 6.57.0 which is optimal for Listeria growth. Relatively stable Listeria populations of approximately 10' CFU/g developed in the cheese after 30-65 days of refrigerated storage in brine containing 8.6% NaC1. Similar Listeria populations were observed when the same starter-free cheese was prepared from pasteurized milk containing 4% NaCl and preserved in a 4% brine solution with addition of 1% potassium sorbate, nisin (25 pg/mL), or 0.1% hydrogen peroxide to the milk not affecting Listeria survival [2]. Ineffectiveness of these antimicrobial agents was attributed to several factors, including loss during processing, degradation during the early stages of cheese ripening, and suboptimal environmental conditions. In contrast, when a 1% lactic starter inoculum was used, L. monocytogenes populations decreased more than 6-orders of magnitude, with the pathogen no longer being detected in cheese after 50 days of ripening (pH 4.6) at 4°C. As was true for the other pickled cheeses, large numbers of listeriae again leached from the cheese into the brine solution; these findings are discussed in detail at the end of this chapter.

Yugoslavian White-Brined Cheese In 1974 Sipka et al. [278] published results from another study in which white-brined cheese was prepared from naturally infected cow's milk containing 240 L. monocytogenes CFU/mL. Following manufacture, the pathogen grew rapidly in cheese, reaching populations of 7.8 X 105 and 1.0 X 106 CFU/g after 7 and 14 days of brining, respectively. Although maximum Listeria populations were similar to those observed in feta cheese [238], the pathogen appeared to be less hardy in white-brined than in feta cheese, with populations decreasing to 1.3 X 105 and 1.2 X 102 CFU/g in 24- and 42-day-old fully ripened cheese, respectively. Increased inactivation of listeriae in white-brined rather than feta cheese apparently is not entirely related to acid development, since the pH of the former cheese, 4.5-5.1, was higher than that of the latter, 4.3.

L. monocytogenes in Fermented Dairy Products

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Twenty-one years later, Katic [200] prepared a similar white-brined cheese from artificially contaminated milk containing 0.8% starter culture and found that after 40 days of storage at 4”C, L. monocytogenes populations had increased 100- and 43-fold in cheeses stored in 10 and 16% brine, respectively. In contrast, numbers of listeriae increased only 4- and 12-fold in identical cheeses that were stored in 10 and 16% brine solutions at 8”C, respectively. Thus, given the same brining conditions, higher storage temperatures were, as expected, more detrimental to Listeria survival.

Ewe‘s and Goat‘s Milk Cheese In areas of the world where cows are not plentiful (i.e., northern Scandinavia, Eastern Europe, Mediterranean, Middle East), ewe’s and goat’s milk have been adapted for use either alone or in combination with cow’s milk to manufacture different types of cheese. Representative cheeses in this group include such well-known varieties as French Roquefort and Greek feta cheese, both of which are tIaditionally prepared from ewe’s milk. Lesser known cheeses typically prepared from ewe’s and/or goat’s milk include Egyptian Kachkaval, Italian Fontina, Spanish Manchego, and Italian Pecorino Romano as well as many varieties of white-brined and ethnic goat’s milk cheese, the latter of which are often produced in mountainous areas of Central Europe and Scandinavia. The occasional presence of L. monocytogenes in milk from healthy ewes and goats has prompted several studies dealing with behavior of this pathogen during manufacture and ripening of some of these less common cheeses.

Kachkaval Cheese Work with this group of cheeses dates back to 1964 when Ikonomov and Todorov [ 1951 examined the behavior of L. monocytogenes in Kachkaval cheese (a relatively soft, brinesalted cheese manufactured from ewe’s milk in Eastern Europe and the Middle East, pH 5.0-5.8) prepared from raw ewe’s milk inoculated with the pathogen. According to these investigators, L. monocytogenes survived in curd immersed in 5 6 % salt brine at 7176°C during cheesemaking and was still present in Kachkaval cheese (pH 5.0-5.4) after 30-50 days of ripening at 18-22°C.

Manchego Cheese The next such study did not appear in the literature until 1987 when Dominguez et al. [ 1491 reported manufacturing four lots of Manchego cheese (a hard aromatic cheese traditionally prepared from ewe’s milk in Spain, pH = 5.8) from a blend of pasteurized ewe’s, goat’s, and cow’s milk (15 :35 :50) inoculated to contain either 4.0 X 103or 1.9 X 105L. monocytogenes CFU/mL. To assess growth of listeriae in cheeses having different rates of acid development, milks were inoculated to contain either 0.1 or 1.0% of a mesophilic lactic acid bacteria starter culture. The coagulum was cut approximately 45 min after addition of rennet; the resulting curd was drained, hooped, and pressed for approximately 10 h. The finished cheese was then brine salted overnight, ripened 10 days at 15”C/85-90% RH, covered with paraffin, and aged an additional 50 days at 15°C. Numbers of L. monocytogenes increased 5 10-fold in all cheeses during the first 10 h of manufacture, primarily from entrapment of the organism within curd particles. The fact that cheeses prepared with either 0.1 or 1.O% starter culture contained similar Listeria populations indicates that behavior of the pathogen was not greatly influenced by the rate of acid production during cheesemaking. After the cheese was brined overnight, popula-

Ryser

474

tions of listeriae decreased approximately 3- to 100-fold, with additional small decreases being observed during ripening of unparaffined cheese at 15°C. However, numbers of listeriae remained relatively constant in cheese at pH 5.1-5.8 after paraffining, with populations in 60-day-old cheese approximating the original inoculum in milk from which the cheese was manufactured.

Goat‘s Milk Cheese Working in Sweden, Tham [291] examined the viability of Listeria in cheese prepared from raw goat’s milk inoculated to contain 105-106L. monocytogenes CFU/mL. A mixture of mesophilic lactic acid bacteria served as the starter culture. Approximately 45 min after addition of rennet, the coagulum was cut into cubes which were cooked at 37”C, drained, hooped, pressed for I h, and brine salted for 10 h. The finished cheese was then ripened at 12°C for 22 weeks. Actual numbers of L. monocytogenes could be determined in cheeses from only two of six lots using a blood agar/pour plate method. As shown in Fig. 13, Listeria populations decreased approximately 10-fold in goat’s milk cheese during the first 14 weeks of ripening at 12°C. Extended survival, along with slight growth of listeriae in cheeses ripened longer than 14 weeks, probably is related to an increase in pH from 5.55 to 6.20 during ripening as well as a decrease in numbers of competitive microorganisms that were initially present in the raw milk. Although large numbers of enterococci and other microbial competitors interfered with the quantitative recovery of L. monocytogenes in the remaining four cheeses, the pathogen survived 10-16 weeks in three of these cheeses as determined by cold enrichment. The impact of an active starter culture on the fate of Listeria in Swedish raw milk goat cheese was also later confirmed by Eilertz et al. [152], with L.

L . m o n o c y t o g e n e s in cheese VI -4 L . m o n o c y f o g e n e s in cheese V -A Total aerobic count in cheese V 4 Total aerobic count in cheese VI -0

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FIGURE13 Survival of L. rnonocytogenes and total aerobic flora during manufacture and ripening of raw goat’s milk cheese. (Adapted from Ref. 291.)

L. monocytogenes in Fermented Dairy Products

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rnonocytogenes populations increasing 10- and 10,000-fold in cheeses prepared with and without starter culture, respectively, after 4 weeks of ripening at 4°C. As you will recall from Chapter 10, a middle-aged English woman developed listerial meningitis in February of 1988 after consuming 2-3 oz of commercially produced Anari-type goat’s milk cheese containing 107L. rnonocytogenes CFU/g [63]. During a series of follow-up investigations [224], L. monocytogenes populations of < 10 CFU/g were discovered in one 2- to 3-day-old Anari cheese and in three 2- to 3-day-old Halloumi cheeses produced by the same manufacturer. After these naturally contaminated cheeses were stored at 4°C for 4-5 weeks Listeria populations as high as 8 X 104 and 1 X 106 CFU/g developed in Anari (pH 5-6) and Halloumi cheese (pH 6), respectively. Assuming a lag time of zero and an original L. rnonocytogenes population of 1-9 CFU/g, these authors calculated generation times of 47-56 and 32-37 h for this pathogen in Anari and Halloumi cheese, respectively. Both of these generation times are similar to those previously reported for L. rnonocytogenes in refrigerated fluid milks (see Chapter 1 1, Table 7). Assuming that these cheeses were on sale for up to 3 months after distribution, potentially hazardous levels of listeriae easily could have developed in such products during retail storage. Hence, as with cheeses prepared from cow’s milk, it is imperative that goat’s milk and ewe’s milk cheeses also be manufactured from high-quality pasteurized milk under the best possible hygienic conditions.

Soft Unripened Cheese Soft unripened cheeses include such high moisture, white-curd varieties as cottage, baker’s, cream, and American-type Neufchitel cheese. Unlike the groups of cheeses discussed thus far, milk to be manufactured into soft unripened cheese is coagulated through production of acid by the starter culture (or alternatively, by direct acidification of milk to pH 4.6-4.7 with gluconic acid, glucono-delta-lactone, or a mineral acid plus the lactone) rather than by the addition of a coagulant. Hence, these products are sometimes referred to as acid-curd cheese. Since the refrigerated shelf life of most soft unripened cheeses is typically less than 60 days, these varieties must be prepared from pasteurized milk or cream in the case of cream cheese. Although pH values of 4.6-5.0 provide an unfavorable environment for microorganisms that may contaminate soft unripened cheese before, during, or after manufacture, the fact that these cheeses can be consumed immediately after production may pose a public health risk, particularly if psychrotrophic, acid-tolerant organisms such as L. rnonocytogenes are present.

Cottage Cheese Nearly 1 year before the famed cheeseborne listeriosis outbreak in California, Ryser et al. [265] used the short-set procedure to prepare cottage cheese from pasteurized skim milk inoculated to contain l 04- 105L. rnonocytogenes CFU/mL. Following manufacture, half the curd was creamed to contain 2 4 % milk fat and half remained uncreamed. Both products were examined for numbers of listeriae during 28 days of storage at 3°C. Numbers of L. rnonocytogenes remained relatively constant during the initial 5-6 h of cheesemaking, during which the pH of milk decreased from 6.65 to 4.70. These findings agree with those of Schaack and Marth [269], who later demonstrated that growth of L. rnonocytogenes in slum milk at 30°C is completely suppressed by a 5% inoculum of S. crernoris. After increasing the temperature of the curd/whey mixture to 57.2”C (135°F) over 90 min and cooking the curd at this temperature for an additional 30 min,

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L. monocytogenes was not detected in samples of curd or whey that were directly plated on McBride Listeria Agar. However, following cold enrichment in Tryptose Broth, L. monocytogenes was detected in four of eight, two of eight, one of eight, and two of eight samples of cooked curd, whey, wash water, and washed curd, respectively, which suggests that some Listeria cells were only sublethally injured during cooking of the curd at pH 4.6-4.7. As indicated in Chapter 7, such injury may preclude growth on McBride Listeria Agar which contains both lithium chloride and phenylethanol as selective agents. Examination of the finished product indicated that L. monocytogenes survived in both creamed and uncreamed cottage cheese at levels generally <100 CFU/g during 28 days of refrigerated storage. Although there was no evidence for growth of listeriae in either cheese during storage, probably because of pH values generally <5.5, the pathogen was recovered more frequently and at higher numbers in creamed rather than uncreamed cottage cheese. Higher pH values in 3-day-old creamed (pH 5.32-5.45) rather than uncreamed (pH 5.12-5.22) cottage cheese may have been responsible for increasing the repair rate of injured cells, thereby increasing recovery of listeriae. Although behavior of L. monocytogenes in cheese failed to gain widespread attention until 1985, a search of the scientific literature has uncovered an earlier study by Stajner et al. [281] which examined the viability of Listeria in unsalted small-curd skim milk cheese (similar to uncreamed cottage cheese) manufactured from naturally infected milk containing approximately 5 X 105L. monocytogenes CFU/mL. Results from these Yugoslavian investigators support the findings of Ryser and Marth [265] in that the pathogen survived at least 7 days in finished cheese (pH 4.55-4.75) stored at 3-5°C. More recently, El-Shenawy and Marth [160] studied the behavior of listeriae in cottage cheese prepared from pasteurized skim milk that was inoculated to contain 106 L. monocytogenes CFU/mL and then coagulated over a period of 3 h using hydrochloric acid, gluconic acid, or bovine rennet rather than a lactic acid bacteria starter culture during which time the temperature of the milk was gradually increased from 2 to 32°C. The resulting coagulum was then cut and cooked using the aforementioned procedure of Ryser et al. [265]. As might be expected, acidification of the milk to a pH of 4.7-4.8 followed by heating was again detrimental to survival of listeriae during manufacture of cottage cheese. Overall, L. monocytogenes populations decreased -4.5 and >6.0 orders of magnitude in fully cooked (57.2"C/30 min) curd obtained by adding hydrochloric and gluconic acid, respectively (Fig. 14). However, using cold enrichment, Listeria was recovered from samples of fully cooked gluconic acid curd. Numbers of listeriae decreased faster in acidified whey than curd, with cold enrichment results indicating that the pathogen was eliminated from gluconic but not hydrochloric acid whey after 30 min of cooking at 57.2"C. Nonetheless, direct acidification of milk (pH 4.7-4.8) for cottage cheesemaking followed by cooking the resultant curd at 572°C for 30 min should be more than sufficient to eliminate expected numbers of listeriae (<10 CFU/mL) that might inadvertently enter pasteurized milk as postprocessing contaminants. In contrast to acid curd/whey, populations of listeriae in freshly cut rennet curd and whey were virtually identical to those initially observed in milk (Fig. 14). Furthermore, slight increases in numbers of listeriae were noted midway through manufacture with fully cooked rennet curd (pH 6.6) and whey still containing 104 and 103 L. monocytogenes CFU/g or CFU/mL, respectively. Thus, listericidal effects associated with cooking were greatly enhanced under acidic conditions. Subsequent experiments with selective plating media confirmed that substantial numbers of L. monocytogenes cells were sublethally in-

L. monocytogenes in Fermented Dairy Products

3, 2,

0

477

- Rennet Curd

0 - -- Rennet Whey A HC1 Curd - - HC1 Whey I- GACurd 0 - - GA Whey

A 1,

0 A

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1

I

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C

D

I

E

F

FIGURE 14 Survival of L. monocytogenes in curd and whey obtained during preparation of cottage cheese made with rennet, HCI, or gluconic acid (GA). A: Immediately after cutting; B: after temperature was increased to 48.9"C; C: after temperature was increased to 54.4"C; D: after temperature was increased to 57.2"C; E: after 15 minutes of cooking; and F: after 30 minutes of cooking. (Adapted from Ref. 160.) jured during manufacture of cottage cheese, as was suggested by Ryser and Marth [265] five years earlier. Sublethal injury was far more evident in whey rather than curd samples, probably because curd afforded some thermal protection to listeriae. Not surprisingly, the degree of sublethal injury was also closely related to coagulant type (i.e., acidity) and cooking temperature, with less injury being observed in rennet rather than acid curd/whey and partially rather than fully cooked samples of curd and whey. Heat alone was primarily responsible for rennet-associated injury, whereas the combined effects of heat and acid led to injury of listeriae in acid curd and whey. With the exception of cottage cheese prepared from milk acidified with gluconic acid, both of these studies demonstrated limited survival of L. monocytogenes in cottage cheese. However, the fact that the pathogen failed to grow in the product and decreased drastically in numbers during manufacture suggests that cottage cheese poses far less of a public health threat than do those varieties that are surface ripened with molds or bacteria. Lower health risks associated with consumption of cottage cheese are also supported by the extremely low incidence of L. monocytogenes in commercially produced cottage cheese examined in American and European surveys. The likelihood of L. monocytogenes entering cottage cheese during creaming and/ or packaging of the product is far greater than having listeriae present in pasteurized milk at sufficiently high levels to survive in the cooked curd. Although most recently published work has addressed the fate of L. monocytogenes in cottage cheese as a postmanufacturing contaminant, results from these efforts have been somewhat conflicting. Based on the findings of three studies conducted in the United States [240] and England [169,192],L.

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monocytogenes failed to grow in artificially contaminated, commercially prepared creamed cottage cheese (pH 4.5-5. I), with populations generally decreasing 0.5- 1.5 orders of magnitude during 1-5 weeks of storage at refrigeration or abusive temperatures. According to Moir et al. [230], numbers of L. monocytogenes remained relatively stable in commercial, Australian-produced creamed cottage cheese during 1 month of storage at 15°C. This behavior is similar to that observed by Hicks and Lund [ 1921 when creamed cottage cheese was inoculated with an acid-adapted strain of L. monocytogenes previously cultured in Tryptose Phosphate Broth at pH 5.5. In contrast to these findings, at least four additional studies attest to growth of L. monocytogenes [ 132,143,1761and L. innocua [ 1681 in similar samples of creamed cottage cheese, with populations increasing 0.5-3 .O orders of magnitude during refrigerated storage. Although not readily apparent, such behavioral differences in creamed cottage cheese may be related to differences between Listeria strains as well as differences in acid tolerances [ 1721 and abilities to readily compete with the native microflora. Since L. monocytogenes can persist beyond the normal shelf life of cottage cheese, several options also have been examined for minimizing growth and/or survival of Listeria in this product during refrigerated storage. A few chemical additives, including sorbate [240], 3% sodium lactate [240], 3% calcium lactate [240], and 0.04% lysozyme, were shown to be, at best, only minimally effective, with Listeria populations in inoculated creamed cottage cheese decreasing 5 10-fold during the product’s normal refrigerated shelf life. Addition of various bacteriocins, including PA- 1 (an inhibitory substance produced by one strain of Pediococcus acidilactici) and nisin (produced by certain strains of S. Zactis), appears to be a far more promising means of ridding cottage cheese of viable listeriae that may have inadvertently entered the product after cooking. Using commercially prepared dry cottage cheese curd to which 7.5 X 103 L. monocytogenes CFU/g were added during creaming, Pucci et al. [245] found that the product (pH 5.1) still contained 1 X 102 L. monocytogenes CFU/g after 7 days of storage at 4°C. In contrast, addition of bacteriocin PA-1 powder to identical samples led to complete inactivation of the pathogen within 24 h. However, inability of bacteriocin PA-1 to prevent growth of the pathogen in commercially prepared cheese sauce (pH 6.0) and half-and-half (pH 6.6) shows that listericidal activity of this bacteriocin is strongly dependent on the pH of the food system. Using a different brand of dry cottage cheese curd that was inoculated to contain 3 X 105L. monocytogenes CFU/g during creaming, Benkerroum and Sandine [ 1161 detected very few viable listeriae in the product (pH 4.9-5.0) after l or more days of storage at 4°C. This antagonistic effect of cottage cheese (presumably from natural flora or byproducts in cottage cheese) toward listeriae was enhanced by adding nisin (2.5 X 103IU/ g), with viable listeriae completely being eliminated from such cheese after I day of refrigerated storage. The efficacy of nisin has since been confirmed by Ferreira and Lund [ 1691, who reported that L. monocytogenes populations decreased about 1000-fold in creamed cottage cheese (pH 4.6-4.7) containing 2000 IU nisin/g after 3 days of storage at 20°C. In these studies, nisin and bacteriocin PA-1 remained active in creamed cottage cheese during prolonged storage. Thus it appears that these bacteriocins may also prove useful in limiting Listeria survival in other fermented dairy products, including natural cheeses, cold-pack cheese food, and various cheese spreads [65,72] during the normal shelf life.

L. monocytogenes in Fermented Dairy Products

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Additionally, modified atmosphere packaging also has been explored as a means for both extending the refrigerated shelf life of cottage cheese and minimizing Listeria growth. According to Chen and Hotchkiss [ 1321, L. monocytogenes populations in creamed cottage cheese (pH 5. I ) packaged under 35% CO2remained stable during 9 weeks of storage at 4"C, whereas the pathogen increased 1000-fold in aerobically packaged cheese. At 7"C, numbers of listeriae failed to change in CO:-packaged cheese during the first 4 weeks of storage. However, rapid growth occurred within 16 days in aerobically packaged cheese. Fedio et al. [ 1681 also reported that L. innocua failed to grow in creamed cottage cheese (pH 5) during 28 days of storage at 5°C when the product was packaged under 50 or 100% CO2. However, this organism began growing rapidly in identical samples packaged aerobically or under 100% N2 after 7 days of incubation. Although modified atmospheric packaging appears to be useful in minimizing Listeria growth in cottage cheese, inactivation of the pathogen will not occur under such conditions, with the results of Chen and Hotchkiss [132] also indicating that the effectiveness of CO2 is strongly temperature dependent.

Cream Cheese In the only other study dealing with the behavior of listeriae in soft unripened cheese, Cottin et al. [ 1401 prepared cream cheese from a chemically acidified mixture of milk and cream that was inoculated to contain 10'- 10' L. monocytogenes CFU/mL. Using the lowest inoculum, Listeria grew in the finished product (pH 5 6 ) and attained a stable population of 10' CFU/g within 2 days of storage at 4°C. Thus, unlike cottage cheese, the pH and moisture content of cream cheese are both sufficiently high to permit limited growth of listeriae in the product during refrigeration.

-

Whey Cheeses A few cheeses such as ricotta, Broccio, and Ricotone are prepared from sweet whey derived from the manufacture of mozzarella, Cheddar, Swiss, Tilsiter, and feta cheese. Manufacture of these whey cheeses is based on the direct acidification of whey, whey/milk, and whey/cream mixtures to pH 5.9-6.0 using food-grade acids (i.e., citric, acetic), lactic starter cultures, or acid whey powder followed by cooking at 180-190°F to precipitate the whey protein. The fine precipitate which eventually rises to the vat surface is then removed, drained, matted, and either marketed as whey cheese or a dairy ingredient. Several additional extremely low-moisture ( I 3 to 18%) whey cheeses, including Gjetost, Mysost, and Gudbrandsdalsost, are unique to Norway and are prepared by thermally concentrating and then boiling a blend of goat's and cow's milk whey until the mixture carmelizes and becomes viscous. This plastic mass is then cooled, extruded, and cut into extremely dense blocks for marketing. As was true for mozzarella cheese, L. monocytogenes will be completely inactivated during manufacture of these whey cheeses. However, the potential still exists for contamination during packaging as is evidenced by at least one Class I recall of ricotta cheese in July of 1991 and the report of a small cluster of listeriosis cases traced to Anari whey cheese produced in Cyprus (see Chap. 10).Consequently, Papageorgiou et al. [239] examined the fate of L. monocytogenes as a postprocessing contaminant in Greek Myzithra (identical to Anari cheese), Anthotyros, and Manouri cheese, all of which are starter culture-free, soft (50-70% moisture), low-acid (pH 6.0-6.5) whey cheeses. Immediately after commercial manufacture, these cheeses were inoculated to contain -500 L. monocyto-

Ryser

480

genes strain Scott A or CA CFU/g. Regardless of cheese type or Listeria strain, the pathogen grew rapidly and attained maximum populations of 107-108CFU/g after 24-30 days, 5-12 days, and 56-72 h of storage at 5, 12, and 22OC, respectively. Consequently, strict hygienic practices must be followed to prevent Listeria contamination during packaging, with postpackaging pasteurization of the product also being recommended as an added safeguard.

Cold-Pack C h e e s e Food Unlike the natural cheeses discussed thus far, cold-pack cheese food is typically prepared by comminuting and blending aged Cheddar cheese (or another variety) with nonfat dry milk, dried whey, water, cream, plastic cream (composition similar to butter), salt, acidulants (i.e., lactic and/or acetic acid), preservatives (i.e., potassium sorbate and/or sodium propionate), and other optional ingredients into a homogeneous mass without heating. Since all of the dairy ingredients and some of the optional ingredients used in manufacturing cold-pack cheese food can potentially harbor L. monocytogenes, Ryser and Marth [263] investigated the behavior of this pathogen in nine different formulations of coldpack cheese food inoculated to contain approximately 500 L. monocytogenes strains Scott A, V7, CA, or OH CFU/g. During 182 days of storage at 4OC, populations of all four Listeria strains decreased less than 10-fold in nonacidified, (pH 5.20) preservative-free cheese food, with the pathogen surviving throughout the product's entire 6-month shelf life (Fig. 15).In sharp contrast to these findings, addition of preservatives with or without acidifying agents led to the

1.0

-

-5

-

--a

Scott A

--0

v7

- * CA --+

OH

10

FIGURE15 Survival of four strains of L. monocytogenes in nonacidified cold-pack cheese food manufactured without preservatives. (Adapted from Ref. 263.)

L. monocytogenes in Fermented Dairy Products

481

eventual demise of listeriae in cheese food stored at 4°C (Fig. 16). In nonacidified cheese food (pH 5.20) preserved with 0.3% sodium propionate, L. monocytogenes survived an average of 142 days as compared with 118, 103, and 98 days in the same product adjusted to pH 5.0-5.1 with lactic, acetic, and lactic plus acetic acid, respectively. Using 0.3% sorbic acid in place of sodium propionate, the pathogen survived an average of 130 days in nonacidified cheese food (pH 5.45) as compared with 112, 93, and 74 days in cheese food acidified to pH 5.0-5.1 with lactic, lactic plus acetic, and acetic acids, respectively. Thus, sorbic acid was consistently more antagonistic to listeriae than sodium propionate. In addition, antilisterial effects of both preservatives were more pronounced in cheese food acidified with acetic rather than lactic acid. Since organic acids are far more bactericidal in the undissociated than dissociated state, increased inactivation of listeriae in the presence of acetic acid probably resulted from the higher proportion of undissociated acetic (-36%) rather than lactic acid (-5.9%) in cheese food acidified to pH 5.0-5.1. These findings indicate that it would be prudent to consider (a) adding preservatives, particularly sorbic acid, to cold-pack cheese food and (b) reducing the pH of the product to 5.0 by adding small amounts of lactic and/or acetic acid to minimize L. rnonocytogenes survival in the finished product. Additional information on conditions leading to inhibition and/or inactivation of this pathogen by sorbic, propionic, lactic, and acetic acids can be found in Chapter 6. 150

-

140

S SorbicAcid p NaPropionata A Acetic Acid L LacticAcid

120 130

01

110

6

100-

'$

?

90

-

vI

80

-

70

-

60

-

50

I

I

I

-

h

P

S

P+L

S+L

P+A

P+L+A

S+L+A

S4A

Additive

FIGURE16 Maximum length of survival of L. monocytogenes in acidified and nonacidified cold-pack cheese food containing preservatives.Each bar represents the average maximum length of survival of all four Listeria strains in one of eight different formulations of cheese food manufactured in duplicate. Any two differing by >20.24 days (length of bar) are significantly different ( p < 0.05). (Adapted from Ref. 263.)

482

Ryser

Behavior of L. monocytogenes in Cheese as Affected by Cheese Composition As suggested earlier, the fate of L. rnonocytogenes and other foodborne pathogens during cheese ripening is determined by the microbiological, biochemical, and physical properties of the particular cheese. Thus cheese is a very complex system, with the following factors acting simultaneously to determine the behavior of L. monocytogenes during ripening: (a) type, amount, and activity of the starter culture; (b) pH as determined by concentrations of lactic, acetic, formic, and other acids; (c) presence of hydrogen peroxide, diacetyl, and various antimicrobial agents (i.e., nisin, diplococcin, and other bacteriocins); (d) levels of nutrients, salt, moisture, and oxygen; and (e) the temperature at which the cheese is ripened. All of these factors act together to produce a particular outcome, however, a few conclusions concerning the ability of L. monocytogenes to grow and/or survive in some of the aforementioned cheeses prepared from contaminated milk can be drawn by examining the behavior of this pathogen in relation to the combined effects of moisture content, water activity (a,), and salt in the moisture phase as well as the pH of the cheese and the temperature(s) at which the cheese is ripened (Table 15). Although fully ripened Camembert and feta cheese have widely differing pH values of 7.5 and 4.4, respectively, both cheeses are very similar in terms of moisture content, water activity, percentage of salt in the water phase, and ripening temperature. Thus rapid growth of L. rnonocytogenes in Camembert cheese can be largely attributed to the increase in pH of the cheese during ripening, whereas a pH value of 4.4 appears to be responsible for preventing growth of the bacterium during ripening of feta cheese. Inability of L. rnonocytogenes to multiply in blue cheese during ripening and storage is probably related to the high concentration of salt in the water phase (which results in a low a,), since other workers have confirmed that this organism will not grow in laboratory media [275] and skim milk [236] containing > I 0 and 12% salt, respectively. As with Camembert cheese, growth of two of four L. rnonocytogenes strains in brick cheese appears to be directly related to the high pH that the cheese attained during extended ripening. However, a general inability of the remaining two strains to grow in brick cheese of similar composition is as yet unexplained. When comparing the behavior of L. monocytogenes in Cheddar and Colby cheese, the initial inactivation rate for the pathogen was somewhat slower in the latter cheese. At first glance, it appears that increased viability of Listeria in Colby cheese during the early stages of ripening may be related to the lower percentage of salt in the water phase in this cheese than in Cheddar cheese. However, data in Table 15 show that L. monocytogenes was inactivated at similar rates in Colby cheese and cold-pack cheese food, the latter is compositionally similar to Colby cheese except for a higher concentration of salt in the water phase. Hence, factors other than a low concentration of salt in the water phase also must be involved in enhancing the viability of listeriae during ripening of Colby cheese. Lack of growth and decreased survival of L. rnonocytogenes in Parmesan cheese and in an unidentified hard Italian cheese as compared with other varieties in Table 15 correlate well with the lower moisture content/water activity of these cheeses during ripening. Barring thermal and/or acid injury of L. rnonocytogenes, which is likely to occur during manufacture of cottage cheese and other varieties that undergo substantial heat treatments during manufacture (i.e., mozzarella, Swiss), factors outlined in Table I5 are

L. monocytogenes in fermented Dairy Products

483

TABLE 15 Behavior of L. monocytogenes During Cheese Ripening as Affected by Cheese Composition

Cheese Camembert Blue Brick Feta Cheddar Cheddar Colby Parmesan Hard Italian Cold-pack cheese foodb

Moisture @)

Estimated a,

54.4 38.9 43.0 54.7 37.2 37.2 40.0 32.0 NR' 41.4

0.975 0.950 0.990 0.975 0.975 0.975 0.975 0.935 0.950 0.975

-

Approximately 24 h after the start of cheesemaking. Prepared without preservatives or acidifying agents. Not reported. Percentage of salt in solid and water phase.

Estimated salt in water phase (%)

Initial"

Final

Ripening temp. ("C)

4.72 11.52 1.89 4.57 4.61 4.61 3.91 4.96 2.12d 4.90

4.6 4.6 5.3 4.7 5.1 5.1 5.1 5.1 5.3 5.3

7.5 6.3 7.3 4.4 5.1 5.1 5.1 5.1 5.7 5.1

15/6 9-12/4 15/10 22/4 6 13 4 13 4 4

PH

Log,, of L. monocytogenes CFU/g Initiala

Maximum

Final

Survival (days)

Ref.

3.1-3.6 4.0-5.0 3.0-4.7 5.2-6.2 2.5-3.2 2.6-3.4 3.5-4.5 3.3-4.3 4.5-5.1 2.4-2.8

6.7-7.5 4.0-5.0 4.6-6.7 5.7-6.2 2.6-3.8 3.0-3.7 3.6-4.6 3.3-4.3 4.5-5.6 2.4-2.8

6.7-7.5 1.0-2.3 2.7-6.1 2.8-4.6 0-1.5 0 2.3-4.1 1.0-1.3 2.0 1.1-2.0

65 120 168 90 70-434 70-224 112-140 21-1 12 35 180

260 237 264 238 259 259 306 307 I35 263

484

Ryser

useful in predicting whether or not this pathogen will grow in other cheeses having similar microbiological, biochemical, and physical characteristics. In addition to being present in milk at the time of cheesemaking, Listeria also can easily contaminate the finished cheese during packaging, ripening, and storage. Consequently, Genegeorgis et al. [ 1761 evaluated the fate of Listeria as a postprocessing contaminant by inoculating 49 retail cheeses (24 typed28 brands) with L. monocytogenes and then storing these cheeses at 4-30°C for up to 36 days. As expected, Listeria growth was primarily confined to high-moisture varieties, including Brie, Camembert, ricotta, and the soft Hispanic cheeses, all of which had a pH 2 6 and low to moderate levels of salt in the moisture phase (Table 16). Back et al. [ 1081 also reported temperature-dependent surface growth of L. monocytogenes on several additional retail European soft cheeses including Cambazola, English Brie, Blue Lymeswold, and White Lymeswold, with Listeria populations remaining relatively stable on Blue Stilton, White Stilton, Mycella, and Chaume cheese during short-term storage. In addition, commercial [ 1311 and experimentally produced [170] Arzua cheese (a soft, low-acid Spanish cheese prepared from raw cow's milk) supported growth of Listeria as evidenced by populations >loo0 CFU/g in the finished product. All of these findings again point to the high-moisture, low-acid cheeses as being of primary public health importance.

Feasibility of Preparing Cheese from Raw Milk According to current FDA regulations, milk pasteurization or use of a similar heat treatment during cheesemaking is required for the manufacture of 16 cheese varieties, including Brie, cottage, cream, Neufchgtel, Monterey, mozzarella, Scamorza, Muenster, Gammelost, Koch Kaese, and Sapsago [197]. Seven varieties of manufacturing cheese (i.e., for use in pasteurized processed cheese, cheese foods, cheese spreads) require neither pasteurization of the cheesemilk nor a 60-day minimum ripening at 21.7"C (235"F), whereas the 34 remaining varieties of cheese recognized under current standards of identity must either be manufactured from pasteurized milk or held a minimum of 60 days at 2 1.7"C (135°F) to eliminate pathogenic microorganisms. Although statistics on milk pasteurization for cheesemaking are scarce, available evidence indicates that a major portion of the natural cheeses sold in the United States are prepared from pasteurized milk. The mandatory holding period of 60 days at 21.7"C (235°F) for cheeses manufactured from raw milk was adopted in 1949 [6,197] after researchers demonstrated that viable Brucella abortus, the causative agent of brucellosis, was eliminated from cheese by such an aging process. Although this 60-day holding period was generally deemed adequate to eliminate most foodborne pathogens, later studies demonstrated that Salmonella typhimurium and other hazardous microorganisms can survive such a cheese-ripening process [ 183,1981.Furthermore, results in Table 15 indicate that L. monocytogenes can survive well beyond 60 days in many natural cheeses held at 21.7"C (235OF). In keeping with the grave nature of listeriosis as compared with most other foodborne illnesses, the FDA has continued to maintain a policy of zero tolerance for L. monocytogenes in all ready-to-eat foods. Thus far no well-documented cases of listeriosis have been associated with consumption of cheeses that were legally prepared from raw milk and held a minimum of 60 days at 21.7"C (235°F). However, since -4% of the raw milk supply can be expected to contain L. monocytogenes, it would be prudent to manufacture cheeses from pasteurized milk whenever possible. Although Yousef and Marth [307]

L. monocytogenes in Fermented Dairy Products

485

TABLE 16 Growth and Inactivation of L. monocytogenes in SurfaceInoculated Retail Cheeses During Storage at 4-30°C Cheese category and type

Soft mold ripened Brie Camembert Blue Bacterial surface ripened Limburger Muenster Soft Italian Provolone String cheese Semisoft and hard ripened Monterey Jack Colby Cheddar Swiss Hispanic Queso Fresco Queso Rancher0 Queso Panella Cotija Pickled cheese Feta Ewe’s milk cheese Kasseri Soft unripened Cottage Cheese Cream Cheese Whey cheeses Ricotta Processed cheese American Monterey Jack Piedmont

PH

6.0-7.7 7.3 5.1

% NaCl in moisture phase

2.5-3.6 2.5 6.1

7.2 5.5

4.8 3.8

5.6 5.5

4.6 4.4

5.0-5.2 5.5 4.9-5.6 5.5

1.O-3.0 4.9 2.6-5.4 2.7

6.5-6.6 6.2 6.2-6.7 5.5-5.6

4.5-6.6 4.1 2.5-3.9 9.6- 12.5

4.2-4.3

2.2-7.5

4.8-5.3

5.5-5.8

4.9-5.1 4.8

1.0-1.2 <0.9

5.9-6.1

<0.7

5.7 5.7 6.4

Growth

+ +

+/-

+ + -

+/-

+

2.1 4.4 5.1

Source: Adapted from Ref. 176.

demonstrated that ripening Parmesan cheese for 10 months, as legally required, is sufficient to produce a high-quality, Listeria-free product, desirable flavor and texture characteristics are not easily attainable in sharp Cheddar and Swiss cheese prepared from pasteurized milk. Hence, alternative means should be developed to enhance the safety of these products. Such methods might include cold sterilization of the milk via microfiltration or addition of various flavor- and texture-enhancing enzymes (or microorganisms) to pasteurized milk, which would allow the cheesemaker to obtain a higher quality product [199]. However, as important as it is to manufacture cheese from Listeria-free milk, it is equally important to prevent contamination of the product during manufacture, ripening, and storage by using good manufacturing practices. Information concerning problem areas

486

Ryser

and safeguards during manufacture of dairy products and other, foods can be found in Chapter 17.

Whey Listeria monocytogenes has not yet been isolated from commercially produced cheese whey. However, studies have shown that when various cheeses were experimentally produced from pasteurized milk inoculated with L. monocytogenes, between 1-5% of the original inoculum was lost in the whey during cheesemaking (Table 17). These findings again demonstrate that the pathogen is concentrated 8- to 10-fold in curd during milk coagulation. Unlike other wheys, populations of L. monocytogenes in acid whey (pH 4.6) obtained from the manufacture of cottage cheese were reduced more than 10,000- fold after cooking the curdlwhey mixture at 57.2"C (1 35°F) for 30 min. However, as previously noted, Listeria was detected in several whey samples after 6 weeks of cold enrichment [265], which, in turn, suggests that some cells were only sublethally injured during cooking of the curd/whey mixture. These observations prompted Ryser and Marth [262] to examine the behavior of L. monocytogenes in wheys from Camembert cheese that were filter-sterilized and adjusted to pH values of 5.0-6.8. All whey samples were then inoculated to contain -100-500 L. monocytogenes strains Scott A, V7, CA, or OH CFU/mL and incubated at 6°C. Although the four L. monocytogenes strains failed to grow in wheys having pH values 15.4, the pathogen survived in all samples with populations decreasing 110-fold during 35 days of refrigerated storage. In contrast, L. monocytogenes grew in all remaining samples after a 3-day lag period and attained average maximum populations of 7.48, 7.87, and 7.84 log,, CFU/mL in wheys adjusted to pH 5.6, 6.2, and 6.8, respectively, following 35 days of incubation. As previously noted, these Listeria populations in whey were slightly higher than those that would be expected to develop in skim or whole milk during refrigerated storage. Generation times for L. monocytogenes in wheys adjusted to pH 5.6, 6.2, and 6.8 ranged between 25.2-31.6 h, 14.8 and 21.1 h, and 14.0 and 19.4 h, respectively, depending on the individual strain. Although doubling times were similar for all strains at the same pH value, generation times were significantly longer at pH 5.6 than

TABLE17 Number of Listeriae Recovered from Whey During Manufacture of Various Cheeses Prepared from Pasteurized Milk Inoculated to Contain -500-5000 L. rnonocytogenes CFU/mL % of original

Cheese

L. monocytogenes CFU/mL of whey

inoculum in whey

Camembert Blue Brick Feta Gouda Colby Cheddar

8 43 12 15 5 21 22

1.3 3.6 2.5 3.2 1 .o 2.4 5.0

Average

18

21.7

Ref.

26 1 237 264 238 235 306 259

L. monocytogenes in fermented Dairy Products

487

at pH 6.2 and 6.8. Interestingly, Hughey et al. [193] observed that L. monocytogenes could be inactivated in similar samples of demineralized whey by adding lysozyme. However, this enzyme did not decrease viability of the pathogen in normal whey, which in turn suggests that lysozyme activity is neutralized by whey minerals and/or proteins. Using a different approach to examine the behavior of Listeria in whey, Northolt et al. [235] inoculated heat-treated (68"C/lO s) wheys (pH 6.5) to contain 500-1000 L. monocytogenes CFU/mL and incubated the samples at temperatures between 7-30°C. Following a 6- to 24-h lag period, the pathogen grew in all samples with doubling times of 12 h, 6 h, 4 h, and 40 min in wheys incubated at 7, 12, 20, and 30"C, respectively. Although incubation of all whey samples was terminated before L. monocytogenes reached the stationary growth phase, the pathogen did attain populations of 104-106 CFU/mL at the conclusion of the experiment. In the only study thus far reported dealing with nonsterile whey, researchers in France [ 1131 produced whey containing 2-7 L. monacytogenes CFU/mL from previously inoculated milk and examined samples for listeriae after 101, 156, and 25 1 days of storage at 4°C. Moderate growth and extended survival of the pathogen were observed in whey collected immediately after coagulation of the milk (pH 5.4), with 156- and 251-day-old whey samples at pH 4.8 containing 2.5 X 104 and 7.0 X 102 L. monocytogenes CFUI mL, respectively. As predicted by Ryser and Marth [262], the pathogen also failed to grow in more acidic wheys (pH 5.2-5.3) collected after hooping with listeriae no longer observed in 101- and 156-day-old wheys having pH values of 3.28 and 4.26, respectively. Not surprisingly, increasing the incubation temperature to 6, 14, and 20°C led to faster demise of listeriae in 21 similar wheys (pH 3.75-5.72) initially containing 60-96 L. monocytogenes CFU/mL, with the pathogen being eliminated from all but one sample (pH 5.72) examined after 114 days of incubation at 6°C. These findings demonstrate that L. monocytogenes can grow to high numbers in fluid wheys having pH values of 25.4 and remain viable in more acidic wheys during many months of refrigerated storage. Given results of a study described in Chapter 11 in which numbers of L. monocytogenes decreased only 1-5 orders of magnitude during manufacture of nonfat dry milk [ 1501, it appears that this organism also is likely to survive the spray-drying process used in converting fluid whey into whey powder. However, to our knowledge, no Listeria spp. have yet been isolated from dried whey manufactured commercially in Europe or the United States. Although Gabis et al. [171] failed to find any Listeria spp. in 23 environmental samples from whey processing factories, these authors did isolate L. monocytogenes from a floor drain that was located within a raw milk receiving room of a dry milk processing factory. Additionally, Listeria spp. other than L. monocytogenes were isolated from several drains and trenches in the powder production area of a second factory that manufactured dry milk products. Considering the widespread use of dried whey (and nonfat dried milk) as an ingredient in numerous products including cheese food, ice cream, sherbet, candy, beverages, and baked goods, strict enforcement of good manufacturing practices for dried whey and nonfat dry milk should be continued to prevent a possible public health problem involving listeriae.

Brine Solutions Since L. monocytogenes is quite halotolerant, it is not surprising to learn that brine solutions in which cheeses are salted and/or ripened also can serve as potential reservoirs for this organism. Current evidence suggests that these brine solutions may become contami-

488

Ryser

nated with L. monocytogenes through directhdirect contact with the cheese factory environment (i.e., equipment, condensate on walls, floors, and ceilings) as well as actual shedding of L. monocytogenes into the brine solution from Listeria-contaminated cheese. Breer [ 1231 and Terplan [28] reported isolating L. monocytogenes from commercial brine solutions in Europe. In one instance, the pathogen was detected in brine tanks 4 days after soft/semisoft cheeses were removed from the salt brine. Since one 1991 recall of mozzarella cheese in the United States [80] was presumably traced to a contaminated brine tank, interest in the incidence of listeriae in brine solutions will likely increase. Migration of L. monocytogenes into brine solutions and salted whey during salting of artificially contaminated cheese has been well documented. Ryser and Marth [26] brine salted brick cheese containing 103-104 L. monocytogenes CFU/g at 10°C. Cold enrichment of membrane filters through which 50-mL portions of 22% brine solution were filtered indicated that the pathogen leached from the cheese into the salt solution during 24 h of brining. Furthermore, viable listeriae were detected in samples of 22% brine stored at 10°C at least 5 days after blocks of cheese were removed from the brine. When Abdalla et al. [ l ] brine salted Sudanese white pickled cheese in whey containing 8% NaCI, L. monocytogenes once again leached from the cheese into the salted whey, with populations increasing 2.5-3.5% orders of magnitude during 65 days of storage at 4°C. In conjunction with their study on the fate of L. monocytogenes during manufacture, ripening, and storage of feta cheese, Papageorgiou and Marth [238] also examined Listeria viability in the brine solution in which cheese was salted and stored. After 1 day of salting, a 12% brine solution contained an average Listeria population of 2.63 log,, CFU/g, which again indicates that the pathogen leached from cheese into the brine (Fig. 17). However, no growth of L. monocytogenes was observed in the 12% brine solution despite ample migration of cheese nutrients into salt brine as well as favorable values for temperature and pH. After transferring feta cheese to a 6% brine solution, the pathogen leached from cheese into salt brine and grew rapidly, with similar populations being observed in both cheese and 6% brine after 6 days of incubation at 22°C. Although numbers of listeriae

-

0

I

1

I

I

2

I

I

3

I

I

4

I

,

I

5

I

,<'

1

20

34

I

48

I

62

I

76

Days

FIGURE17 Average populations of L. rnonocytogenes strain CA in 12 and 6% salt brine during ripening and storage of feta cheese. (Adapted from Ref. 238.)

L. monocytogenes in Fermented Dairy Products

489

decreased in both cheese and salt brine during 90 days of refrigerated storage, the pathogen was inactivated slower in 6% salt brine than in feta cheese. Larsen et al. [208] reported that L. monocytogenes began growing in salt-free whey (pH 5.6) and whey containing 3.6% NaCl and/or KC1 after 7-14 days of storage at 4"C, with Listeria growth generally being unrelated to the type of salt used. However, L. monocytogenes populations remained unchanged in identical samples containing 4.8% NaCl and/or KCI. When similar whey samples were incubated at 25"C, L. monocytogenes populations increased 3-4 orders of magnitude, with more rapid growth being observed in wheys containing 3.6% rather than 4.8% NaCl and/or KCI. However, Listeria growth at 25°C could be prevented by adding a L. l a d s subsp. lactis or L. lactis subsp. cremoris starter culture before incubation. In 1994, Rajkowski et al. [249] also reported that addition of 4.5% NaCl to ultrahigh temperature (UHT)-sterilized milk decreased the growth rate of L. monocytogenes in samples incubated at 12-37°C. However, fortification of these same samples with 0.5 or 1 .O% polyphosphate did not alter Listeria growth characteristics. Since feta and other white-pickled cheeses such as Teleme, Halloumi, and Domiati are frequently cured in whey or skim milk containing 6 or 12% salt, Papageorgiou and Marth [2361 examined behavior of Listeria in salted whey and skim milk. Autoclaved samples of skim milk (pH 6.0-6.2) and deproteinated whey (pH 5.5-5.7) containing 6 and 12% NaCl were inoculated to contain 103 L. monocytogenes (strains Scott A or CA) CFU/niL and incubated at 4 and 22°C. After a lag period of 5- 10 days, L. monocytogenes grew rapidly in 6% salted whey and 6% salted skim milk, with the pathogen attaining maximum populations of 107-10R CFU/mL following 50-55 days of refrigerated storage (Table 18). In the study discussed earlier, Ryser and Marth [262] reported that these same Listeria strains had shorter lag periods and generation times but achieved similar maximum populations in unsalted, filtersterilized whey (pH 5.6) after 24 days of incubation at 6°C. Hence these findings suggest that addition of NaCl or KCI to whey and milk plays a major role in decreasing the growth rate of Listeria. Increasing the incubation temperature to 22°C resulted in lag periods of 6-12 h for both Listeritr strains in whey and skim milk containing 6% salt. Generation times were

-

TABLE 18 Generation Times (GT) and Maximum Populations

(MP) of L. monocytogenes Strains Scott A and CA in 6% Salted Whey and Skim Milk Incubated at 4 and 22°C Strain Incubation at 4°C Scott A Scott A CA CA Incubation at 22°C Scott A Scott A CA CA

Product

GT (h)

MP (logl,,CFU/mL)

Whey Skim milk Whey Skim milk

46.8 1 45.23 37.49 49.43

7.97 7.58 8.04 7.69

Whey Skim milk Whey Skim milk

3.67 4.3 1 3.56 4.42

8.02 7.70 8.10 7.89

Source: Adapted from Ref. 236.

Ryser

490 5 ScottA Whey

Scott A Skim milk

4 -

f

0

3 -

2 CAWhey

1 -

CA Skim milk

@-

0 1

I

-25

I

0

I

I

I

I

25

50

75

I

I

I

100

125

I

I 150

Days

FIGURE18 Behavior of L. monocytogenesstrains Scott A and CA in 12% salted whey and skim milk during extended storage at 22°C. (Adapted from Ref. 236.) similarly reduced with both strains exhibiting faster growth rates and higher maximum populations in salted whey rather than salted skim milk (see Table 18). These findings agree with those of other researchers who also observed that L. monocytogenes grew faster and attained higher maximum populations in unsalted whey [262] than in skim milk [255]. Unlike the previous findings obtained with 6% salt, growth of L. rnonocytogenes was completely inhibited in 12% salted whey and skim milk, with populations of both strains decreasing < 10-fold during 130 days of storage at 4°C. Although strain Scott A also persisted >130 days in 12% salted whey and skim milk incubated at 22"C, strain CA proved to be less salt tolerant, surviving only 80 and 105 days in 12% salted skim milk and whey, respectively (Fig. 18). Increased destruction of L. monocytogenes in salt solutions held at ambient rather than refrigeration temperatures has been well documented. Results from several of these studies dealing with viability of listeriae in salted Tryptose Broth 112761 and cabbage juice [ 1391 are discussed in Chapter 6. Based on these observations, acidification of brine solutions used in cheesemaking to pH values <5.0 has been recommended to prevent growth of L. monocytogenes, particularly if such solutions contain 510% salt. In 1989, Hughey et al. [193] also noted that addition of 0.35% H 2 0 2to a 23% brine solution caused numbers of L. monocytogenes to decrease by six orders of magnitude within 24 h. However, unlike organic acids, the bactericidal activity of H 2 0 2 dissipates fairly rapidly. Hence addition of H202to salt brine provides only temporary protection against listeriae that might be present within the cheesemaking environment.

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predicting the temperature evolution of Taleggio, an Italian soft cheese. J. Dairy Sci. 77: 1176-1 182. Glass, K.A., B.B. Prasad, J.H. Schlyter, H.E. Uljas, N.Y. Farkye, and J.B. Luchansky. 1995. Effects of acid type and AltaTM2341 on Listeria monocytogenes in a Queso Blanco type of cheese. J. Food Prot. 58:737-741. Gledel, J. 1986. Epidemiology and significance of listeriosis in France. In: A. Schonberg, ed. Listeriosis-Joint WHO/ROI Consultation on Prevention and Control, Berlin, December 10- 12, Institut fur Veterinkedizin des Bundesgesundheitsamtes, Berlin, pp. 9-20. Gledel, J. 1988. Listeria and the dairy industry in France. Foodborne Listeriosis-Proceedings of a Symposium. Wiesbaden, Germany, Sept. 7, pp. 72-82. Goel, M.C., D.C. Kulshrestha, E.H. Marth, D.W. Francis, J.G. Bradshaw, and R.B. Read, Jr. 1971. Fate of coliforms in yogurt, buttermilk, sour cream, and cottage cheese during refrigerated storage. J. Milk Food Technol. 3454-58. Goepfert, J.M., N.F. Olson, and E.H. Marth. 1968. Behavior of Salmonella typhimuriurn during manufacture and curing of Cheddar cheese. Appl. Microbiol. 16:862-866. Gohil, V.S., M.A. Ahmed, R. Davies, and R.K. Robinson. 1995. Incidence of Listeria spp. in retail foods in the United Arab Emirates. J. Food Prot. 58:102-104. Gohil, V.S., M.A. Ahmed, R. Davies, and R.K. Robinson. 1996. Growth and survival of Listeria monocytogenes in two traditional foods from the United Arab Emirates. Food Microbiol. 13:159-164. Goz, M., and A.T. Cengiz. 1992. Incidence of Listeria monocytogenes in various cheese species. In: Proceedings of the XIth International Symposium on Problems of Listeriosis, Copenhagen, Denmark, May 11- 14, p. 160- 161. Greenaway, C., and P.G. Drew. 1990. Survey of dairy products in Victoria, Australia for Listeria species. In: Posters and Brief Communications of the XXIII International Dairy Congress, Montreal, Canada, Oct. 8-12. Abst. 234. 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. Griffith, M., and K. Deibel. 1988. Survival of Listeria monocytogenes in yogurt and acidified milk. Annual Meeting of American Society for Microbiology, Miami Beach, FL, May 813, Abstr. P-2 1. Gul, K., A. Suay, M.N. Dag, M. Mete, and 0. Mete. 1995. Isolation of Listeria species from cheese samples collected in the province of Diyarbakir. Turkish J. Infect. 9:45-46. Hapke, B. 1989. Personal communication. Hicks, S.J., and B.M. Lund. 1991. The survival of Listeria rnonocytogenes in cottage cheese. J. Appl. Bacteriol. 70:308-314. Hughey, J.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. 63 1-638. Ibrahim, G.A.M., H. Domjan-Kovacs, A. Fabian, and B. Ralovich. 1992. Listeria in milk and dairy products in Hungary. In: Proceedings of the XIth International Symposium on Problems of Listeriosis, Copenhagen, Denmark, May 1 1-14, p. 297-298. Ikonomov, L., and D. Todorov. 1964. Studies of the viability of Listeria rnonocytogenes in ewe’s milk and dairy products. Vet. Med. Nauki, Sofiya 7:23-29. International Dairy Federation. 1989. Pathogenic Listeria-Abstracts of replies from 24 countries to questionnaire 1288/B on pathogenic Listeria. Circular 89/5, March 3 1, International Dairy Federation, Brussels. Johnson, E.A., J.H. Nelson, and M. Johnson. 1990. Microbiological safety of cheese made from heat-treated milk, Part I. Executive summary, introduction and history. J. Food Prot. 531441-452. Johnson, E.A., J.H. Nelson, and M. Johnson. 1990. Microbiological safety of cheese made from heat-treated milk, Part 11. Microbiology. J. Food Prot. 5 3 5 19-540.

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Johnson, E.A., J.H. Nelson, and M. Johnson. 1990. Microbiological safety of cheese made froni heat-treated milk, Part 111. Technology, discussion, recommendations, bibliography. J. Food Prot. 53:610-623. Katic, V. 1995. The survival of Listeria monocytogenes in white brined cheese. Acta Vet. (Beograd) 45:3 1-36. Kaufmann, U. 1990. Behavior of Listeria monocytogenes in raw milk hard cheeses. Revue Suisse Agric. 225-9. Ken-, K.G., N.A. Rotowa, and P.M. Hawkey. 1992. Listeria in yoghurt? J. Nutr. Med. 3: 27-29. Khattab, A.A., A.M. El-Leboudy, and H.M. Ahmad. 1993. Survival of Listeria monocytogenes in yoghurt and acidified milk during storage at 4-5°C. Egypt. J. Food Sci. 21:41-48. Kinderlerer, J.L., and B.M. Lund. 1992. Inhibition of Listeria monocytogenes and Listeria innocua by hexanoic and octanoic acids. Lett. Appl. Microbiol. 14:271-274. Kinderlerer, J.L., H.E. Matthias, and P. Finner. 1996. Effect of medium-chain fatty acids in mould ripened cheese on the growth of Listeria monocytogenes. J. Dairy Res. 63593-606. Kovincic, I., I.F. Vujicic, M. Svabic-Valhovic, M. Vulic, M. Gagic, and I.V. Wesley. 1991. Survival of Listeria monocytogenes during the manufacture and ripening of Trappist cheese. J. Food Prot. 54:418-420. Lafaivre, J. 1988. Personal communication. Larson, A., E.A. Johnson, and J.H. Nelson. 1993. Behavior of Listeria monocytogenes and Salmonella heidelberg in rennet whey containing added sodium and/or potassium chloride. J. Food Prot. 56:385-389. Lewis, S.J., and J.E.L. 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. Loncarevic, S., M.-L. Danielsson-Tham, and W. Tham. 1995. Occurrence of Listeria monocytogenes in soft and semi-soft cheeses in retail outlets in Sweden. Intern. J. Food Microbiol. 26:245-250. Lopez-Diaz, T.M., J.A. Santos, C.J. Gonzalez, B. Moreno, and M.L. Garcia. 1995. Bacteriological quality of a traditional Spanish blue cheese. Milchwissenschaft. 50503-505. Lukasova, J. 1993. Influence of milk cultures on the survival of Listeria monocytogenes in milk. Prumsyl Potravin 44: 158- 160. 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 faeces, sewage and soil from urban sources. Int. J. Food Microbiol. 21:325-334. Mackey, B.M., and N. Bratchell. 1989. The heat resistance of Listeria monocytogenes: A review. Lett. Appl. Microbiol. 9:89-94. Maheswari, R.R.A., and M. Gueguen. 1990. Geotrichurn candidurn inhibiteur de Listeria monocytogenes? Posters and Brief Communications of the XXIIIrd International Dairy Congress, Montreal, Canada, Oct. 8- 12, Abst. 701. Maisner-Patin, S., N. Deschamps, S.R. Tatini, and J. Richard. 1992. Inhibition of Listeria monocytogenes in Camembert cheese made with a nisin-producing starter. Lait 72:249263. Marier, R., J.G. Wells, R.C. Swanson, W. Callahan, and I.J. Mehlman. 1973. An outbreak of enteropathogenic Escherichia coli foodborne disease traced to imported French cheese. Lancet 2: 1376- 1378. Martin, von F., K. Friedrich, F. Beyer, und G. Terplan. 1995. Antagonistische Wirkungen von Brevibacterium linens-Stammen gegen Listerien. Arch. Lebensmittelhygiene 46:7- 1 1. Mas, M., and J. Gonzalez-Crespo. 1993. Control de microorganismos pathogenos en Queso de los Ibores. Alimentaria 3 1:41-44. Massa, C.C. 1996. Microbiological quality of cheese: importance of good processing. Alimentaria 34:69-72.

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13 Incidence and Behavior of Listeria monocytogenes in Meat Products JEFFREY M. FARBER AND PEARL 1. PETERKIN Health Canada, Ottawa, Ontario, Canada

INTRODUCTION Only within the past 10 years have there been cases of human listeriosis traced to meat, with evidence for transmission of listeriosis through consumption of contaminated meats and meat products (see Chap. 10). Products such as piit&, turkey frankfurters, and sausages have been implicated [ 11 1,1151. In a valuable review, Jay [ 1 111 has summarized reports on the prevalence of listeriae in meat products from 1971 to 1994 worldwide. Recently, leading researchers in the United States [13] and the United Kingdom [ 1441 have stated that all Listeria monocytogenes strains should be considered as potentially pathogenic. With this in mind, and given the ubiquity of this pathogen within slaughterhouse and meat-packing environments, it is not surprising that the incidence and behavior of L. monocytogenes in meat products are receiving increased attention worldwide. In the beginning of this chapter, results are given for both the IJ.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) Listeria-testing program for cooked and ready-to-eat meat products and the Agriculture Canada Listeria monocytogenes-monitoring program for ready-to-eat meat products. Thereafter results from nonregulatory, independent surveys of raw meat and of various meat products marketed in North America, 505

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Europe, and elsewhere will be discussed. Information regarding behavior of L. monocytogenes in raw meat, cooked meat, and various sausage products can be found in the second half of this chapter.

INCIDENCE OF L/ST€R/A IN MEAT PRODUCTS Results from USDA-FSIS Listeria-Monitoring Programs The USDA-FSIS monitoring/verification program for L. rnonocytogenes in meat products began in September, 1987, with sampling of domestic corned beef, cooked corned beef, and massaged corned beef, as well as imported cooked meats. This program was later expanded to include a much wider range of products (Fig. l), with meat/poultry salads and spreads being added most recently [93]. Under the revised Listeria policy (Fig. 2), which appeared in the May 23, 1989, issue of the Federal Register [55], 25-g rather than 1-g monitoring samples obtained from intact retail packages (e.g., frankfurters, luncheon meat, sausages) are now examined for L. monocytogenes. If any of these samples contain L. rnonocytogenes, the entire lot manufactured on the day or during the shift the positive monitoring sample was taken is considered adulterated. Unlike the previous policy, which required identification of L. rnonocytogenes in one or more verification samples before initiation of further action, the present policy grants USDA-FSIS officials power to request firms to issue an immediate Class I recall for all tainted lots in the marketplace. However, manufacturers can avoid a formal recall by holding all sampled lots until results of Listeria testing are known. In either event, government officials also will (a) initiate a hold-test program until consistent production of Listeria-free product has been achieved; (b) collect and analyze other potentially contaminated products manufactured at the same facility; (c) encourage firms to review their opera-

I

I I

I

Plans for LIsteda testlng program announced - December 1985

4

Developmentof USDA method and sampltngscheme for detectinglkWa in meat prod= Announcement of I.M&I

.c

I I

1986

monitoringhrerlficationprogram- March 1987

1

I

1

inmate various samplingprograms

IJerky - October 1988to present1

products - September 1987 to present

Cooked beef, roast beef. and cooked c o r m beef September 1987 to present

I

I

I

sausage - September

FIGURE1 Chronological development of the USDA/FSIS testing programs for Listeria in cooked and ready-to-eat meat products. (Adapted from Ref. 93.)

Listeria monocytogenes in Meat Products

MONITORING:

Intact retail packages - e.g., frankfurters, sausage, luncheon

meat

1

507

MONITORING: Non-intact samples from a large unpackaged product - e.g., roast beef, corned beef

1

Homogenize 25-g sample in 225 ml enrichment broth

Homogenize 25-8 sample in 225m1 enrichment broth

L. monocytogenesdetected using USDA Method

L. monocytogenesdetected using USDA Method

+

1

1. FSIS rgquests immediate Class I

recall of the sample production lot

2. FSIS initiates (a) Hold-test program for contaminated product

4

+

VERTFICATTON: Intact sample fiom subsequent

1 + L. monocytogenes detected using lots Homogenize 25-g sample in 225 ml enrichment broth USDA Method

(b) Testing of other potentially contaminated products (c) Microbiological Incident Surveillance Sampling Program

FIGURE2 USDA/FSIS Monitoring/Verification Program for L. rnonocytogenes in cooked and ready-to-eat meat products. (Adapted from Ref. 55.) tions for conditions that may allow growth of listeriae, and (d) take any additional appropriate in-factory action to prevent production of contaminated product. The current policy regarding large products that are not normally available in retailsized packages (e.g., roast beef, corned beef) (see Fig. 2) more closely resembles the September 1 987 version of the monitoring/verification program in that USDA-FSIS officials will not request an immediate Class I recall if L. rnonocytogenes is detected in one or more monitoring samples. In such instances, verification samples (intact samples from subsequent lots) will be collected and analyzed for L. rnonocytogenes using current USDA methodology. Any verification sample that is positive for L. monocytogenes will automatically trigger action similar to that just described for products in intact retail-sized packages weighing 1 3 lb; included is the issuance of an immediate Class I recall in the unlikely event that the firm failed to hold all sampled lots until results of Listeria testing became known.

Raw Meat In January 1987, the USDA-FSIS began a monitoring and sampling program to determine the incidence of L. rnonocytogenes in domestic raw beef. This program was not undertaken to initiate regulatory action against particular firms but rather to provide the agency with critical background information. During the 26-month period from January 1987 to February 1990, L. rnonocytogenes was isolated from 122 of 1726 (7.1%) 25-g samples of domes-

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tically produced raw beef [64]. In 1995, the FSIS reported on the levels of bacteria, including L. monocytogenes, in ground beef [21]. The survey, based on 600 1-lb samples of ground beef from 661 plants, reported an 18% incidence for the organism.

Cooked a n d Ready-to-Eat Meat Products The USDA-FSIS monitoring/verification program, started in September of 1987, is designed to determine the extent of L. monocytogenes contamination in both domestic and imported cooked and ready-to-eat meat products. These products now include beef jerky, cooked sausage (both large and small diameter), cooked/roast/corned beef, meat salads/ meat spreads, and sliced canned ham/luncheon meat (see Fig. 1). Each sample collected, consisting of one to six subsamples, represents one lot of product. A portion of each subsample is then pooled to form a composite, which is analyzed. If a positive result is obtained for a lot, the composite is not reanalyzed to determine the number of positive subsamples in the lot. Results from this program for the years 1993-1996 (Table I ) show that, in general, there is a low incidence of L. monocytogenes in cooked and ready-to-eat meat products in the United States, with overall incidences from 0 to 8.1%. The organism was usually absent from beef jerky, as its presence was reported in this product only once, in 1994, at a 2.2% incidence. Cooked small-diameter sausage showed a higher prevalence of the organism than cooked large-diameter sausage, with the incidences ranging from 3.7 to 5.3%, and 1.O to 2.1 %, respectively. L. monocytogenes was present in only 2.1-3.4% of samples of domestic/imported cooked beef, roast beef, and cooked corned beef over this 4-year period. In most instances, these products were removed from their packages after cooking and then repackaged for sale, thus suggesting that the pathogen most likely entered the product through direct contact with the factory environment, knives, and/or gowns worn by workers [ 1071. At least two other firms handled raw and finished product in the same production area of the factory but at different times, which, in turn, indicates the possibility of cross contamination between raw and finished product. In December 1987, USDA-FSIS officials added sliced canned ham and sliced canned luncheon meat to the monitoring/verification program for the presence of L. monocytogenes (see Fig. 1). From 1993 to 1996, the pathogen was detected in 5.1-8.1% of samples, with these products exhibiting some of the highest incidences among the ready-to-eat meat

TABLE 1 Incidence of Listeria rnonocytogenes in USDA-FSIS Monitoring Samples of Cooked and Ready-to-Eat Meat Products, 1993-1996 Number of lots sampleda (% positive) Meat product Beef jerky Cooked sausage-large diameter Cooked sausage-small diameter Cooked/roast/corned beef Salads/spreads Sliced ham/sliced luncheon meats

1993 39 328 472 426 273 149

(0) (2.1) (5.3) (3.1) (2.2) (8.1)

1994 45 438 602 479 580 232

(2.2) (1.1) (4.8) (2.1) (2.4) (5.6)

1995

50 438 611 560 597 99

Each sample, consisting of one to six subsamples, represents one lot of product. Source: From Ref. 25. a

(0) (1.1) (4.1) (2.7) (4.7) (5.1)

1996 43 420 562 507 554 91

(0) (1.0) (3.7) (3.4) (2.2) (7.8)

Listeria monocytogenes in Meat Products

509

products examined (see Table 1). In June 1988, monitoring of meat/poultry salads and spreads was begun, with incidences of 2.2-4.7% being reported from 1993 to 1996. After a request from the food industry that the FDA reconsider its policy of “zero tolerance” for the presence of L. rnonocytogenes in cooked and ready-to-eat foods, the FDA stated that it would need the support of scientific data on infectious dose to justify any change in its regulation that the pathogen should not be present in such foods [ 111. Later, the FDA announced that its pathogen-monitoring program would concentrate on selected high-risk foods, including prepared sandwiches [22]. The agency stated that if a prepared sandwich is found to be positive for L. monocytogenes, then a comprehensive follow-up inspection of the firm is warranted, including sampling of raw materials, food processing surfaces, and finished product.

Recalls and Other Regulatory Actions Between 1990 and 1997, there were at least 12 separate Class I or voluntary recalls of cooked and ready-to-eat meat contaminated with L. rnonocytogenes (Table 2). Of these, seven recalls were for prepared sandwiches, three were for ham products, and the remaining two for frankfurters. Thus, over half of all recalls issued for cooked and readyto-eat meat products since 1990 have involved prepared sandwiches, with at least 60,000 sandwiches being removed from the marketplace. In all but one of these recalls of prepared sandwiches, the distribution involved at least four states. Product losses may be higher than indicated, since firms holding tested products until results of Listeria analyses become known can “recall” all contaminated lots internally, thereby avoiding a formal Class I recall. Considering the wide variety of tainted sandwiches that have been withdrawn from sale, it appears that L. monocytogenes entered these products during cutting, slicing, and/ or packaging rather than being initially present in the many different types of sandwich fillings. The two recalls of ham salad in 1991, one originating in Minnesota and the other in West Virginia, were each confined to one state (see Table 2). The quantities involved were smaller than those for recalls of prepared sandwiches, with 460 and 600 Ib of products being recalled, respectively. The frankfurter recalls in 1991 and 1992 were also confined to one state each; namely, Michigan and Connecticut. These quantities were larger than for the ham salad, with over 3000 Ib of product being recalled in both instances.

Results from Agriculture Canada Listeria monocytogenesMonitoring Programs

Cooked and Ready-to-Eat Meat Products Agriculture Canada started its L. rnonocytogenes monitoring program on processed and ready-to-eat meat products from registered processing plants in 1987. At the same time, the Health Protection Branch (HPB), Health Canada, started a similar testing program for L. rnonocytogenes in ready-to-eat meat products from nonregistered establishments [7 11. Five subsamples of 150 g (or a convenient package size) each were collected. The quantity analyzed depended on the product: For meats supporting growth of Listeria spp., 25 g was tested, whereas for meats not supporting growth of Listeria spp., 5 g was tested. During 1989- 1990, the incidence of L. monocytogenes in the ready-to-eat meat samples tested by the Agriculture Canada monitoring program was high: an average of about 24% (Table 3). During this period, samples taken at establishments with Listeria-

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TABLE 2 Chronological List of Recalls (Class I or Voluntary) Issued in the United States for Cooked and Ready-to-Eat Meat Products Contaminated with Listeria rnonocytogenes, 1990-1997 Product Sandwiches-BBQ beef, ham and cheese, etc. Ham salad Sandwiches-roast beef, etc. Skinless hot dogs Ham salad Frankfurters-beef, pork Sandwiches-roast beef, turkey, subs, ham and cheese, BBQ beef, etc. Sandwiches-beef, ground beef, pork, ham and cheese, ham salad, hot dog, BBQ beef, etc. Sandwiches-ham and cheese, ham salad, etc. Sandwiches-roast beef, hot dog, burgers, sausage, meat ball, ham and cheese, etc. Sandwiches-hot dog, ham and cheese, sausage, beef, salami, etc. Smoked ham NA. not available.

Date recall initiated

Origin

Distribution

Quantity

Ref.

-4860 units 460 lb -8057 units 3700 lb 600 lb 3578 lb NA

16 16 16 14 15 17 18

NA

19

20, 563 units 20-25,000 units

16 16

1991 1991 07/03/9 1 1991 1991 03/20/92 06118/92

LA MN LA MI

LA, FL,AL, MS MN LA, FL, AL, MS MI

CT LA

CT LA, FL,AL, MS

09/25/92

TN

09/08/95 02/16/96

MI LA

TN, NC, SC, GA, AL, MS, KY, VA, AS MT, IN, OH, IL, WI LA, FL, AL, MS

0 1122197

MS

MS

NA

23

07131/98

NH

NH, VT

NA

25a

wv

wv

Listeria monocytogenes in Meat Products

51 1

TABLE 3 Incidence of Listeria monocytogenes in Agriculture Canada Testing Samples of Domestic and Imported Ready-to-Eat Meat Products and in Processing Plants, 1989- 1994 Domestic products no. of samples Year 1989 1990 1991 1992 1993 1994

(% positive)

396 66 19 35

(12) (35) (0) (3)

NDb ND

Imported products no. of samples (% positive) USA

50 1279 984 469

(4) (2) (2) (3)

417 (3) 382 (5)

Plant environment

Others

5 3 14 9

(0) (0) (0) (0)

1 (0) 1 (0)

no. of samples (% positive) 677 (17) 2267 (8) 3314 (13) Phase Ia ~3808 (10) 2522 (6) 3629 (2)

Phase I1 ~

Phase I11 ~

430 (9) 124 (19) 138 (6)

164 (13) 20 (0)

a Phase 1-10 composited postprocess samples of product contact surfaces. Phase 11-if Phase I positive, 10 individual environmental samples. Phase 111-if Phase I1 positive, review of plant procedure prior to further individual sampling. ND, not determined. After 1992, environmental testing only was done for domestic products. Source: From Ref. 8.

positive products showed an incidence of 46% for meat products (data not shown) and an average of 12% for environmental samples. The incidence dropped sharply during 1991-1992 to levels of 0-3% for meat products, but the contamination in the factory environment remained about the same. After 1992, only factory environmental samples were taken for testing domestic products, but a more rigorous testing procedure involving three phases was instituted. Phase I of the program consisted of taking 10 postprocess environmental samples of a product contact surface. These 10 samples were composited into one sample for analysis. Any positive Phase I analysis triggered entrance into Phase 11, consisting of 10 repeat environmental samples, analyzed individually, and a review of good manufacturing practices (GMP) throughout the plant. Phase 111 was entered if one or more analyses in Phase I1 were positive, in which case a thorough review of all manufacturing procedures was undertaken before collecting further environmental samples for individual analysis. Between 1992- 1994, the incidence of L. monocytogenes in food processing environments dropped sharply, indicating the value of this approach. The presence of L. monocytogenes in imported ready-to-eat meat products during this period remained low, with incidences ranging from 0 to 5% (see Table 3). The importance to Canadian consumers of imports from the United States compared with other countries is evident from the the relatively large number of American samples tested.

Recalls and Other Regulatory Actions The current Canadian policy on control of L. monocytogenes in foods is risk based. Thus, for high-risk meats such as liver pit6 and jellied pork tongue, which have been causally linked to listeriosis, a Class I recall would be initiated at the retail level. However, all other ready-to-eat meats that support growth of L. monocytogenes and have a refrigerated shelf life of >10 days would only be subject to a Class I1 recall. In Canada between

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TABLE 4 Class II Recalls issued in Canada for Processed and Ready-to-Eat Meat Products Contaminated with Listeria monocytogenes, 1989-August 1997 Number of recalls

Year

wieners

1989 1990 1991 1992 I993 1994 1995 1996 1997 Totals

4 1

dry fermented sausages

sandwiches1 subs

1 1

7

2 20

sliced roast beef

cooked ham

2 1

1

8

2 15 1 19

4

3

12

1 9

1 1

miscellaneousa

2 3 1

2 8

Pizza, burrito, cretons, pork ribs, ham salad Source: From Refs. 9 and 24. a

1989-1997, 20 recalls were ordered for dry, fermented sausages, some of which were smoked (Table 4). An almost equal number of recalls were initiated for sandwiches, including submarine sandwiches, containing meat. Sliced cold meats of various types also were found to contain L. monocytogenes during this period, possibly because of contamination from retail slicers. Wieners and miscellaneous products comprised the balance of the recalls.

Incidence of Listeria spp. in Raw Meat Slaughter animals are a recognized reservoir of human pathogens, including L. monocytogenes. Numerous studies have shown that, in general, the incidence of listeriae is low (09%), both in feedlot cattle [166] and in pork carcasses [2,82,150,159]. However, a 96% incidence for listeriae was reported on pork carcasses from one slaughterhouse [82], thus suggesting poor sanitation. The incidence of listeriae on slaughterhouse equipment was also low (0-3%) [82,154] except for the factory mentioned above where the incidence was reportedly 42% [82]. Levels of the organism were low (from 10 to 100 CFU/g) [82]. The only factory area yielding L. monocytogenes was a cold room maintained at 5"C, at which temperature this psychrotrophic organism is able to grow.

North America In addition to the government monitoring programs in North America, there are several reports of nonregulatory surveys of both raw and ready-to-eat meat products (Table 5). Other authors also found that in raw meat products such as roasts and steaks sampled in the United States, the incidence of Listeria spp. ranged from 0 to 6%, with L. monocytogenes being in the same range (Table 5). In contrast, comminuted raw meats showed a much higher incidence of listeriae, from 24 to 1OO%, with L. monocytogenes ranging from 0 to 25%. Reported levels of listeriae ranged from 4.1 X 103to 2.1 X 104 CFU/g. In raw meat products sampled in Canada (principally ground meats), the incidence

Listeria monocytogenes in Meat Products

513

TABLE 5 Incidence of Listeria monocytogenes in R a w M e a t a n d Ready-to-Eat M e a t Products in t h e USA a n d Canada, 1990-1997

Product Raw meats-USA beef roast pork roast lamb roast ground beef ground pork pork sausage lamb patty beef pork loin Raw meats-Canada ground beef’ ground pork ground veal ground meat meat cuts wild animal meatmoose, deer, bear RTE products -USA wieners wieners wieners RTE products -Canada fermented sausages cooked meat wieners luncheon meats sausages

Number of samples analyzed

Percentage of positive samples

Listeria spp.

50 50 10 39 20 17 2 658 135

-

LM

Levels of Listeria spp. (CFU/g)

LM- 10 4-560 4-240 240-2 1000 4-56

Ref. 116 1 I6 116 192 192 192 192 111 159

22 19 3 11 18 10

73 73 73 135 135 135

93 24 30

181 181 20

30 16 38 67 9

73 170 I70 170 170

LM, L. monocytogenes; ND, not determined; RTE, ready-to-eat.

of Listeria spp. is high (ranging from 66 to 100%).The incidence of L. monocytugenes ranged from 44 to loo%, indicating that contamination of raw meat with this pathogen is relatively common (see Table 5). This may be the result of contaminated equipment at either the wholesale or retail level. It is noteworthy that there was a much lower incidence of listeriae in wild rather than domesticated animals.

Europe Since 1990, many reports have been published on the prevalence of Listeria spp. and L. monocytogenes in raw meat and raw meat products produced in various European countries (Table 6). In the United Kingdom, MacGowan et al. [ 1361 reported incidences of Listeria spp. ranging from 59 to 88% in beef, lamb, and pork, with L. monucytugenes being preseni in 28-40% of these samples. Based on a larger sampling of beef, lamb, and pork in Northern Ireland, Wilson [184] reported an incidence of about 4% for listeriae.

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TABLE 6 Incidence of Listeria monocytogenes in Raw Meat in European and Other Countries, 1990-1997

Country United Kingdom

Ireland

Norway Germany Italy

Switzerland

Spain Poland Bosnia/Hercegovina

Product

Number of samples analyzed

Pork sausage Beef Lamb Pork Sausage Meat Beef Lamb Pork Beef Ground beef Pork Lamb Sausage Frozen beef burgers Ground meat Ground meat Rinderhack Calf Horse Pork Mutton Sausage casing surface Pork Beef Ground beef Pork Ground meat Beef Raw meat Sausage Ground meat Beef Pork Sausage meat Dried meat Ham-uncooked Ground meat Ground pork Ground beef Pork Beef Beef Pork

59 26 20 32 23 15 1295 37 794 20 85 20 20 20 94 40 21 59 19 19 19 18 156 116 67 99 148 153 308 174 82 30 85 18 31 102 44 19 168 42 41 245 114 20 50

% of positive

samples

Listeria spp.

LM

Ref. 79 136 136 136 136 96 184 184 184 163 163 163 163 163 163 157 111 111 138 138 138 138 43 43 179 179 52 52 53 53 131 131 38 38 38 38 38 38 61 139 139 126 126 133 133

Listeria monocytogenes in Meat Products

515

TABLE 6 Continued ~-

Country

Product

Bulgaria Trinidad

Beef and pork Beef Ground beef Mutton Goat meat Pork Beef Goat Sheep Camel Beef Lamb Pork Beef Cattle Buffalo Sheep Goat Beef Ground beef Pork Ground pork Pork Beef Lamb Beef steak Pork

United Arab Emirates

Australia Malaysia India

Japan

China Taiwan

Number of samples analyzed

234 76 35 66 70 71 15 17 24 14 50 50 50 12 54 54 54 54 15 5 18 6 25 10 14 25 34

% of positive .

samples

Listeria spp.

LM

Ref.

151 1 1 1

1 1 86 86 86 86 110 110

110 26 37 37 37 37 158 158 158 158 182 182 182 187 187

LM, L. rnonocytogenes; ND, not determined. a 5-500 Listerra CFU/g. 5- 10,000 Lisferia CFU/g.
According to Gilbert 1791, 49% of raw pork sausage harbored L. monocytogenes, with MacGowan et al. [136] reporting a similar incidence of 35%. Working in Ireland, Sheridan et al. [ 1631 observed high incidences of listeriae ranging from 45 to 85% in 20 samples each of beef, pork, and lamb, with L. monocytogenes being present in 1 5 5 0 % of the samples. Interestingly, less contamination was observed in comminuted meats (94 samples of frozen beef burgers, 85 samples of ground beef, and 20 samples of sausage), with L. monocytogenes being present in only 5- 18% of the samples. An incidence of 5% for L. monocytogenes in ground meat was found in Norway [157], with the pathogen belonging to serotype 1 . In Germany, about 50% of ground beef and raw beef products reportedly contain L. rnonocytogenes 11113. Since raw minced meat is a popular dish in Germany, the German

5 16

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Ministry of Health decided to restrict the sale of contaminated product [7]. For samples containing <100 CFU/g, the factory or retail outlet undergoes further surveillance, and a public warning is issued if numbers of listeriae reach 2 1000 CFU/g. Workers in Italy have examined many types of raw meat for listeriae. Maini et al. [138] reported that in 19 samples each of calf, horse, pork, and mutton, the incidences of listeriae were 63, 26,53, and 56%, respectively. Cantoni et al. [43] showed that the prevalences of the organism on sausages and their casing surfaces were roughly equal; that is, 60 and 47% for listeriae and 13 and 12% for L. monocytogenes, respectively. Other surveys of fresh pork and beef showed incidences ranging from 21 to 47% for listeriae and 13 to 22% for L. monocytogenes, with serotype 4 being found in 7% of these samples [43]. Comminuted meats and sausages showed higher incidences for Listeria spp. (44-54%) than for L. monocytogenes (9- 19%) [52,53,13I], with serotype 1/2c predominating [52]. Listeria-positive samples generally contained < 100 CFU/g. Breer and Schopfer [38] reported that listeriae were recovered from 11-45% of 209 samples of beef, pork, and pork products collected in Switzerland, with an incidence of 0- 15% for L. monocytogenes. In comminuted meat products, the corresponding incidences were 40-65% and 8- 15%, respectively. Reports from Spain showed that of 25 1 samples of ground meat, 63-80% contained listeriae, with L. monocytogenes being recovered from 17-29% of the samples [61,139]. In Eastern European countries, workers have observed similar levels (8-20%) of listeriae contamination in 613 samples of fresh beef and pork to those found in Western Europe [ 126,133,1511. The incidence of L. monocytogenes in these samples ranged from 7 to 10%, with a prevalence (94%) of serotypes 1 to 3 and the remaining being serotype 4 [126].

Other Countries Reports from elsewhere indicate that the problem of Listeria-contaminated meat is worldwide and deserves the serious attention of the industry. In general, the prevalence of Listeria spp., and more particularly, L. monocytogenes, is in the same range as that already discussed for North America and Europe. In Trinidad, Adesiyun [ I ] identified listeriae in 0- 10% of samples of fresh beef, mutton, and goat meat, with L. monocytogenes being present in 0-4% of the samples. The organisms were prevalent in ground beef samples at similar levels: 1 1 and 6%, respectively. L. monocytogenes serotypes 1/2c and 4 were found in both local and imported meat. Similar incidences of listeriae have been reported in the Asian countries. In the United Arab Emirates, Gohil et al. [86] examined fresh beef ( I 5 samples), goat (17 samples), sheep (24 samples), and camel (I4 samples) and found that listeriae were present in 0-21% of the samples. No samples contained L. monocytogenes. Among 50 samples each of fresh beef, lamb, and pork examined in Australia, listeriae were found in 34, 40, and 30% and L. monocytogenes in 24, 16, and 10%, respectively [ 1 101. In Malaysia, 50% of 12 beef samples contained L. monocytogenes [26]. Examination of 54 samples each of cattle, buffalo, sheep, and goat in India by Erahmbhatt and Anjaria [37] yielded a low incidence of L. monocytogenes (4-6%). Ryu et al. [ 1581 reported high incidences for listeriae in Japanese meats, with 40 and 13% of fresh beef and and 61 and 39% of fresh pork yielding Listeria spp. and L. monocytogenes, respectively. In ground beef and pork samples, the prevalence was higher, with 80 and 60% and 100 and 67% of the samples being positive, respectively. A study done by Wang et al. [182] in China showed a high incidence (from 43 to

Listeria monocytogenes in Meat Products

517

70%) of listeriae in pork, beef, and lamb. However, the incidence of L. monocytogenes was much lower, ranging from 0 to 28%, with serotypes 1/2a, 1/2b, and 1/2c being identified. In contrast, meats from Taiwan showed a high incidence, with 24 and 59% of all beef steak and pork samples respectively being positive for L. monocytogenes [ 1871.

Incidence of Listeria spp. in Sausage and Ready-to-Eat Meat Products The prevalence of L. rnonocytogenes in processed, ready-to-eat meats which may be consumed without further heating is obviously of greater concern than contamination of raw meats. Given that one survey of retail meat slicers revealed an L. monocytogenes contamination rate of 13%, such machines can easily serve to cross contaminate sliced meats [ 1071. Two factors, namely, development of improved Listeria isolation methods and a heightened concern about foodborne listeriosis, have led scientists to examine sausage, piti, and other ready-to-eat meat products for listeriae. Consequently, numerous worldwide surveys have been conducted since 1990 to determine the incidence of these organisms in such products.

North America Since the association of sporadic listeriosis with consumption of uncooked frankfurters [ 1611, several nonregulatory United States surveys were initiated to determine the incidence of listeriae in packages of retail wieners (see Table 5). After examining 20 brands of retail wieners, Wang and Muriana [ 18 11 reported that 19 brands (93 samples) showed a 10% incidence of listeriae, with an 8% incidence of L. monocytogenes. However, one brand contained listeriae in 83% of its 24 packages, with a 7 1% incidence of L. monocytogenes. Most listeriae were found in the liquid exudate at a level of 1-3 CFU/mL, rather than in the meat itself, thus indicating probable postprocessing contamination. A survey commissioned by the Los Angeles Times found that, in a sample of 30 packages of retail wieners, 20% contained listeriae, with a 17% incidence for L. rnonocytogenes [20]. A Canadian study [ 1701 reported that wieners (38 samples) and sliced meats (67 samples) showed incidences of 13-26% for Listeria spp., and 13-21% for L. monocytogenes (see Table 5). In contrast, Listeria spp. were not isolated from cooked meat (16 samples) anti sausages (9 samples). However, fermented sausages (30 samples) studied by Farber et al. [73] yielded a 20% incidence for L. rnonocytogenes.

Europe Numerous surveys have been conducted to determine the incidence of listeriae in readyto-eat meat and sausage products manufactured in Europe (Table 7). Public Health Laboratory Service workers in England and Wales surveyed a large number of samples to determine the incidence of listeriae in various foods. McLauchlin and Gilbert’s report [145] on pit6 (696 samples) and ready-to-eat meat and poultry (3939 samples) showed listeriae present in 20 and 15% and L. monocytogenes present in 17 and 8% of the samples, respectively. A report on 605 samples of sliced meats showed a lower incidence for listeriae (6%) and L. monocytogenes (3%), with levels of <200 to <500 CFU/g [ 1771. Most (91%) L. rnonocytogenes isolates were serotype 1/2, with the remainder being serotype 4. In an extensive study conducted in Yorkshire 121, cooked meats (1 55 1 samples), pit6 (239 samples), meat salad (1 5 samples), and meat sandwiches (237 samples) revealed incidences of 13, 11, 33, and 20% for listeriae and 5, 7, 13, and 8% for L. rnonocytogenes,

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TABLE 7 Incidence of Listeria monocytogenes in Sausages and Ready-to-Eat Meat Products in European and Other Countries, 1990-1997

Country /Product United Kingdom pit6 RTE meat and poultry sliced meat ham salami tongue corned beef mixed meats prepared sandwiches cooked meats pit6 salad with meat sandwiches with meat cook-chill-meat and poultry salami pit6 pit6 cook-chill food-meat cook-chill food-meat pit6 cured or smoked meat cooked tripe Denmark sliced ham sliced rolled sausage sliced smoked pork loin frankfurters Norway VP processed meat France sausages, pitis, ham Italy pork sausage sausage-mixed meat ground beef rissoles sausage wiirstel pit6 Switzerland sliced cured dried beef salami mettwurst

No. of samples analyzed

% of positive

samples

Listeria spp.

LM

Levels of Listeria spp. (CFUlg)

Ref.

696 3939

145 145

303 128 28 27 119 91 1551 239 15 237 736

177 177 177 177 177 108 12 12 12 12 79

67 1834 626 992 854 216 29 44

<20- 1000 <20- 100 <20-100 20- 100

LM-<200-106 LM-<200- 105 LM-< 10- 103 LM-< 10-103

79 80 80 101 101 79 10 109

80 80 78 67

156 156 156 156

35

157

990

128

55 20 45 82 118 48

132 132 132 47 47 47

26 59 14

LM- 10-224

LM-20 LM-20

172 172 172

Listeria monocytogenes in Meat Products

5 19

TABLE 7 Continued

Country/Product Spain cured sausilge cooked ham or sausage luncheon meat pit6 Hungary dry cured sausage fermented sausage smoked sausage Yugoslavia fermented sausage “hot smoked” sausage VP hot smoked sausage South Africa Vienna sausage ham cervelat Australia piit6 luncheon meat pit6 processed meat luncheon meat VP salami VP corned beef VP ham VP luncheon meat salami ham corned beef pit6 luncheon meat New Zealand RTE pork RTE beef RTE lamb

No. of samples analyzed

% of positive

samples

Listeria spp.

LM

Levels of Listeria spp. (CFU/g)

17 15 60 36

1-100

1-100

Ref.

30 30 127 127

136 21 23

124 124 124

21 15 14

40 40 40

47 43 44

180 180 180

7 28 25 25 20 19 72 71 13 132 90 39 7 16

162 162 98 98 171 91 91 91 91 176 176 176 176 176

34 18 3

103 103 103

LM, L. monocytogenes; RTE, ready-to-eat; ND, not determined; VP, vacuum-packed.

respectively. Levels of the organism in this study ranged from <20 to 1000 CFU/g. Another report on 91 samples of preprepared sandwiches showed a 17% incidence for L. rnonocytogenes [ 1OS]. Of 13 strains examined, 10 were serotype 1 /2 and 3 were serotype 4. A survey of 1834 and 626 pi% samples in 1989 and 1990 showed incidences of 10 and 4%, respectively, for L. rnonocytogenes with levels of the organism up to 106CFU/ g [80]. Both serotypes 1/2 and 4 were present. In another study, L. rnonocytogenes was

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present in 16% of 67 salami samples [79]. Cooked tripe, which is often eaten without further heating, showed a 9% incidence of L. monocytogenes [ 1091. In cook-chill catering, often used by hospitals, food is prepared and cooked in a traditional manner, cooled very rapidly, and maintained chilled (0-3°C) for up to 5 days until reheated for use. In three surveys of cook-chill foods containing meat (736, 992, and 854 samples), the incidence of L. monocytogenes was 2, 3, and 9%, respectively [79,101]. Of 28 strains typed, 7 were serotype 1/2, 17 were serotype 3, and 4 were serotype 4 [ l o l l . These results must give rise for concern in light of the many highly vulnerable patients in hospitals. In Denmark, 305 samples of wieners and sliced meats showed similar incidences, 10% of which contained L. monocytogenes, at a level of < 100 CFU/g [ 1561. A Norwegian study of vacuum packaged processed meat showed that 11% of 35 samples contained L. monocytogenes [ 1571. In France, 22% of 18 dry sausages harbored L. monocytogenes, whereas in Germany, 9% of 11 mettwurst samples contained the organism [ 1111. Several surveys of sausages in Italy showed similar incidences to the above. Levrk et al. [132], reporting on 120 samples of pork sausage/mixed chicken, turkey, pork sausage/beef rissoles, found that overall as many as 90% of these samples contained listeriae, with 33% being positive for L. monocytogenes. In another study carried out over a 2-year period (1990- 1991), Casolari et al. [47], demonstrated similar incidences in sausage, with 88% and 28% of 82 samples showing the presence of Listeria and L. monocytogenes, respectively. Additionally, 36% of 166 meat product samples (wurstel, p2t6) contained listeriae, with L. monocytogenes being present in 4% of these samples at a level of 10-224 CFU/g. A survey in Switzerland taken during the production of cured and air-dried meat products [ 1721 uncovered a substantial number of Bundnerfleisch (cured, air-dried beef), salami, and mettwurst samples that contained listeriae. Overall, of samples taken during production, Bundnerfleisch (19 samples), salami (30 samples), and mettwurst (3 samples) contained 26, 50, and 100% of listeriae and 11, 10, and 0% of L. monocytogenes, respectively (data not shown). The endproducts showed listeriae at incidences ranging from 15 to 93%, in contrast to incidences ranging from 0 to 5% for L. monocytogenes. The organism was present at levels of <20 CFU/g. Listeriae were isolated only from the surface of Bunderfleisch. L. monocytogenes isolates were of serotype 1/2 (86%), and serotype 4 (14%). Since most L. monocytogenes isolates from human listeriosis patients in Switzerland belong to serotype 4b, these data suggest that transmission of the pathogen via meat is relatively uncommon. Surveys of ready-to-eat meat products in Spain showed a 13% incidence for Listeria spp. in cured or cooked sausages and ham (a total of 32 samples) [30]. Twelve percent of the cured sausages contained L. rnonocytogenes,with the pathogen being absent in ham and cooked sausage [30]. Among sliced meat (60 samples) and pi%&(36 samples), 42 and 14% of samples contained Listeria spp., with 22 and 3% of samples being positive for L. monocytogenes, respectively [ 1271. Both organisms were found at levels of 1- 100 CFU/g. In 1991, a Hungarian researcher, KovAcsn6 Domjan [ 1241, recovered listeriae from 37 of 136 (27%), 7 of 21 (33%), and 15 of 23 (65%) samples of dry-cured, fermented, and smoked pork sausage, respectively. L. monocytogenes was present in 10, 10, and 13% of these samples, respectively. Working in Yugoslavia, Buntid [42] found that 28 and 19% of 2 1 fermented sausage samples contained listeriae and L. monocytogenes, respectively. In contrast, listeriae were not recovered from hot, smoked sausage. Of 14 surface samples of vacuum-packed hot smoked sausage, 35 and 2 1% yielded listeriae and L. monocytogenes, respectively, indicating recontamination before and during vacuum packaging.

Listeria monocytogenes in Meat Products

52 1

Overall, the results from Table 7 indicate that a substantial portion of European processed meat is contaminated with listeriae, including L. monocytogenes. The presence of listeriae other than L. monocytogenes in processed as well as raw meat may be indicative of possible contamination with L. rnonocytogenes.

Other Countries A recent report by Vorster et al. [ 1801 in South Africa showed a low prevalence for listeriae in 134 retail samples of processed, ready-to-eat meats, with an overall incidence of 8% in Vienna sausage, ham, and cervelat (see Table 7). None of the samples yielded L. monocytogenes. Several surveys from Australia show incidences in processed meats which are similar to those reported from North America and Europe. According to Robertson and coworkers [162,171], l of 20 (5%) and 3 of 28 (1 l%) sliced meat samples yielded Listeria spp., with L. monocytogenes being recovered at rates of 0 and 7%, respectively. No Listeria spp. were recovered from seven samples of pgt6. Hobson et al. [98] reported that in 25 samples each of pgt6 and processed meat, L. monocytogenes was present in 8 and 4%, respectively. When 175 samples of vacuum-packaged processed meat were examined, Grau and Vanderlinde [91] reported that 60 of 72 (88%) siimples of corned beef, 29 of 71 (41%) samples of ham, 3 of 13 (23%) samples of luncheon meats, and 1 of 19 ( 5 % ) samples of salami yielded listeriae, with 72,34, 15, and 0% of these samples being contaminated with L. monocytogenes, respectively. Sixteen corned beef samples had listeriae counts >50 CFU/g, and, of these, six counts were >I000 CFU/g. Listeria counts for salami were <50 CFU/g, and no L. rnonocytogenes was found. In another survey involving 284 samples of ready-to-eat meat products, Varabioff [ 1761 found that except for pit&, which was free of listeriae, the incidence of listeriae ranged from 5 to 41% (see Table 7); L. rnonocytogenes was present in 4-19% of the samples. Of the isolates recovered, 62% were serotype 1 and 36% were serotype 4. Additional testing of cutting utensils and cutting boards showed that most of the contamination occurred at the retail level. A New Zealand survey of 55 samples of ready-to-eat meat products showed listeriae incidences ranging from 23 to 67% [ 1031. Interestingly, even though mixed-source products had a lower incidence (23%) of listeriae than products made from a single meat (5067%), at least half of the isolates from mixed meats were L. monocytogenes, whereas the pathogen was seldom recovered from single meat products. The authors also reported a significantly higher incidence for L. monocytogenes in smoked products than in meats preserved by other methods. Additional information concerning the various habitats, niches, and relative incidence of listeriae in all facets of the meat industry is needed, as it is now evident that the presence of L. rnonocytogenes in processed meat products may pose a serious health hazard to certain individuals. Therefore, it is necessary to control all listeriae in meat-processing facilities and to design procedures and treatments that will eliminate L. rnonocytogenes from ready-to-eat processed meat products.

BEHAVIOR OF L. MONOCYTOGENES IN MEAT PRODUCTS Although L. monocytogenes was found during the 1950s in European meat products destined for human consumption, a general failure to positively link cases of human listeriosis to foods other than raw milk provided little incentive to examine the behavior of listeriae in meat. This situation was further complicated by a lack of reliable and convenient methods to isolate listeriae selectively from heavily contaminated samples of animal origin,

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including organ as well as muscle tissue. Thus, the first definitive studies on the behavior of L. monocytogenes in raw meat were not begun until the 1970s. Even then, to quantitate this organism in meat products during extended storage accurately, it was generally deemed necessary to use specially treated “sterile” meat rather than raw meat products containing a normal microbial background flora. Outbreaks of foodborne listeriosis linked to consumption of contaminated cheese prompted concerns about the microbiological safety of raw and, particularly, ready-to-eat meat products marketed in North America and Europe. These outbreaks also demonstrated an urgent need for methods to detect listeriae rapidly and accurately in a wide range of foods. Subsequent development of the USDA procedure to detect listeriae in meat and poultry products provided researchers with a method by which to determine the growth and survival of L. monocytogenes in raw and ready-to-eat products. Recent meat-oriented epidemiological studies along with several large listeriosis outbreaks linked to meat products suggest that the behavior and control of L. monocytogenes in meat products is likely to remain an active area of research for some time to come. As in the previous section of this chapter, information concerning the behavior of L. monocytogenes in raw meat will be presented first, followed by a discussion of the fate of this pathogen in processed meat products.

Listerial Infections in Domestic Livestock As you will recall from Chapter 3, domestic farm animals such as cows, sheep, and pigs not only succumb to listerial infections but also asymptomatically shed L. monocytogenes in their feces for many months. Although virtually all meat from animals exhibiting obvious signs of listeriosis will be condemned and destroyed immediately after slaughter, meat from subclinically infected animals will likely be passed by inspectors as being fit for consumption. Since between 2 and 16% of all healthy cows, sheep, and pigs passively shed L. monocytogenes in their feces, ample opportunity exists for contamination of muscle tissue during slaughter, evisceration, and dressing of the animals. In all likelihood, meat from domestic livestock will first be exposed to listeriae in the slaughterhouse environment. However, various organs from apparently healthy animals can also occasionally contain L. monocytogenes. In 1972, Hohne [99] reported that approximately 13% of the parotid glands from clinically healthy pigs contained L. monocytogenes. Three years later, Hohne et al. [IOO] detected L. monocytogenes in intestinal lymph nodes from eight apparently healthy slaughtered animals (five small ruminants, two pigs, one cow) destined for human consumption. Subsequently, Cottin et al. [54] identified L. monocytogenes in 15 of 514 (3.1%) spleen and/or lung tissue samples obtained from apparently healthy cattle. According to Amtsberg et al. [3], L. monocytogenes was isolated from the spleen and muscle tissue of an apparently healthy animal. These findings suggest that L. monocytogenes might be transported to tissue via the blood stream in animals suffering from symptomatic as well as asymptomatic septicemic listerial infections. Hence, to understand the behavior of L. monocytogenes in meat products, it is fitting to begin this section by first examining the localization of L. monocytogenes in organ and particularly muscle tissue from infected animals.

Localization In Tissues In 1988, Johnson et al. [ 1 141 reported results from a study in which samples of muscle, organ, and lymphoid tissue as well as feces and blood from several Holstein cows were

Listeria monocytogenes in Meat Products

523

examined for L. monocytogenes 2, 6, or 54 days after intravenous inoculation (lO'o-lO1l L. monocytogenes cells) using a combination of direct plating and cold enrichment. As expected, recovery of listeriae varied among animals and was strongly influenced by the time that elapsed between inoculation and slaughter. Overall, 94% of all samples obtained from cows slaughtered 2 days after inoculation contained L. monocytogenes, with 23 of 32 (72%) samples being positive by direct plating. More important, the pathogen was routinely detected in muscle tissue from the same animals, frequently at levels of 120280 CFU/g. Despite a marked decrease in recovery of L. monocytogenes from animals examined 6 and 54 days postinoculation, the pathogen was still present at levels of 140675 CFU/g in kidney tissue as well as in mesenteric and mammary lymph nodes, with populations being about two orders of magnitude lower in liver and spleen tissue. Following 2 weeks of cold enrichment, the pathogen also was detected in plate-flank tissue taken from an animal 6 days after slaughter. These findings suggest that consumption of animal organs may constitute a greater health hazard than muscle tissue, and they also readily explain how evisceration of domestic animals can lead to surface contamination of muscle tissue. Assuming that most contamination occurs during handling of carcasses after slaughter, Chung et al. [49] investigated the ability of L. monocytogenes to attach to (or become entrapped) and proliferate on muscle and fat tissue both alone and in the presence of Pseudomonas aeruginosa. Immersion of lean muscle and fat tissue in broth cultures containing 10' or 108 L. monocytogenes CFU/mL for various times followed by thorough rinsing resulted in large numbers of listeriae being attached to (or entrapped in) both types of samples within the first 10 mins. Despite large differences in hydrophobicity, pliability, and surface qualities, L. monocytogenes adhered (entrapped) equally well to lean muscle and fat tissue during 50-60 min of incubation at ambient temperature. According to an earlier report by Herald and Zottola [97], attachment of L. monocytogenes to stainless steel and presumably meat surfaces is related to flagellae, fibrils, and exopolymeric substances (i.e., polysaccharides), all of which are readily produced by Listeria during extended incubation at room temperature. However, lean muscle tissue supported faster growth of the pathogen than fat tissue when samples were stored 1 day at ambient followed by 7 days at refrigeration temperature. Following attachment of L. monocytogenes and P. aeruginosa to lean meat at a concentration of 106 CFU/4 cm2, Listeria populations increased approximately 100-fold during 24 h of incubation at room temperature and remained at this level during 7 days of refrigerated storage regardless of the presence or absence oft'. aeruginosa. In contrast, populations of P. aeruginosa on lean meat increased > 100-fold during initial storage at room temperature, but then decreased with levels frequently 10 times lower than the Listeria population following 7 days of refrigerated storage. Dickson [62] simulated contamination of raw beef during processing, handling, and storage by placing surfaceF of heavily inoculated lean and fat beef tissue (-2 X 106 L. monocytogenes CFU/cm2) in direct contact with uninoculated tissue. Overall, transfer of listeriae was largely dependent on the type of tissue, with minimum and maximum transfer being observed from fat-to-fat and lean-to-fat tissue, respectively. However, bacterial transfer was also influenced by adsorption time of the original inoculum and contact time with uninoculated tissue, Adsorption times of <60 min generally led to the highest Listeriu transfer rates, particularly between inoculated and uninoculated lean beef tissue. These findings are readily explained when one considers that most listeriae are likely to be freely suspended (unattached) in water films on tissue surfaces shortly after inoculation. Follow-

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ing an adsorption time of 30 min, approximately 30 and 50% of the original Listeria inoculum migrated from lean and fat tissue, respectively, to lean tissue after 5 min of direct contact at ambient temperature. When contamination between fat and lean tissue was simulated using shorter contact times of 15-60 s, a greater percentage of listeriae migrated from inoculated fat to uninoculated lean tissue, which in turn likely reflects the transfer of cells in unadsorbed water from hydrophobic fat to hydrophilic lean tissue. More important, the fact that bacterial transfer also occurred at 5°C with 0.6-9.5% of the original Listeria population migrating from inoculated to uninoculated lean and/or fat tissue after an 18-h adsorption period provides a reasonable explanation for the spread of this pathogen to Listeria-free meat during storage in walk-in coolers. These findings attest to the hardy nature of L. monocytogenes on the surface of raw meats and to the need for effective means of reducing surface contamination on carcasses. Regarding the latter, Chung et al. [50] reported that wash solutions containing nisin effectively delayed growth of L. monocytogenes on surfaces of raw meats, particularly when such products were incubated at refrigeration, rather than ambient temperatures. Although nisin-producing bacterial starter cultures have been used in the dairy industry for many years, with the exception of certain types of cheese spread, present laws in North America still forbid direct addition of nisin to most foods, including raw meat. Populations of enteric pathogens (i.e., Salmonella spp., enteropathogenic Escherichia coli, and Yersinia spp.) on raw meats can be sharply reduced by exposing the water phase of meat surfaces to 0.2 M lactic acid (pH 2.5) at 21°C [ 1141. Although L. monocytogenes is generally recognized as being more acid tolerant than the previously mentioned enteric pathogens, this organism is nevertheless inactivated at pH values <4. Hence, provided that L. monocytogenes is exposed to lactic acid for sufficient time, acid washes may be somewhat helpful in decreasing Listeria populations on the surface of animal carcasses. As noted by Johnson et al. [ 1141, L. monocytogenes was routinely detected in muscle tissue from cows that were killed 2 days after being inoculated intravenously with the pathogen. Although some contamination of muscle tissue might have occurred during sampling, results from this study suggest that L. monocytogenes can enter muscle tissue via the blood stream. To further investigate this hypothesis, Johnson et al. [ 1121 examined muscle, liver, and spleen tissue from two lambs and one calf that had been inoculated intravenously with L. monocytogenes. Microscopic examination of tissues stained with immunoperoxidase or Azure A revealed L. monocytogenes cells at levels of 103-104CFU/g in muscle tissue and 103-106 CFU/g in both liver and spleen tissue. Although L. monocytogenes appeared to be associated with phagocytes in liver and spleen tissue, the pathogen was observed in loose connective tissue between muscle fibers and also within the muscle fibers themselves. Using the USDA enrichment method, Johnson et al. [ 1 161 subsequently detected L. monocytogenes serotype 1/2a at 5 10 CFU/g in aseptically removed interior samples from 2 of 50 (4%) and 3 of 50 (6%) retail whole muscle beef and pork roasts, respectively. One beef sample also was positive for L. innocua and L. welshimeri. Although the presence of these two nonpathogenic (i.e., noninvasive) Listeria spp. within a whole muscle roast suggests possible contamination during sampling, other results from Johnson et al. [ 1141161 strongly support at least limited transmission of L. monocytogenes from the blood stream into muscle tissue.

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525

Raw Beef

Growth and Survival Interest in behavior of Listeria in raw meat products dates back to at least 1966 when Sielaff [ 165al inoculated beef, pork, and rabbit with L. rnonocytogenes immediately after slaughter and examined these samples for listeriae during extended storage at 3-4°C. Under these conditions, the pathogen survived at least 15 days in all three products. Although this study was among the first to examine the ability of L. rnonocytogenes to survive in raw beef, information concerning actual growth of this pathogen in raw beef was not available until 1978. In that year, Gouet et al. [87] reported results from a study which examined multiplication of L. monocytogenes in “sterile” minced beef alone and in combination with a defined microflora. L. monocytogenes failed to grow alone in minced beef (pH 5.8) stored at 8”C, and populations decreased < 10-fold during 17 days of incubation. In contrast, numbers of listeriae decreased approximately 100-fold in samples of minced beef (pH 5.8) that were simultaneously inoculated with Lactobacillus plantarurn and held at 8°C for 17 days. These researchers also found that higher concentrations of L. rnonocytogenes (106 CFU/g) enhanced growth of L. plantarurn. When samples of minced beef were simultaneously inoculated to contain equal numbers of L. rnonocytogenes, Pseudornonasjluorescens, and Escherichia coli, Listrria populations decreased approximately 10-fold after 24 h of incubation at 8OC, with numbers rapidly increasing after day 7 (Fig. 3). Rapid growth of listeriae during the latter half of incubation was likely caused by proteolysis of meat proteins by P. jluorescens, which in turn gradually increased the pH of the meat from 5.8 to 6.8. With a complex microflora consisting of 103CFUI g each of L. plantarurn, P. jluorescens, E. coli, Micrococcus sp., Clostridiurn perjhkgens, and Enterococcus (Streptococcus)faecalis, behavior of L. rnonocytogenes was similar to that previously observed for the pathogen in the presence of P.jluorescens and E. coli, with listeriae populations reaching approximately 6 X 105CFU/g in minced beef following 17 days at 8°C (Fig. 4). A rapid increase in numbers of P. jluorescens before growth of L. plantarurn again appeared instrumental in raising the pH from 5.8 to 7.2 which in turn stimulated growth of listeriae. Hence, results from this early study suggested that L. rnonocytogenes can grow in temperature-abused retail ground beef, since the microbial composition of this product was fairly similar to that found in the ground beef inoculated with the seven different organisms. In a similar investigation completed 11 years later, Kaya and Schmidt [ 1201 also found that growth of L. rnonocytogenes in artificially contaminated sterile minced meat during extended incubation at 8-20” C was suppressed by adding 106Lactobacillus CFUI g but was unhindered in the presence of 1O6 Pseudornonas sp. CFU/g. However, in contrast to the previous study by Gouet et al. [87], this L. monocytogenes strain readily grew in the absence of other microorganisms, with populations increasing approximately 2 and 2 4 orders of magnitude in sterile minced meat after 10 and 1-5 days of storage at 4 and 8-2OoC, respectively. In this study, proteolysis of meat proteins by pseudomonads apparently was not a prerequisite for growth of L. monocytogenes in sterile minced meat having an initial pH value of 5.8-6.0. During 1988 and 1989, three additional studies were done to determine the behavior of listeriae in ground beef; however, unlike previous investigations, the meat was not pretreated to eliminate the normal background flora. When ground beef at pH 5.6-5.9 was inoculated to contain 105and 106L. rnonocytogenes Scott A or V7 CFU/g, packaged

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526

- 7.0 4

l90 1

7iI

/

/

/

-

6.5 'd

=e

-

'1

0 '

1

I

I

3

7

I

I

11

17

6.0

- 5.5

Days

FIGURE3

Behavior of L. monocytogenes alone (H) and in combination with fscherichia c o l i ( 0 )and Pseudomonas fluorescens (+) in minced beef stored at 8°C. Dashed line (-A-)designates the change in pH during storage. (Adapted from Ref. 87.)

in oxygen-permeable or impermeable film and examined for numbers of listeriae during extended storage at 4OC, Johnson et al. [ 1131 found that the populations and pH remained relatively constant in both products during 14 days of incubation regardless of the film's degree of permeability. In contrast to the previous study by Gouet et al. [87] in which the pH of minced beef containing a complex microflora increased from 5.8 to 7.2 during extended incubation at 8"C, all packaged ground beef samples in this study remained in the range of pH 5.6-5.9 throughout storage. Thus, the lower pH of the meat in this study along with a lower incubation temperature (4 vs 8OC) are likely to have been at least partly responsible for inhibiting growth of listeriae in packaged samples of "retail-like" ground beef. In keeping with these findings, Shelef [163] also reported that L. monocytogenes failed to grow in artificially contaminated ground beef or ground liver during 1 and 40 days of incubation at 25 and 4OC, respectively. However, in contrast to the findings of Johnson et al. [ 1131, the pathogen failed to grow in ground beef despite a final pH value of 7.8. Although the reason(s) for this behavior remain(s) obscure, inability of L. monocytogenes to multiply in ground beef under these conditions is likely related to the type and load of inherent microflora in the product. This hypothesis is supported by a West German study in which Kaya and Schmidt [120] showed that an increase in the natural bacterial flora of ground beef from 105 to

Listeria monocytogenes in Meat Products

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10

9

8 7

6 5 4

3

2

1

I

I

I

I

0 1 1'

3

7

11

17

1

Days

FIGURE 4 Behavior of L. monocytogenesalone (m) and in combination with Lactobacillus plantarum (O), Pseudomonas fluorescens (+I, Escherichia coli (A),Microoccus sp. (A),Clostridium perfringens ( O ) ,and Streptococcus faecalis (U) in minced beef stored at 8°C. (Adapted from Ref. 87.) 1O7 CFU/g led to increased inhibition of L. rnonocytogenes in artificially contaminated meat. When ground beef was inoculated to contain 10s L. rnonocytogenes CFUlg, the pathogen grew after 1-5 days at 7-20°C in samples harboring -105 non-Listeria contaminantslg but failed to multiply in corresponding samples containing higher levels of naturally occurring organisms. Although a similar growth pattern was observed for samples naturally contaminated with 1O2 Listerialg and 1O 5 non-Listerialg, growth was markedly hindered in naturally rather than artificially contaminated samples, with Listeriapopulations remaining constant in the former during 14 days at 8°C. However, behavioral differences were no longer observed at lower temperatures, with the organism failing to grow during 14 days of incubation at 4°C in both artificially and naturally contaminated ground beef containing similar background populations. Work done by Barbosa et al. [29] with vacuum-packaged ground beef supports previous findings that the organism grows better in beef having a pH > 6. This study also showed that besides pH, strain type also can affect the fate of Listeria in ground beef. The authors used four different strains of L. monocytogenes for inoculation and observed slightly different responses with each organism. For example, in normal pH (5.47) ground beef, L. rnonocytogenes serotypes 3a and 3b increased 2.3 and 1.8 logs, respectively after 35 days of storage at 4"C, whereas after 56 days, the levels of strains 1/2a and Scott A (serotype 4b) remained constant and decreased 1 log in number, respectively. In general, the organism multiplied slowly on all vacuum-packaged samples, but growth was better

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on ground beef of pH 6.14 than of pH 5.47. For example, after 28 days of storage at 4"C, serotypes 3b, 3a, and 1/2a had increased by 2.87, 2.64, and 2.24 logs, respectively, in high-pH ground beef, whereas strain Scott A did not change significantly in numbers [29]. The authors felt that the antimicrobial effect of low pH may also impact Listeria indirectly, since altering the natural microbial flora could possibly promote growth of bacteriocinproducing lactic acid bacteria. L. monocytogenes appears to behave similarly on the surface of fresh intact beef muscle. In two studies conducted shortly before L. monocytogenes emerged as a serious foodborne pathogen, Lee et al. [129,130] dealt indirectly with the incidence and subsequent behavior of Listeria spp. along with many other psychrotrophic and mesophilic organisms present on the surface of hot-boned and conventionally boned beef. In this study, hot-boned beef was obtained from five steers 2 h after slaughter, vacuum packaged, and cooled from 32 to 21°C. In contrast, conventionally processed beef was obtained from carcasses that were hung in a cold room at 2°C for 2 days after slaughter. Both types of beef were then examined for mesophilic and psychrotrophic organisms at day 0 when the surface temperature had decreased to 2 1"C (<1 h for conventionally processed beef) and again following 14 days of storage at 2°C. Nearly 1250 bacterial isolates were subsequently identified by computer analysis of 116 miniaturized testdisolate. Although Listeria spp. were never isolated from slow or moderately chilled, hot-boned beef at day 0, 9.1-13.5% of all microorganisms present on the surface of such beef after 14 days were identified as Listeria spp., some isolates of which were likely L. monocytogenes (Table 8). Overall, only one isolate from conventionally processed beef was identified as belonging to the genus Listeria. From these data one can infer that Listeria can grow on the surface of vacuum-packaged hot-boned beef but not on the surface of unpackaged conventionally processed beef during 2 weeks of storage at 2°C. Since the high water-binding properties

TABLE 8 Generation (GT) and Lag Times (LT) of L. monocytogenes in Meats

Roast beef Corned beef Cooked meat Ham Cooked beef Piit6 Piit6

- 1Sa

3a 3b 0 5 5 10 15 5 10 7 12Sa 10 a

100.0 26.7 80.9 110.0 44-6 1 33.2 13.4 6.1 18.6-22.6 8.5-9.0 19.7 1.6 9.12

Vacuum packs. CO2 packs. Source: Adapted from Refs. 46, 104, and 167.

a

173.7 59.0 477.1

80.6-83.4 22.6-30.4 48 24 27.6

104 104 91 167 91 91 91 102 60 75

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of hot-boned beef make this product particularly well suited for sausage making, widespread use of hot-boned beef by the processed meat industry may be partially related to the relatively high incidence of listeriae in ready-to-eat meat products. In a more definitive Australian study reported in 1988, Grau and Vanderlinde [90] examined the ability of L. monocytogenes to grow on the surface of artificially contaminated (102--l O3 CFU/cm2) vacuum-packaged, nonsterile beef striploin during extended incubation at 0 and 5.3"C. Although L. monocytogenes populations increased in all samples (and in product exudates that developed in packages) during storage, the extent of Listeria growth was markedly influenced by incubation temperature, pH of the sample (5.6 vs 6.01, and type of tissue (lean vs fat). Overall, higher Listeria populations consistently developed on fat tissue, with growth also being more rapid at the higher of the two incubation temperatures and pH values. Numbers of listeriae on fat tissue of pH 5.6 increased from 5 X 103to 3 X 107 CFU/cm2 during 16 days of incubation, whereas the pathogen was just beginning to grow on corresponding samples after 7- 14 days of storage at 0°C. These researchers also noted that Listeria populations increased < 10- and 1000fold on vacuum-packaged meats of pH 5.6 and 6.0, respectively, after 10-1 1 weeks of storage at 0°C. Thus, it appears that two conditions, (a) a storage temperature of 0°C and (b) a product pH value of 55.6, must be met simultaneously to prevent significant growth of L. monocytogenes in vacuum-packaged raw meats destined for export. Grau and Vanderlinde [92] extended their studies by using two models: the modified Arrhenius and square-root model to examine aerobic growth of L. monocyiogenes on lean and fatty raw beef tissue. For both lean and fatty tissue, the modified Arrhenius model gave better fits and estimates of the growth rates. The effect of temperatures between 0 and 30°C on the growth rate could be described by a modified Arrhenius equation: Ln (gen/h) = -205.73 1.2939 X 105/K - 2.0298 X 107/K2,where K = OK. The combined effect of temperature and pH on the growth rate of the organism on lean beef was best described by the following equation: Ln (gen/h) = -232.64 + 1.4041 X 105/K - 2.1908 X 107/K2 1.1586 X 102/pH - 4.0952 X 102/pH2.For lean meat at pH values of about 5.5-5.6 and 6.0-7.0, the latter equation applied at 2.5 to 35°C and 0 to 35"C, respectively. There was considerable scatter in the measured lag periods for both types of meat, and therefore a poor fit was observed for both models. In two trials, both models predicted growth on lean tissue where no growth was observed experimentally. In the first, L. monocytogenes failed to grow on lean tissue with a mean pH of 5.61 during 13 weeks of storage at OOC, whereas in the other no growth was observed after 48 h at 43.2"C and pH 5.46. However, growth of the organism was observed on lean tissue having a mean pH of 5.61 at 2.5"C, on lean meat of pH values 16.0, and all fatty tissue (pH 5.5-5.7) regardless of storage temperature. One should be aware, however, that these investigators used only one strain of L. monocytogenes (which was not a meat strain), and that meats where the contaminating flora exceeded 10% of the Listeria count were discarded, thus partially eliminating the effects of background microflora on growth of the organism. Growth rates predicted by the best equations for lean tissue were compared with literature data for aerobic growth of L. monocytogenes on several foods. Predicted growth rates for lean meat were higher than those for corn, clarified cabbage juice, and milk, whereas food supporting growth rates similar to lean beef included UHT milk, raw and cooked chicken, and cooked ground meat [92]. Other researchers have found that some models derived from the growth of L. monocytogenes in broth cannot reliably predict growth of the organism in raw pork [83]. For

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example, L. monocytogenes grew on pork fat tissue without lag at -0.3"C. At higher temperature, growth rates were greater than those in unacidified Tryptic Soy Broth (TSB) held at lower temperatures. Faster growth of the organism on fat tissue as compared with TSB suggests that fat tissue may contain micronutrients which are not present in TSB [83]. In addition, the organism grew slower on pork muscle tissue at temperatures 2 15.4"C than in acidified (pH 5.5) TSB, implying that pork muscle tissue may contain growth inhibitors which are either absent or present in very low levels both in TSB and fat tissue. All models published thus far predict that the organism will grow in broth at pH 5.5 and 5°C in contrast to the failure of L. monocytogenes to grow on normal pH raw pork muscle even at temperatures as high as 15°C. Only one published study has shown that the organism can grow on raw meat (beef muscle) of normal pH at low temperatures [92].

Raw Lamb and Pork Most investigations have focused almost exclusively on the behavior of Listeria in raw beef; however, several reports on the fate of this pathogen in raw lamb and pork can be found in the scientific literature. As early as 1973, Khan et al. [121] published results from a study in which aseptically obtained "sterile" raw lamb meat was inoculated to contain 105CFU/g of L. monocytogenes, packaged in gas-permeable or gas-impermeable film, and examined for numbers of listeriae during extended storage at 0 and 8°C. Although Listeria populations remained relatively constant in lamb meat packaged in gaspermeable film during 20 days at OOC, numbers of listeriae decreased approximately 10fold in corresponding samples that were packaged in gas-impermeable film. These results are similar to those obtained by Johnson et al. [ 1 131, who concluded that L. monocytogenes also was unable to grow in refrigerated ground beef that was packaged in gas-permeable or gas-impermeable film. Following 12 days of storage at 8 rather than OOC, populations of listeriae in lamb increased > 1000-fold; however, unlike meat packaged in gas-permeable film, the pathogen exhibited a 2-day lag period and grew markedly slower in gasimpermeable packages. Various physical differences of the meat, combined with an increased concentration of CO2 (and presumably a lower pH) in gas-impermeable packages, may have been responsible for partially inactivating the pathogen at 0°C and delaying the onset of growth at 8°C. Two years later, Khan et al. [ 1221 examined the behavior of L. monocytogenes in preparations of sarcoplasmic pork, beef, and lamb protein that were inoculated to contain 104CFU/mL of L. monocytogenes and then held at 4°C. According to these investigators, the pathogen grew readily in preparations of pork and beef protein, reaching levels of approximately 105and l O7 CFU/mL, respectively, after 12 days of refrigerated storage. Although Listeria populations remained relatively constant in corresponding preparations of lamb protein held at 4"C, the pathogen grew readily in lamb meat stored at 8°C. Hence, it appears that raw pork, beef, and lamb can support rapid growth of listeriae, particularly when these products have undergone temperature abuse, as frequently occurs at the retail level. These early observations were confirmed by Lovett et al. [134], who found that L. monocytogenes reached levels of at least 108CFU/g in inoculated samples of retail lamb, pork, and beef after 14 days at 7°C. L. monocytogenes exhibited both a 2-day lag period and a slower rate of growth in beef and pork than in lamb. Such variations in growth rate might be related to differences in concentrations of various amino acids (particularly ly-

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sine, serine, and valine), which are reportedly essential for growth of L. monocytogenes, as first suggested by Khan et al. [ 12 1 ] in 1973.

Cooked and Ready-to-Eat Meats

Cured Ham Investigators in Europe and the United States determined the behavior of L. monocytogenes in ham during processing and extended storage. Working in The Netherlands, Stegeman et al. [ 1681 examined the thermal resistance of listeriae in experimentally produced hams to which 2 or 3% NaCl and 120 or 180 ppm sodium nitrite were added during manufacture. After inoculating the product to contain 1O4 CFU/g of L. monocytogenes, all hams were canned, heated to an internal temperature of 68.9-71.0 (or 64.0"C) within 5 h to simulate normal and underprocessing, respectively, cooled, and sampled after 5 days of storage at 4°C. According to these authors, three different enrichment procedures failed to recover viable listeriae from any samples. Results from the study just described indicate that standard thermal treatments are more than sufficient for producing Listeria-free ham. However, once removed from protective packaging, all cooked/ready-to-eat meats can become contaminated with listeriae during slicing and further handling. To simulate postprocessing contamination, Glass and Doyle [84] inoculated the surface of commercially produced ham slices and five other meat products (to be discussed shortly) to contain approximately 0.2 or 500 CFU/g of L. monocytogenes. All samples were then vacuum packaged and periodically examined for numbers of listeriae during prolonged incubation at 4.4"C. Regardless of the original inoculum, L. monocytogenes attained populations of 1OS- 1Oh CFU/g on organoleptically acceptable ham (pH 6.3-6.5) after 4 weeks of refrigerated storage, indicating that manufacturers cannot rely on the combination of vacuum packaging and refrigeration for control of listeriae on ham.

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Cooked Roast Beef Glass and Doyle [84] evaluated the potential for growth of L. monocytogenes on the surface of vacuum-packaged samples of cooked roast beef having initial pH values of -5.90. Unlike sliced ham, cooked roast beef supported far less growth of listeriae, with populations increasing 2 2 orders of magnitude on organoleptically acceptable product after 4 weeks of refrigerated storage. Slower growth of the pathogen on precooked roast beef correlated well with a decrease in product pH to 55.15 in 4-week-old samples.

Luncheon Meats Since previous studies found that luncheon meat, cooked ham, and cooked breast meat were the most frequently contaminated cooked meats in The Netherlands, these products were used in a study to determine the survival and growth of L. monocytogenes [34]. Products were inoculated with low levels (10 CFU/g) of the organism and then stored under vacuum or an atmosphere of 30% c o 2 / 7 0 % N 2 at 7°C for 4-6 weeks. Growth on vacuum-packed product was similar to that of modified-atmosphere packaged (MAP) stored meats, with counts increasing up to 108CFU/g after 35 days (Fig. 5). High numbers of lactic acid bacteria were present but did not affect growth of the organism. However, the pathogen decreased in number on saveloy (fermented sausage; pH 5.5-5.7) and raw

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A 0

chicken breast luncheon meat cooked ham

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$ 4

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2 I

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Time (days)

FIGURE5 Growth of Listeria rnonocytogenes on MAP (30% CO2/7O% N 2 )luncheon meat, cooked ham, and chicken breast at 7°C. (Adapted from Ref. 34.)

Coburger ham (pH 4.3-4.5), most likely as a result of the acidity of these products. Grau and Vandelinde [91] examined growth of L. monocytogenes on both naturally contaminated and artificially inoculated processed corned beef and ham. For corned beef stored at O"C, L. monocytogenes grew at about half the rate of the other microflora, whereas at 9"C, both groups of organisms grew at similar rates. On ham stored at 5OC, the organism grew at only one third the rate of the other flora. Meat composition, that is, pH, salt, and residual nitrite, played a role in determining the growth potential for this pathogen. For example, at OOC, the organism failed to grow on ham containing 170 ppm residual nitrite, but it did grow on ham with 11 ppm nitrite. Although L. monocytogenes grew at similar rates on ham stored at 15"C, as the storage temperature decreased, the organism again grew slower on ham containing the higher level of residual nitrite. Fastest growth was observed on corned beef of pH 6.2, a, 0.97, and <5 ppm nitrite, with slowest growth being seen on ham of pH 6.6, a, 0.97, and 170 ppm nitrite. From these inoculated pack studies, equations were developed to describe the growth of both L. monocytogenes and the other flora; that is, lactic acid bacteria and Brochothrix thermospacta on ham and corned beef (Fig. 6). For naturally contaminated corned beef, good agreement was obtained between predicted and actual growth of the organism. As predicted, L. monocytogenes could not grow on naturally contaminated ham stored at 0.loC, although slight growth (i.e., from -0.3 to 6.2 CFU/g) of the organism was observed on product stored at 4.8"C. Juneja et al. [ 1171 examined the potential for outgrowth of various foodborne pathogens on cooked ground beef during cooling from 54.4 to 7.2"C within 6, 9, 12, 15, 18 or 21 h. L. monocytogenes was inoculated into ground beef at a level of 103 CFU/g, the meat was then heated linearly to 60°C within 1 h, and then cooled as described above. The organism was not detected in any beef sample examined. Presumably, the slight heating and cooling regime was sufficient to reduce levels of the organism below the detectable level; levels at which the organism remained during the duration of the cooling period [117]. The fate of L. monocytogenes on unirradiated and irradiated cook-chill roast beef

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I

I 4

7.5

3 2

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0

3.5

p

6 I J

.-cd

-2

0

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30 20 Time (Days)

40

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3

bD

sB 0

1.5

and of other (lactic acid FIGURE6 Changes in the number of L. monocytogenes (II) bacteria and B. thermosphacta) flora ( 0 )o n the lean tissue of commercial vacuum packs of corned beef stored at (a) 4.8"C or (b) 0.1"C. Note the different vertical scales for Listeria and other flora. Off odors first detected (?I. (Adapted from Ref. 91.)

and gravy was examined at 5 and 10°C [89]. The organism grew well on both products, increasing 5 logs in number on unirradiated beef and gravy over 15 days at 5°C and by 6 logs in irradiated products stored at 10°C for 23 days. Although the observed lag phase for L. rnonocytogenes was longer on irradiated as compared with unirradiated product, the specific growth rates were similar at each storage temperature, suggesting that the background microflora of beef and gravy did not interfere with growth of L. rnonocytogenes. The authors concluded that there would be no increased risk of listeriosis if cookchill roast beef and gravy were to be irradiated with 2 kGy [89]. L. rnonocytogenes also grows well on cooked beef [102] (Table 8). Interestingly, the organism grew at similar rates both aerobically and anaerobically, although the authors claimed that samples stored under aerobic conditions probably became anaerobic during the course of the experiment [102]. L. rnonocytogenes also survived pasteurization (e.g., 91 and 96°C for 3 or 5 min) of precooked beef roasts and then grew when samples were stored at 4 and 10°C for up to 56 and 12 days, respectively [94]. Products stored at 10°C supported far better growth of the surviving listeriae than those stored at 4°C; that is, at IOOC, L. rnonocytogenes reached levels in 12 days that took up to 56 days to attain at the lower storage temperature. It is well known that the organism can repair itself much better at higher temperatures. Van Laack et al. [ 1751 examined the effect of three packaging treatments, that is, vacuum packed directly after hot boning (hot packaged), vacuum packed after chilling for 1 day (cold packaged), or unpackaged, on survival of L. rnonocytogenes on pork loins stored at 1°C for up to 9 days. Populations increased about 1 log (Table 9), demonstrating that the organism can grow on raw refrigerated pork in the presence of numerous competitors. Although the data were not analyzed statistically, the type of packaging did not appear to greatly influence growth.

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TABLE 9

Influence of Packaging Treatment on Numbers of L. rnonocytogenes on Pork Loins During 9 days storage at 1 1°C

*

Sampling day

Packaging treatment HP CP

cu HP CP

cu HP CP

cu HP CP

cu HP CP

cu

Average log,,, CFU/cm2a Trial 1

Trial 2

2.68 (5/8)h 2.48 ( 5 / 8 ) 2.65 (618) 2.98 (6/8) 2.48 (5/8) 2.91 (7/8) 3.36 (8/8) 2.68 ( 5 / 8 ) 2.64 (6/8) 3.77 (7/8) 2.92 (7/8) 2.73 (8/8) 3.73 (8/8) 3.19 (7/8) 3.48 (5/8)

2.42 (8/8) 2.86 (8/8) 2.86 (8/8) 2.86 (818) 2.86 (8/8) 2.73 (8/8) 3.1 1 (8.8) 2.77 (7/8) 2.91 (7/8) 3.1 1 (8/8) 3.36 (8/8) 3.19 (7/8) 3.23 (8/8) 3.61 (7/8) 3.23 (5/8)

HP, hot packaging; CP, cold packaging; CU, cold unpackaged. Average of MPN values from “positive” samples. The number between brackets indicates the proportion of samples from which a given pathogen was recovered. Source: Adapted from Ref. 175. a

The issue of whether L. rnonocytogenes can grow on raw and cooked meats is complex. As seen in this section, factors such as pH, a,, background microflora, sodium nitrite and NaC1, length and temperature of storage, strain type, and history all play a role in determining the fate of Listeria on a meat surface. Another seemingly important factor which is often overlooked is the initial inoculum on the product. Although early work from modeling experiments suggested that initial numbers of organisms present on a food surface had little or no influence on subsequent outgrowth and growth rate, more recent work does not substantiate this theory. For example, Farber and Daley [70] found that when L. monocytogenes was present in very low numbers on meats such as sliced ham, turkey breasts, wieners, and pSt6 stored at 4OC, its numbers did not increase. Similar results have been observed for foods other than meats and poultry (J.M. Farber, unpublished results).

Unfermented Sausage Even though potentially contaminated raw meats find their way into enormous quantities of sausage products, with over 200 varieties manufactured in the United States alone, no information pertaining to the behavior of L. monocytogenes during manufacture and storage of these popular meat products appeared in the scientific literature before 1988. Although the California listeriosis outbreak of 1985 eventually led to the aforementioned surveys in which Listeria was detected in raw and processed meats, including sausage,

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the early consensus was that consumption of such products did not pose a serious threat to public health, as shown by a lack of any confirmed cases of meatborne listeriosis. However, this situation changed in December, 1988, following the report of a breast cancer patient who developed listerial meningitis and eventually died after consuming turkey frankfurters that were contaminated with L. monocytogenes. Unfermented sausages are best classified according to the following five categories, which are based on the method of manufacture: (a) fresh sausage (e.g., fresh pork sausage, bratwurst), (b) cooked sausage (e.g., liver sausage, Braunschweiger), (c) cooked smoked sausage (e.g., frankfurters, bologna), (d) uncooked smoked sausage (e.g., Mettwurst, smoked country-style pork sausage, kielbasa), and (e) cooked meat specialty items (e.g., head cheese). Research efforts have dealt primarily with the behavior of Listeria in sausages belonging to the first three categories.

Fresh Sausage Although fresh pork sausage is by far the most widely manufactured type of fresh sausage, this category also includes other well-known varieties such as fresh Italian, breakfast, and beef sausage as well as fresh bratwurst, Thuringer, and bockwurst. The last two are most popular in Germany. All varieties of fresh sausage are normally prepared from coarse or finely comminuted pork, beef, or veal to which water is added along with an array of spices which varies with the type of sausage. In the United States, certain varieties of fresh sausage also may contain binders and/or extenders (e.g., cereal, vegetable starch, nonfat dry milk, dried whey) at levels not exceeding 3.5% by weight. After being stuffed into natural or artificial casings, the product is twisted and cut to form individual sausage links which are cooled rapidly to preserve freshness and flavor. Unlike cooked and fermented sausages, fresh sausages have a short shelf life and must be kept refrigerated to prevent growth of spoilage organisms, including lactic acid bacteria and micrococci. When commercially prepared fresh bratwursts were surface-inoculated to contain approximately 0.1 or 600 L. monocytogenes CFU/g, vacuum packaged, and stored at 4.4OC, Glass and Doyle [84] found that the pathogen attained populations of 106CFU/g on organoleptically acceptable 4-week-old bratwursts regardless of the initial inoculum. As with ham, profuse growth of listeriae on fresh bratwurst was attributed to a pH value >6 which was maintained by the product throughout the first 4 weeks of refrigerated storage. In another study involving fresh sausage, Hughey et al. [ 1061 investigated the ability of lysozyrne to prevent growth of L. monocytogenes in bratwurst prepared from coarsely ground pork. After addition of commercial bratwurst spice, distilled water was added with or without 100 ppm lysozyme and 5 mM ethylenediaminetetraacetic acid (EDTA), a generally recognized as safe chelating agent that enhances the antibacterial activity of lysozyme. This meat mixture was then inoculated to contain -4 X 103 CFU/g of L. monocytogenes and stuffed into natural hog casings which were subsequently linked, separated, vacuum packaged, and stored at 5°C for 45 days. As expected from the previous study, Listeria populations increased rapidly in fresh bratwurst (pH z 6) without added lysozyme or EDTA, reaching levels of > 106CFU/g following 10 days of refrigerated storage (Fig. 7). L. monocytogenes also behaved similarly in bratwurst containing lysozyme alone; however, the presence of EDTA alone resulted in a 15-day lag period, thus preventing the pathogen from reaching populations of 10'

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FIGURE7 Effect of lysozyme (Lys) and EDTA on growth of L. monocytogenes in fresh bratwurst. (Adapted from Ref. 106.)

CFU/g until nearly 30 days of storage. In contrast, lysozyme and EDTA acted synergistically to retard growth of L. monocytogenes in fresh bratwurst. Under these conditions, the pathogen exhibited a lag period of nearly 2 1 days, that is, approximately 7 days beyond the normal shelf life of the product, and reached populations of <105 CFU/g following 44 days of refrigerated storage. Although only listeriostatic, the combined use of lysozyme and EDTA appears to be an effective means of controlling Listeria growth during the normal shelf life of fresh bratwurst. Furthermore, once growth is prevented, low levels of L. monocytogenes that occasionally appear in fresh bratwurst (<103CFU/g) should be readily eliminated by proper cooking.

Cooked Smoked Sausage This group of sausages, which includes the ever-popular frankfurter (hot dog) as well as bologna and various luncheon meats, is prepared from mixtures of comminuted beef and/ or pork to which salt, sugar, sodium nitrite, and spices are normally added. When making frankfurters, this meathgredient mixture, commonly referred to as the sausage emulsion, is stuffed into natural or artificial casings, which are then twisted to form sausage links. This string of frankfurter links is cooked to an internal temperature of 7 1.1"C (160'F) to (a) coagulate protein, (b) fix the color, and (c) pasteurize the product. Although not absolutely

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necessary, frankfurters and other similar sausages are frequently hung in smoking rooms either before or after cooking. Alternatively, commercially available liquid smoke products can be added to the sausage emulsion or applied directly to the frankfurter surface before or during heating. In either event, beside imparting a pleasant smoked flavor to the finished product, some smoke components (i.e., formaldehyde, acetic acid, creosote, and phenols with high boiling points) possess bacteriostatic and/or bactericidal properties. After cooking, frankfurters are carefully cooled, packaged, and refrigerated during shipment to wholesale and retail markets. Skinless frankfurters, which are very popular, are produced in a similar manner except that the artificial casings are mechanically peeled from the sausage after cooking or smoking. Epidemiological data from the Centers for Disease Control and Prevention (CDC) showing an apparent association between listeriosis and undercooked frankfurters prompted several thermal resistance studies. Zaika et al. [ 1931 prepared frankfurters from a sausage emulsion inoculated to contain 1O8 CFU/g of L. monocytogenes. After stuffing, all frankfurters were thermally processed (without smoke) according to a standard commercial heating schedule. These USDA officials found that L. monocytogenes populations decreased approximately 1000-fold in frankfurters that were heated to an internal temperature of 7 1.1"C (160°F). Based on these data, cooking frankfurters to an internal temperature of 7 1,I "C would probably eliminate maximum levels of L. monocytogenes (<103 CFU/g) that could conceivably occur in raw frankfurter eniulsions. Data gathered by the American Meat Institute in 1988 pointed to frankfurters as being likely carriers of L. monocytogenes and also suggested that poor environmental conditions before packaging could play a major role in contaminating the finished product [4]. Moreover, Glass and Doyle [84] reported that this pathogen can proliferate on vacuumpackaged, artificially contaminated (-0.0 1 L. monocytogenes CFU/g) retail frankfurters at 4.4"C with populations two to five orders of magnitude higher on organoleptically acceptable samples after 4 weeks of refrigerated storage. Similarly, L. monocytogenes increased in number from 5 X 10' to 2.1 X 105MPN/g on vacuum-packed frankfurters stored at 4°C for 20 days. Interestingly, uninoculated control samples which were initially negative for Listeria contained 1.2 X 102MPN/g after the 20-day storage period [40]. In a more detailed study examining growth of L. monocytogenes on vacuum-packaged allbeef, poultry or beef/pork wieners at 5°C for up to 28 days, McKellar et al. [ 1431 found that of 61 wieners analyzed, 40 (65.6%) supported growth of L. monocytogenes. For those samples supporting growth, an average increase of 1.26 logs was observed within a 14day period. Unlike NaCl levels, concentrations of phenol, nitrite and lactic acid bacteria varied considerably during storage. In addition, average pH levels decreased significantly by 0.19 pH units during storage. Several statistical models were derived in an attempt to describe growth and death of the organism in all wiener samples. Although no single model was completely adequate, the best model implicated initial and final lactic acid bacteria counts and initial pH as factors influencing growth of L. monocytogenes. Preventing Listeria contamination and subsequent growth is further complicated by present consumer demands for reduced levels of salt and preservatives, along with longer shelf life, smaller packages, and greater convenience, all of which will require the processed meat industry to develop even stricter requirements for processing, cooking, handling, packaging, and refrigeration of products. Several studies were initiated by the food industry to examine the feasibility of using heat to eliminate L. monocytogenes from the surface of finished frankfurters. In one such study [6], frankfurters were dipped in a broth culture of L. monocytogenes ( 106-108 CFU/

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mL) to simulate postprocessing contamination. Listeria populations on the surface of the frankfurters decreased only 100-fold after 8 min of heating at 86.1-873°C (1 87- 190°F). Furthermore, this heat treatment rendered the sausages organoleptically unacceptable for most consumers. Hence “postprocess pasteurization” may not be a viable means of eliminating L. monocytogenes from the surface of frankfurters that have been contaminated after manufacture. Additional efforts to control Listeria contamination on the surface of frankfurters have focused on the bactericidal properties of commercially available liquid smoke products. In one study [147a], beef frankfurters were immersed in a culture containing 1 X 103CFU/mL of L. monocytogenes, removed, thoroughly air dried, and then dipped in fullstrength commercially available liquid smoke solution (CharSol C- 10). Although Listeria populations were unchanged in control frankfurters that were dipped in phosphate buffer, vacuum packaged, and analyzed after 72 h at 4”C, numbers of listeriae had decreased 60 to 299.9% 15 min after the frankfurters were treated with liquid smoke. Furthermore, the pathogen was never detected in smoke-treated sausage following 72 h of refrigerated storage. However, although dipping frankfurters in full-strength liquid smoke eliminated L. monocytogenes, this treatment produces an extremely intense smoke-flavored product that is no longer organoleptically acceptable to most consumers. Results from several additional experiments dealing with the antilisterial effects of less concentrated liquid smoke solutions were reported by Wendorff [ 1831. Initially, beef frankfurters were dipped in a concentrated broth culture of L. monocytogenes, removed, thoroughly dried, dipped into aqueous liquid smoke solutions containing 10-40% CharSol C- 10 or CharSol Poly- 10 (concentrations normally used in frankfurter production) and then analyzed for listeriae after 72 h of refrigerated storage using the Gene-Trak DNA Hybridization assay. Although L. monocytogenes was eliminated from the surface of frankfurters dipped in 40% solutions of CharSol C- 10 or Poly- 10, the pathogen was still detected on frankfurters treated with 10 and 25% solutions of CharSol C-10. After demonstrating that these liquid smoke compounds lost their activity against Listeria on the surface of frankfurters when added directly to sausage emulsions before stuffing, Wendorff [ 1831 assessed inactivation of listeriae on surface-inoculated skinless frankfurters that were sprayed with five levels of CharSol Poly-10 and CharSol Supreme (twice the strength of CharSol C-10) just before vacuum packaging (Table 10). When used at organoleptically acceptable concentrations, CharSol Supreme was more effective than CharSol Poly- 10, with Listeria populations on the surface of frankfurters decreasing >40% following 72 h of refrigerated storage. Using a realistic L. monocytogenes inoculum level (- 10’ CFU/g), approximately 89% of the Listeria population was inactivated on frankfurters treated with CharSol Supreme or CharSol Poly-10 at levels of 2 and 4 oz./ 100 lb of frankfurters, thus indicating that none of these treatments can guarantee a Listeria-free product. Hence, to avoid contamination of finished product, efforts must be made to develop microbial monitoring, sampling, and hazard analysis critical control point (HACCP) programs that can effectively address problems pertaining to a lack of separation between raw and finished processing areas, as well as procedures used to clean and sanitize the factory environment and equipment such as grinders, mixers, and particularly sausage peelers. In the only other published study involving cooked smoked sausage, Glass and Doyle [84] examined the behavior of L. monocytogenes on vacuum-packaged, artificially contaminated slices of commercially produced bologna. As was true for ham and bratwurst, the pathogen also grew well on bologna (pH 6.1 -6.4), with populations generally

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TABLE 10 Fate of L. monocytogenes o n the Surface o f Beef Frankfurters Sprayed with CharSol Poly-I0 or CharSol Supreme Liquid Smoke and Stored at 4°C for 72 h

L. monocytogenes

Treatment Control CharSol Poly - 10

CharSol supreme

Level (oz/ 100 lb frankfurters)

Initial inoculum (CFU/g)

Inactivation after 72 h (%)

0 1.7a 3.4a 5.1a 8.5 12.0 1 3.6a 5.4 9.0 12.6

5.28 5.30 5.28 5.16 4.84 4.67 5.02 5.04 4.73 4.4 1 4.30

0 Growth 0 23.6 63.1 75.2 44.7 42.1 71.5 86.3 89.5

Organoleptically acceptable concentration of liquid smoke. Source: Adapted from Ref. 183. a

three to four orders of magnitude above initial levels on organoleptically acceptable samples after 4 weeks at 4.4"C. These findings stressed the importance of following good manufacturing practices, which will in turn greatly reduce the possibility of listeriae contaminating ready-to-eat meats during slicing and packaging.

Uncooked Smoked Sausage According to incidence data in Table 7, one variety of western European uncooked smoked sausage, namely Mettwurst, appears to be particularly prone to contamination with Listeria spp., including L. monocytogenes. These observations prompted a 1989 study by Triissel and Jemmi [ 1731 in which an experimentally produced Mettwurst emulsion (pH 5.3) containing -2.6% NaCl and 100 ppm sodium nitrate was inoculated with L. monocytogenes at levels of 103or 107CFU/g and examined for numbers of listeriae during manufacture after 7 days of ripening at 14"C, and after 3 weeks of subsequent storage of 4°C. Overall, Listeria populations remained relatively constant during manufacture and ripening, which in turn reflects the apparent inability of this organism to multiply in ready-to-eat meat products having pH values <5.5. As predicted from the European surveys, this pathogen also survived well in Mettwurst during the product's entire refrigerated shelf life, with 4week-old samples still containing approximately 102and 1O6 CFU/g of L. monocytogenes. Given these findings, it would be prudent for Mettwurst producers to review their manufacturing practices and develop procedures for decreasing Listeria contamination during all facets of production.

Cooked Meat Specialty Items A significant amount of beef jerky is consumed annually in the United States, with its popularity primarily resulting from the product's ease of preparation and stability at ambi-

Farber and Peterkin

540

ent temperature. However, some health concerns have arisen with beef jerky following linkage to several outbreaks of salmonellosis [48]. This situation prompted Harrison and Harrison [95] to examine the fate of E. coli 0 157 :H7, Salmonella typhimurium, and L. monocytogenes in beef jerky during preparation and storage. Half of the inoculated beef loin strips were marinated at 4°C overnight and then dried at 60°C for 10 h. The remaining samples were heated in marinade to 7 1.7"C and then dried. L. monocytogenes populations decreased by 1.8 and 6.0 logs after 3 and 10 h of drying, respectively. Cooking to 7 1.7"C before drying led to a 4.5-log decrease in numbers of the organism, with a further 2-log reduction in numbers occurring during the 10-day drying period (Fig. 8). After 8 weeks of storage at 25"C, none of the beef jerky samples yielded pathogens. Much attention has been given to p2t6 following its incrimination in at least two foodborne listeriosis outbreaks. However, the growth potential of L. monocytogenes in p2t6 still remains controversial. De Boer and van Netten [60] reported that inoculated retail pit6 was a good growth menstruum for L. monocytogenes, with the pathogen reaching levels as high as >8.3 log,, CFU/cm2 after 7 days at 12.5"C when the background microflora was low (<2.3 CFU/cm2). An inverse correlation was observed between Listeria growth and the presence of lactic acid bacteria. When high numbers of lactic acid bacteria were present, the pH was low (average of 4.9), and L. monocytogenes increased slightly or decreased in numbers after 1 week of storage at 7 or 12.5"C. Morris and Ribeiro [ 1481 also found that L. monocytogenes grew on some naturally contaminated pgtis, reaching levels as high as 2 X 108CFU/g after 21 days of refrigerated storage. However, other samples failed to support growth. Several other investigators also reported that L. monocytogenes failed to multiply on retail inoculated p2t6 stored at 4°C for up to 3 weeks [70]. To more fully assess the potential health hazards posed by L. monocytogenes in liver pit6 products, multifactorial design experiments were conducted to examine the influence of temperature (4 and lO"C), NaCl (1 and 3%), sodium nitrite (0 and 200 ppm), sodium erythrobate (0 and 550 ppm), and spice (0 and 0.4%) on growth of L. monocytogenes on experimental pit6 [75]. A total of 16 different liver p2t6 formulations were prepared and

7

0 Lm (unheated)

6 1 5-

Lm (heated)

. Y 3

Y 4a E

5

321-

0

3

"

-

-

6 Drying time (h) in dehydrator

10

FIGURE8 Survival of L. monocytogenes on beef jerky during drying at 60°C (140°F). (Adapted from Ref. 95.)

Listeria monocytogenes in Meat Products

54 1

stored at 4 and 10°C. When analysis of variance was usecl to assess the impact of the various factors on maximum growth rate, temperature was the only factor that affected the growth rate, with L. monocytogenes growing well in all experimental piit6s. The generation and lag times for the organism in piit6 can be seen in Table 8. Overall, potential growth of L. rnonocytogenes in pgt6 appears to be related to pSt6 composition and pH, initial numbers of lactic acid bacteria, and storage temperature and time.

Fermented Sausage Sausages classified as fermented undergo a controlled lactic acid-type fermentation, usually through the action of a commercially produced starter culture added to the meat. Although all fermented sausages can be further classified according to moisture content as either semidry or dry, manufacturing procedures for both types are generally similar until the point of drying. Fermented sausages are normally prepared from comminuted beef and/or pork to which sugar and various spices are added along with sodium or potassium nitrate and/or nitrite. This meat preparation, known as a mix rather than an emulsion, is inoculated with a commercial lactic acid bacteria starter culture, which frequently includes species of Pediococcus (particularly P. cerevisiae and P. acidilactici), Lactobacillus, and Leuconostoc. After stuffing the inoculated sausage mix into natural or artificial casings, the strings of sausage links are hung in ripening or "green-rooms" at 27-40" C/ 80-90% RH. Within 2-3 days, sugar added to the mix is fermented to lactic acid by the starter culture, which in turn decreases the pH to -5.1 and produces the characteristic tangy flavor found in fermented sausages. As in cheese making, controlled lowering of the sausage pH to levels near the isoelectric point of meat protein is crucial for proper removal of water during later stages of sausage manufacture. Following fermentation, sausages destined to become semidry varieties containing -50% moisture (e.g., Cervelattype sausages and Lebanon bologna) zre normally placed in smokehouses where they are smoked and cooked to internal temperatures of 60-68°C. In contrast, dry sausages which will ultimately contain -35% moisture (e.g., pepperoni, Genoa, and Milano salamis) are moved to drying rooms (10-17"C/65-80% RH) where they remain for various times, depending on the type and size of sausage. Some varieties also may be exposed to cool smoke before drying; however, unlike semidry varieties, dry sausages are never cooked. Although fermented sausages keep well because of their relatively high salt content, low pH, and low moisture (a,) content, both varieties, particularly semi-dry types, should be refrigerated.

Semidry Fermented Sausage The relatively severe heat treatment that semidry sausages receive during manufacture is generally sufficient to eliminate most commonly encountered non-spore-forming foodborne pathogens. Hence, despite concerns regarding the presence of listeriae in meat products, behavior of L. monocytogenes during manufacture and storage of semidry fermented sausage has received relatively little attention. Although L. monocytogenes is unlikely to survive during manufacture of semidry sausage, ample opportunity exists for this pathogen to contaminate the finished product during slicing and packaging. Hence, to simulate postprocessing contamination, Glass and Doyle [84] inoculated slices of commercially produced, fermented semidry sausage to contain approximately 0.01 or 100 L. monocytogenes CFU/g, after which all samples were vacuum-packaged and quantitatively examined for listeriae during prolonged incubation

542

Farber and Peterkin

at 4.4"C. Unlike ham, bologna, and frankfurters, the pathogen failed to grow on fermented semidry sausage of pH 4.8-5.2, with populations generally decreasing 5 10-fold on organoleptically acceptable samples after 6- 12 weeks of refrigerated storage. Recognizing the likelihood of listeriae being introduced into semidry sausage during slicing/packaging and surviving throughout the normal shelf life of the product, Cirigliano et al. [5 11 investigated the possibility of eliminating L. monocytogenes from inoculated slices of German-type and Polish-type beef sausage ( 104- 105 L. monocytogenes strain Scott A or V7 CFU/g) by exposing vacuum-packaged product to temperatures of 32.25 1.7"C (90- 125°F) for up to 72 h. According to the authors, L. monocytogenes populations failed to change in product stored at 32.2"C (90°F); however, numbers of both Listeria strains decreased approximately 100-fold after product was held at 373°C (100°F) for 72 h, with a slightly faster rate of inactivation being observed in Polish-type than Germantype sausage. Although increasing the temperature to 43.3"C (1 10°F) eliminated strain V7 from Polish-type and German-type sausage after 8 and 48 h, respectively, strain Scott A was not eliminated from either product until completion of a 72-h heat treatment. When exposed to 48.9 and 51.7"C (120 and 125"F), strain V7 was inactivated in Polish-type and German-type sausages within 4 and 24 h, respectively. Although somewhat more heat resistant, strain Scott A was eliminated from both products after 24 h at 51.7"C (125°F). In some instances, objectionable fat losses were observed for both product types; however, the authors concluded that mild heat treatments can be used to salvage Listeria-contaminated German-type or Polish-type sausage without seriously affecting product quality. In fermented "tea" sausages inoculated with 8 X 106MPN/g of L. monocytogenes, the organism decreased about 1.5 logs during the initial 4-day ripening period where the pH dropped from 5.47 to 4.80, decreased another 1.5 logs during the 9-day drying period, and then remained constant in number during the 20 days of storage at 18-22°C [41]. Similar results were obtained when sausages were inoculated with lower levels of the organism. The initial a, value as well as those after drying and after storage were 0.974, 0.933, and 0.861, respectively.

Dry Fermented Sausage Unlike semidry varieties, dry fermented sausages are never exposed to Listeria inactivation temperatures. Consequently, dry sausages have attracted more attention, as shown by several studies that examined the fate of L. monocytogenes during the fermentation, drying, and storage of hard salami, pepperoni, and several other sausages. In the first such study, Johnson et al. [ 1141 prepared hard salami from naturally contaminated (i.e., meat from cows inoculated intravenously with L. monocytogenes) as well as artificially inoculated ground beef, both of which contained -104 CFU/g of L. monocytogenes. Glucose, spices, sodium nitrite, and salt were added to the ground beef along with a glucose-fermenting strain of Pediococcus acidilactici. After stuffing the mix into casings, all sausages were fermented at 40°C for 24 h, dried at 13°C for 9 days, vacuum packaged in gas-impermeable film, and stored at 4°C for 12 weeks. As shown in Fig. 9, listeriae populations decreased approximately 10- and 100-fold during fermentation (40°C/24 h) of hard salami prepared from naturally contaminated and artificially inoculated ground beef, respectively. Inactivation of listeriae during fermentation was primarily attributed to production of lactic acid (or other metabolites) by the starter culture, with pH values decreasing from approximately 5.7 to 4.4 by the end of fermentation. Although numbers of listeriae remained relatively constant in naturally contaminated hard salami following 9 days of drying (13"C/65% RH), populations in product prepared from artifi-

Listeria monocytogenes in Meat Products

543

5.0

Ferm ,

Drying

Refrigerated Storage

I

I

I

4.0

t-- Naturally Contaminated &-

--

Artificially Inoculated

3.0 \

\

2.0

a

e----.

1.0

A t -0- , 0

1

2

4

6

8

1

0

'

14

a

0

A

A

A

1

1

I

I

28

42

56

70

A A

A I-

84

Days

FIGURE9 Behavior of L. rnonocytogenes during fermentation (Ferm), drying, and storage of hard salami prepared from naturally contaminated and artificially inocu.,A) and open (0) symbols at 1 log,, CFU/g represent posilated ground beef. Solid ( tive and negative results, respectively, after 8 weeks of cold enrichment. (Adapted from Ref. 114.) cially inoculated ground beef decreased nearly 100-fold during drying. L. monocytogenes was detected in both products during 8 weeks of refrigerated storage; however, higher levels of listeriae were recovered from naturally contaminated rather than artificially inoculated hard salami, thus suggesting that behavior of this pathogen is best studied using sausage prepared from naturally contaminated rather than artificially inoculated ground beef. Compositionally, both products were very dry, having a, values of 0.79-0.81 as compared with -0.9 1 for commercially produced hard salami. Although L. monocytogenes might be expected to survive more readily in higher moisture commercial products, growth of the pathogen in retail hard salami appears unlikely given the presence of 57% NaCl and 100- 150 ppm sodium nitrite combined with a pH of 4.3-4.5 and a relatively low storage temperature. In contrast to the study just described, Triissel and Jemmi [I721 reported that L. monocytogenes populations in salami prepared from a mix inoculated to contain approximately 103or 107CFU/g of L. monocytogenes decreased 5 10-fold in product of pH 5 5 . 6 during 7 days of ripening at 12-22"C/82-95% RH. After 8 weeks of drying at 10- 17"Cl 78-82% RH, numbers of listeriae in salami of pH 5.4-5.7 decreased to <10 CFU/g regardless of the initial inoculum. However, using an enrichment procedure, the pathogen was detected in these sausages after an additional 6-1 1 weeks of drying at 10-17"C/3550% RH to a, values of 0.68-0.69. Thus, as was true for certain fermented dairy products discussed in Chapter 12, small numbers of L. monocytogenes cells also can persist in fermented dry sausages for at least 14-19 weeks. Farber et al. [74] examined the fate of

544

Farber and Peterkin

L. monocytogenes during production of uncooked German, American, and Italian-style fermented sausages. Similar results were obtained for both German and American sausage, with Listeria populations decreasing about 2-3 logs after fermentation and smoking and a further 1-2 logs after drying. In contrast, Listeria populations in Italian-style fermented sausage prepared without starter culture increased slightly during fermentation, remained constant during drying, and then decreased slightly during the 4-week holding period at 4°C. This study points to the importance of using starter cultures for production of dry fermented sausages and emphasizes the necessity of having a well-designed and operating HACCP plan for each type of sausage. In another study examining the fate of L. monocytogenes in fermented sausage prepared from poultry [ 1421, meat supplemented with two levels (2.0 and 2.5%) of sodium nitrite and glucono-delta-lactone (GdL) was inoculated to contain 104CFU/g of L. monocytogenes, and then fermented at 20°C both with and without a starter culture. L. monocytogenes survived the 28-day ripening period, with numbers decreasing 1 log in formulations containing 2.5% sodium nitrite and either GdL or starter culture. Except for a possible 2-log decrease in batches containing 2% sodium nitrite, L. monocytogenes populations remained constant in all other formulations. Thus, the microbiological hurdles implemented, including preservatives, starter culture, a, (0.90-0.92), and pH (final pH ranged from 4.8 to 5.9), failed to completely inactivate Listeria [142]. After recognizing that L. monocytogenes may survive the typical manufacturing process for hard salami, Glass and Doyle [85] attempted to identify various heat treatments that could be used to inactivate L. monocytogenes during manufacture of dry fermented sausage. Work with "beaker sausage" prepared from ground beef/pork containing 3.5% salt, 103 ppm sodium nitrite, and approximately 5 X 103 CFU/g of L. monocytogenes indicated that Listeria populations decreased > 10-fold after fermentation (32.2"C/ 16 h) with P. acidilactici during which the pH decreased from approximately 6.3 to 4.8. Prolific growth of L. rnonocytogenes in a similar lot of beaker sausage prepared without P. acidilactici confirms the importance of an active starter culture in preventing growth of listeriae in fermented sausage. Furthermore, these findings suggest that 3.5% salt and 103 ppm sodium nitrite are of virtually no value in preventing growth of listeriae in sausage mix. Subsequent holding of fermented beaker sausage at 46.1"C for 8 h or heating to an internal temperature of 5 1.7 or 57.2"C failed to eliminate listeriae, with the pathogen still being present in all samples examined by the USDA enrichment method. Although holding samples at 5 1.7"C for 8 h or 57.2"C for 4 h reduced Listeria populations > 100-fold, the pathogen was still detected by enrichment in one of two samples that received each of the two heat treatments. Only after heating beaker sausage to an internal temperature of 623°C was the pathogen no longer detected either by direct plating or enrichment. Subsequently, Glass and Doyle [85] investigated the fate of L. monocytogenes in pepperoni during normal processing and storage and during heating to an internal temperature of 5 1.7"C for 4 h immediately after fermentation or drying. After inoculating commercially prepared pepperoni mix to contain 104 CFU/g of L. monocytogenes, populations of listeriae decreased approximately 100-fold following fermentation (35.6"C/ 12 h) by P. acidilactici which caused the pH to decrease from 6.0 to 4.7. These findings are similar to those of Johnson et al. [ 1141 (Fig. 9), who found Listeria populations decreased 10to 100-fold during fermentation of hard salami. Following 5 days of drying at 12.8"C, numbers of listeriae decreased to < 10 CFU/g in normally processed pepperoni; however, with the USDA enrichment procedure, L. monocytogenes could still be detected in 82day-old refrigerated samples of vacuum-packaged pepperoni. Heating the same pepperoni to an internal temperature of 5 1.7"C between fermentation and drying had relatively little

-

-

Listeria monocytogenes in Meat Products

545

effect on 1,. monocytogenes, with viable populations decreasing only about 10-fold. Although holding pepperoni for 4 h at 5 l .7"C reduced Listeria populations to undetectable levels (as determined by direct plating and enrichment), the pathogen was sporadically recovered from 5- to 22-day-old sausage using the USDA enrichment procedure. Subsequent holding of the same pepperoni (pH 4.6) at an internal temperature of 51.7"C for 4 h immediately after 26 days of drying at 123°C completely inactivated the pathogen as determined by direct plating and enrichment procedures. Additional experiments conducted on pepperoni containing 5.3 X 103CFU/g of L. monocytogenes after 19 days of drying verified that a minimum heat treatment of 4 h at 51.7"C was required to obtain a Listeria-free product. Thus, although normal processes used to manufacture pepperoni will not eliminate L. monocytogenes from heavily contaminated product, holding pepperoni and possibly other dry sausages at an internal temperature of 5 1.7"C for at least 4 h may prove to be a viable means of salvaging contaminated product. Although the antibotulinal properties of nitrate, and particularly nitrite, have been recognized for many years, much remains to be learned concerning the effect of these preservatives on Listeria behavior in dry fermented sausage. Junttila et al. [ 1 181 examined the ability of L. monocytogenes to survive in dry Finnish sausage containing various levels of potassium nitrate, sodium nitrite, and salt. All sausage was prepared from a mixture of ground beef and pork to which sugar, spices, and 3.0 or 3.5% salt were added along with 50- 1000 pprn potassium nitrate and/or sodium nitrite. After inoculation to contain 105CFU/g of L. monocytogenes and a starter culture consisting of Staphylococcus carnosus and Lactobacillus plantarum, the sausage mix was stuffed into casings. All sausage links were fermented 2 days at 23"C, smoked 5 days at 20--22"C, and then dried 1 week each at 18 and 10°C. L. monocytogenes populations in sausage containing commonly used levels of salt (3.0%) and sodium nitrite ( I 20 ppm) decreased 1.14 orders of magnitude over 21 days (Fig. 10). Similar findings also were reported when 3.5 rather than 3.0% salt was used. Increasing the levels of sodium nitrite (200 ppm) and potassium nitrate (330 ppm) to those commonly used 30 years ago led to somewhat faster inactivation of listeriae in dry fermented sausage, with inactivation again being most pronounced during the later stage of drying. Over the same 21-day period, Listeria populations decreased approximately 3.3 orders of magnitude in sausage containing 3.5% salt and 1000 ppm potassium nitrite; however, this concentration of potassium nitrate is no longer permitted in dry fermented sausage. Growth of L. monocytogenes in this product was apparently suppressed by the combination of salt, sodium nitrite, and a pH of 4.7; however, given the pathogen's known tolerance to salt, acid, and low temperatures, addition of commonly used levels of sodium nitrite to fermented sausage was only marginally effective in inactivating listeriae. Thus, although this and other studies have provided valuable information concerning the behavior of L. monocytogenes in sausage products, an understanding of interactions between various factors such as starter cultures, food additives, and various heat treatments is still needed to develop suitable methods to eliminate L. monocytogenes from fermented sausage and other processed meat products.

-

Modified-Atmosphere Packaging Modified-atmosphere packaging (MAP) can extend the shelf life of many perishable foods such as meats and poultry. The C02-enriched atmosphere which is created within a meat pack can inhibit normal spoilage flora and select for certain groups of organisms such as the lactic acid bacteria [69]. Concerns have been raised about the ability of L. monocytogenes to outgrow the normal spoilage flora on MAP foods. In addition, MAP foods have

546

Farber and Peterkin

,

Dryingat 18°C

8

3.0 I 0

1

I

I

I

I

I

1

1

2

3

4

5

6

7

I,

N

,

I

Drying at 10°C

I

I

1

14

21

Days

FIGURE10 Fate of L. monocytogenes during fermentation (Ferm),smoking, and drying of Finnish sausage prepared from ground beef/pork containing 3% NaCl and various concentrations of NaNO, and/or KN03.(Adapted from Ref. 118.) a relatively long shelf life, which in turn can give extra time for psychrotrophic foodborne pathogens such as L. monocytogenes to grow to high levels. Although Listeria can grow on vacuum-packed meats such as beef, lamb, and pork, as discussed earlier, the effect of intermediate to high levels of CO2 on survival and growth of this pathogen on meat and poultry is not clear [77].

Beef Growth of L. monocytogenes was observed on samples of vacuum-packaged high-pH (>6) beef stored at 0, 2, 5, and 10°C but not on those vacuum-packs stored at -2°C. However, long lag periods were usually observed, with the organism growing at a slower rate than the spoilage flora [81]. When samples were packaged under CO2, Listeria only grew at 10°C and not at any of the lower storage temperatures tested. However, when normal ultimate-pH beef (pH 5.3-5.5) was tested, L. monocytogenes was unable to grow on samples stored in CO2 packs at 5 or 10°C [27]. Hence, the lower pH of normal as compared with dark firm dry (DFD) meat, combined with the high CO2environment, was probably sufficient to inhibit growth and partially inactivate the organism. As in the findings of Grau and Vanderlinde [ 8 3 ] ,L. rnonocytogenes grew well on vacuum-packaged meat stored at 5 and 10°C. It is interesting that in the study by Avery et al. [27], L. monocytogenes outgrew the spoilage flora on vacuum-packaged beef, which is in contrast to the results obtained by Gill and Reichel [81]. Perhaps L. monocytogenes can compete better with spoilage organisms at a lower pH. A follow-up study by Avery et al. [28] was designed to assess the effects of previous high CO2 exposure of Listeria to its subsequent growth

Listeria monocytogenes in Meat Products

547

during abusive retail display. Beef steaks of normal pH were inoculated with L. monocytogenes, individually packaged in CO2 packs, and then stored at - 1.5"C for <3 h and 5 or 8 weeks. At the end of each storage period, samples were removed, overwrapped, and placed on retail display at 12°C for up to 140 h. Even after only a brief (<3 h) exposure to COz, L. rizonocytogenes grew slightly, if at all, during retail display, with demonstrated lag phases of >75 h. However, no comparative controls were used to confirm growth of the organism following inoculation and storage at 12°C. Experiments were also done whereby steaks were removed from storage and then rinsed to remove some cells of L. monocytogmes, which were reinoculated onto freshly cut beef steaks to simulate cross contamination. Under these conditions, Listeria cells previously exposed to CO, for 5 or 8 weeks did not grow on cross-contaminated steaks. Consequently, it was concluded that (a) prior exposure of L. monocytogenes-contaminated beef steaks to high CO, environments will not increase the likelihood for growth of the organism when the steaks are placed on retail display and possibly temperature abused; and (b) the risk of growth is minimal when cross contamination occurs between high-C02 stored beef and fresh raw beef before retail display. Additional experiments have examined survival and growth of this pathogen on sliced roast beef stored under vacuum or CO2 at - I .5 and 3.0"C. Although unable to grow under CO, at - l SoC, L. monocytogrwes did grow under all other test conditions. At 3"C, the organism grew three times faster on vacuum-packaged as compared with CO2-stored roast beef (see Table 8). When growth occurred, maximum populations were attained only at the end of product shelf life.

Sausages Experiments were done to determine survival of L. monocytogenes on sliced frankfurters incubated under 20-80% CO, at 4,7, and 10°C for up to 6 weeks [ 1251. Although numbers of L. mono~ytogenesin vacuum packs, 20% CO,, and 30% CO2 increased by 2.5, 1.0, and 0.5 logs, respectively, during the commercial minimum shelf life of 3 weeks at 4"C, the pathogen was inhibited in the presence of both 50 and 80% CO,. During an additional 3 weeks of storage, the organism grew in the presence of 50% but not 80% COz. However, increasing the storage temperature to 7°C permitted growth of the organism during this 3-week storage period even in the presence of 80% COz. Therefore, under commercial conditions (4-10°C, 3-week shelf life), only 80% CO2prevented growth of the organism. However, since this level of CO2,caused undesirable organoleptic changes in the product, CO, levels between 50 and 80% may be more appropriate [ 1251.

Lamb Sheridan et al. [165] examined growth of L. monocytogenes on both raw minced lamb and lamb pieces stored at 0 and 5°C under various atmospheres (vacuum; 80% 02:20% CO,; 50% 0,:50% CO,; and 100% CO,). No growth was observed on lamb stored at 0°C. At 5"C, L. monocytogenes grew on aerobically packaged and vacuum-packaged lamb pieces but not on minced lamb (Table 1 1 ) . Disregarding aerobically packaged samples, highest Listcria populations were observed on pieces and minced lamb stored under 80% 0,:20% CO1 and 50% Oz:5O%CO2,respectively, after 42 days of storage at 5°C. Again, L. monocytogenes failed to grow on lamb stored under 100% CO2.

Pork When the microbial ecology of fresh MAP pork was assessed at various storage temperatures, listeriae were one of the predominant organisms on product stored at - I "C but not on samples stored at 4.4 or 10°C [ 1461. In fact, most bacteriocin-producing organisms

Farber and Peferkin

548

TABLE11 Mean Total Aerobic and L. monocytogenes Counts (loglo CFU/g) at 42 Days on Lamb Pieces and Mince Packaged i n Different Gas Atmospheres at 5°C

Pieces

Meat type

TA

Gas atmosphere: Air Vacuum pack 80% 02/20% CO2 50% C02/50% N2 100% CO2

8.86 7.53 8.91 8.15 7.55

Mince

LM"

TA

LM

6.07 3.81 4.91 4.22 1.35

9.20 8.65 8.97 7.75 8.07

5.37 1.74 2.68 3.68 0.58

TA, total aerobic: LM, L. rnonocytogenes. a Although not clearly stated, the initial total LM count appears to be 2.8 X 10' CFU/g. Source: Adapted from Ref. 165.

were isolated from samples stored at the two higher temperatures. No growth of L. rnonocytogenes was observed on fresh pork longissimus dorsi at 1°C regardless of storage atmosphere. At 7"C, L. rnonocytogenes grew on aerobically stored samples but not on those stored under 100% N,, 80% 02:20% CO, or 60% 0,:40% CO, [140]. No additional hazards were identified using modified atmosphere (MAs) for packaging fresh pork of normal pH. According to Davies [59], under an atmosphere of 80% 02:20%CO,, growth of L. rnonocytogenes on cooked ham was no greater than that observed in aerobically stored control samples. Manu-Tawiah et al. [ 1411 examined the influence of 20 and 40% CO2on growth of L. rnonocytogenes and Yersinia enterocolitica on fresh pork chops stored at 4°C for 35 days. Levels of L. rnonocytogenes on air- or vacuum-packaged pork generally did not differ significantly from the numbers on chops packaged in 20 and 40% CO2, with populations increasing 1.5 to 2.0 logs (from 3.7-106 CFU/cm2) after 35 days. The growth rates for Listeria and the aerobic psychrotrophic spoilage flora were faster in air as compared with C02-packaged samples, with the aerobic psychrotrophic spoilage flora always outgrowing L. rnonocytogenes. In contrast to the results of Wimpfheimer et al. [185], no differences were observed in the numbers of L. rnonocytogenes on chops packaged in 60% N2:40% CO, or 50% N,:lO% 02:40% CO2. Y. enterocolitica outgrew L. rnonocytogenes in all MA-packaged samples, increasing to nearly 108CFU/cm2 after 35 days of storage at 4°C. The fact that Yersinia grew much better in MA-packaged than in aerobically stored chops is disconcerting from a public health standpoint. The application of additional hurdles in addition to MAP is a strategy that will be more commonly used in the future. As a case in point, the combined effect of nisin and MAP on growth and survival of L. rnonocytogenes on cooked pork tenderloin was examined during storage at 4 and 20°C [67]. The organism grew on pork tenderloin packaged under 100% CO, at both storage temperatures. However, when nisin ( 104 IU/mL) was added, the combination proved to be bactericidal (Table 12). Although a lower concentration of nisin ( 103IU/mL) also was listericidal in pork stored at 4O, but not at 20"C, simultaneous growth of Pseudornonas fragi, a spoilage organism, was observed. Fewer pseudomonads were generally seen in MA-stored samples, with these levels being unaffected

Listeria monocytogenes in Meat Products

549

TABLE12 Cumulative Changea in Log CFU/g for L. monocytogenes on Cooked Tenderloin Stored in MAP at 4 and 20°C MAP

Time‘ (days) 6 12 18 24 30

100% air with nisinb

100% CO;! with nisin

0

104

0

2.78 4.28 5.03 4.97 4.81

-2.11 -2.10 -2.12 -2.15 -2.18

0.65 2.16 4.97 4.74 4.67

104 -2.18 -2.18 -2.19 -2.18 -2.18

Timed (days) 1 2 4 7 10

100% air with nisinb 0 104 4.20 5.24 5.36 5.64 6.07

-2.10 -2.11 -2.13 -2.14 -2.15

100% CO2 with nisin 0

104

1.90 4.32 5.57 5.67 5.53

-2.10 -2.11 -2.10 -2.13 -2.14

MAP, modified-atmosphere packaging. Log CFU/g at day X -log CFU/g at day 0 for two samples per treatment per day. The concentration of L. rnonocytogenes on the cooked tenderloin surface at 0-day was 3.18 log,, CFU/g. The unit for nisin activity was IU/mL. Storage temperature of 4°C. Storage temperature of 20°C. Source: Adapted from Ref. 67. a

by nisin. The authors used the general concept of a “safety index,” which compares the relative numbers of spoilage organisms with pathogens. In the MAP samples containing nisin, numbers of P. fragi increased during storage relative to the growth of L. monocytogenes. However, such growth was not observed in nisin-free samples. MAP also has been used in conjunction with irradiation to minimize growth of foodborne pathogens on raw pork [88]. In nonirradiated pork stored under an atmosphere of 75% N2: 25% COz, L. monocytogenes populations increased about 2 logs after 9 days of storage at 10°C, with the pathogen being undetected in irradiated (1.75 kGy) control samples. Similar results were obtained using a higher initial inoculum (106 versus 103 CFU/g). After 9 days of incubation at 10°C, Listeria counts numbered about 4.5 X 104 and 1 X 10s CFU/g in irradiated and nonirradiated treated samples, respectively. Potential benefits of using irradiation after meat packaging were also evident, since lactic acid microflora outgrew L. monocytogenes and several other pathogens tested during storage [88]. Since Lactobacillus sake‘ usually predominates the microflora of irradiated MAP pork, sakacin A (a bacteriocin produced by this organism) may have been responsible for inhibiting the growth of L. monocytogenes. However, L. monocytogenes grew to similar levels in both irradiated and nonirradiated samples. Given these findings, L. monocytogenes will likely grow in the presence of up to 50% CO2, with growth at higher CO2 concentrations mainly dependent on the interplay between the gas atmosphere, pH, temperature, and other microbial competitors.

Thermal Inactivation in Meats Interest in the heat resistance of listeriae in raw meat products dates back to at least 1980 when Karaioannoglou and Xenos [ 1191 reported on survival of Listeria in grilled meatballs. In their experiments, minced beef inoculated to contain 102, 103, 104, or 105 L. rnonocytogenes CFU/g was combined with eggs, bread, onion, garlic, salt, and spices and then fashioned into meatballs weighing 35-40 g each. Meatballs were then placed on a

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coal-fired grill at 110-120°C and cooked for 15 min to an internal temperature of 7885°C. After grilling, L. monocytogenes was isolated from all meatballs that originally contained 104-1OS listeriae/g. However, the pathogen was recovered from only one of four meatballs inoculated to contain 103listeriae/g and was absent from meatballs that originally contained 1O2 listeriae/g. Since data from the European surveys discussed earlier indicate that retail raw beef may occasionally contain up to 103Listeria CFU/g (some of which are likely to be L. monocytogenes), thorough cooking of raw meat is presently advised to eliminate L. monocytogenes as well as E. coli 0157:H7, salmonellae, Campylobacter spp., and other organisms that have been associated with foodborne illness. Although these findings attest to the hardy nature of listeriae in fresh ground beef, L. monocytogenes also was equally tenacious in artificially contaminated frozen ground beef, with populations remaining unchanged at 10' CFU/g during 6 months of storage at - 18°C. Concern about possible resistance of L. monocytogenes to pasteurization of milk, along with recovery of this pathogen from cooked meats, prompted interest in the possibility of L. monocytogenes surviving thermal processing steps commonly used to convert raw meat into ready-to-eat products. Boyle et al. [36] investigated the thermal destruction of L. monocytogenes strain Scott A in ground beef (-20% fat) by submerging sealed tubes containing ground beef with 10' listeriae/g in a water bath at 75°C until the internal temperature of samples reached 50, 60, 65, or 70°C. Samples then were examined for listeriae by direct plating as well as selective and cold enrichment. According to these researchers, Listeria levels remained constant in samples heated to an internal temperature of 50°C over 6.2 min. Numbers of listeriae decreased 4.4-6.1 orders of magnitude in samples of ground beef during 8.4 and 10.6 min of heating to 60 and 65"C, respectively, with similar results also being reported in 1988 by Farber et al. [72]. Heating ground pork to 62°C over a period of 25 min was sufficient to inactivate 5.8-7.35 log CFU/g of L. monocytogenes depending on the pork formulation. Most additives, such as kappa-carrageenen, sodium lacate, and algin/calcium binders used in the ground pork formulations, did not influence thermal resistance of the organism [188]. Similarly, Yen et al. [190] found that sodium phosphate, sodium erythorbate, and added water had little or no effect on survival of various L. monocytogenes strains in ground pork during heating. Interestingly, although an added cure decreased Listeria inactivation by 2.0-2.2 logs in ground pork cooked to 62°C as had been noticed previously [69a, 1891, this protective effect was only seen at temperatures below 67°C. Several studies were done to determine the D- and z-values for L. monocytogenes in various ground and whole meat products (Table 13). D,,,o,-values for most products range from 1.8 to 8.3 min. Carlier et al. [45] examined destruction of L. monocytogenes in whole hams cooked to an internal temperature of 583°C. When stored for 2 months at 9"C, survivors were found among hams inoculated to contain 4 X 10s CFU/g of L. monocytogenes but not in those inoculated with <10 CFU/g. It was suggested that a of at minimum core temperature of 65°C be attained in these products, with an F700c-value least 40 min. Another study examined survival of L. monocytogenes in vacuum-packaged, nitrite-free beef roasts prepared with brines containing selected antimicrobial agents [ 1741. The brines used included sodium chloride, sodium tripolyphosphate, Brifisol 4 14, acetic acid, sodium lactate, Lauralac, and potassium sorbate. Meats were cooked in a bag once or twice to 623°C under conditions simulating product contamination from pumping brines or from slicing/cutting after cooking. Survival of L. monocytogenes on the surface of beef roasts was surprisingly high considering that cells had possibly been exposed to temperatures 280°C for more than 30 min. Irrespective of the number of cookings, Liste-

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TABLE 13 Heat Resistance of L. rnonocytogenes in Meats Product Ground beef Fermented sausage mix Ground beef roast Ground beef Beaker sausage Meat slurry Meat slurry Beef Beef steak Liver sausage slurry Lean ground beef Fatty ground beef Meats (predicted value) Ham Ground pork Ground pork

Temp. ("C)

D-value ("C; min.)

z-value

("Cl

Ref.

60 60 60 60 60 60 70 60 60 70 60 70 60 62.8

3.12 16.7 4.47 1.62 9.13 2.54 0.23 7.3 3.8 0.14 8.32, 6.27 0.20, 0.14 2.42 0.6 1.2 3.82 0.13 1.82 3.48a 6.5-7.7 1.14- 1.7

5.3 4.6

72

60 70 60 60 62 60

160 36

6.8 7.2

155 137

5.98, 5.98

78

6.2 9.3 11.4 6.8

35 68

5.05 6.74

5.05-5.45

I37 44

123 152

aCells heat shocked at 42°C for 1 h.

ria survival rates in internally cooked inoculated roasts were similar for all brine treatments except NaC1-sodium tripolyphosphate, raising the possibility that sublethal heating may have induced a heat-shock response in these organisms. The highest number of Listeriapositive samples was seen among roasts processed with standard NaC1-phosphate brines, which most closely resembles product currently being marketed. In contrast, greatest destruction of the organism was observed with brines containing a phosphate blend and sodium lactate or glycerol monolaurin in combination with one, and especially two, cookings [174]. Steam pasteurization is gaining acceptance as a means of reducing surface levels of pathogenic microorganisms on meats. Some advantages of using steam pasteurization include its ability to uniformly heat the entire carcass surface, and uniformly cover irregularly shaped surfaces. Furthermore, waste water accumulation is not an issue, and, by virtue of automation, the process is not subject to operator misuse. In one study, frankfurters were inoculated with L. innocua and steam pasteurized in a small pasteurizer designed in-house. The heating chamber was evacuated for 15 s after which the product was steam treated at a set time, temperature, and pressure. Treatment times of 32 and 40 s at 136 and I15"C, respectively, led to a &log reduction in counts of L. innocua on the meat surface while only slightly affecting color and weight [58]. Another study compared steam pasteurization (S; 15-s steam pasteurization) to traditional methods for reducing pathogens on meat surfaces [ 1531. The latter methods, which were tested both individually and in combination, included knife trimming (T), water washing (W; 35"C), hot water/steam vacuum spot cleaning (V), and spraying with 2% vol/vol lactic acid (pH 2.25 at 54°C)

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TABLE 14 Effectiveness of Combination and Individual Decontamination

Treatments in Reducing L. monocytogenes on Surfaces of Freshly Slaughtered Beef

Treatmenta TW TWS

ws vw vws

TWLS VWLS

Initialh 5.52 5.57 5.46 5.56 5.49 5.51 5.51

f. 0.22

f 0.15 f. 0.03 k 0.12 t 0.04 k 0.19 & 0.13

Mean reductionc 4.96 4.56 4.40 3.49 3.84 5.07 5.01

f. 0.34d

t 0.34d' -t 0.34def 2 0.34f ? 0.34" f. 0.34d t 0.34d

Treatmenta T W V S VWLS*5 VWLS*lO

Initialb 5.26 5.27 5.37 5.38 5.18 5.21

f 0.33 t 0.35 f 0.20 f. 0.16 f. 0.35 f. 0.33

Mean reductionc 2.54 f. 0.33' 1.28 2 0.33g 3.33 f. 0.33ef 3.44 k 0.33ef 4.51 k 0.33d 4.23 -+ 0.33de

Order of treatment within abbreviation indicates order of application: T, trim; W, 35°C water wash; S, 15-s steam pasteurization; V, hot water/steam vacuum spot cleaning; L, 2% lactic acid spray; S* 5 and S* 10, 5and 10-s exposure time, respectively, for steam pasteurization. Mean initial pathogen population (log CFU/cm') from four replications standard error of mean. Mean reduction in pathogen population (log CFU/cm') from four replications 5 standard error of mean. d,ef.g Means having the same superscript within columns are not significantly different ( P < .05). Source: Adapted from Ref. 153. a

*

(L). All combination treatments reduced numbers of L. monocytogenes on meat surfaces, with TWLS, VWLS, and TW (the order of treatment within the abbreviation indicates the order of application) being most effective and VW and VWS the least effective (Table 14). The authors concluded that greater pathogen reduction can be attained by using combinations of decontamination treatments, with knife trimming and/or steam vacuum spot cleaning (to remove visible contamination), followed by steam pasteurization, being a very effective intervention strategy for the meat industry [ 1531. Dorsa et al. [63] also examined the efficacy of using steam vacuuming (SV) and a hot water spray wash of 74 t 2°C (W) to eliminate L. innocua from the surface of beef carcass tissue. Initial reductions of 2.0, 2.2-2.5, and 2.6-2.7 CFU/cm2 were observed after the SV, W, and SV + W treatments, respectively. Although numbers of L. innocua on all treated meat samples were the same as those on the untreated control following 21 days of storage at 5°C (Fig. 1 l), it should be noted that the initial loads of listeriae on meat were high (106 CFU/cm2) and that with lower initial levels, outgrowth of the organism may not have occurred or been minimal. Regarding heat resistance in meat and poultry, we can conclude that L. monocytogenes is about four times more heat resistant than salmonellae in meats. Attention should be given to those meats which are heated slowly, since such processes may induce production of heat-shock proteins which would in turn enhance thermal resistance of the pathogen. D-values obtained for meats can vary quite widely depending on the prior history of the food and organism, recovery method, and strain type; however, in general, L. monocytogenes exhibits higher D-values in meat rather than dairy products: Cured meats require greater heating than noncured products to assure the same level of protection, with multiple decontamination treatments working best to eliminate this pathogen from carcass surfaces.

Bacteriocins for Controlling Listeriae in Meat Several articles have reviewed the potential benefits of using bacteriocins and/or lactic acid bacteria to control Listeria spp. in foods [149] and more specifically in meats

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*

2*o 1Before .o

After

treatment

treatment

2

7

14

21

Time (Days)

FIGURE11 Effects of moist heat interventions on the initial levels and subsequent outgrowth of Listeria innocua (least squares means, log CFU/cm2:error bars denote standard error of the mean) during a combination of aerobic and vacuum storage at 5°C for 21 days. (Adapted from Ref. 63.) [147,169]. Since the general use of bacteriocins to control listeriae has been discussed earlier in this book (Chap. 6), the following discussion of bacteriocins will be confined to meat applications.

Raw Ground Meat In one of the first bacteriocin-related studies, Buchanan and Klawitter [39] assessed the ability of Curnobacterium piscicola strain LK5 to inhibit growth of L. monocytogenes in a variety of different foods. Foods were inoculated with 103CFU/g of L. monocytogenes either with or without 104CFU/g of strain LK5. In sterile raw ground beef, strain LK5 inactivated L. monocytogenes at 5°C and prevented its growth at 19°C. C. piscicola had no effect on L. monocytogenes in nonsterile ground beef (or chicken roll), with no growth of the pathogen being observed in control samples. The bacteriocin-producing strain generally was most effective in foods containing a background microflora. Similar studies using sterile and nonsterile ground beef were done with Lactobacillus casei and its associated bacteriocin, lactocin 705 [ 1781. Meat was inoculated with either L. casei or the pure bacteriocin and then stored at 20°C for 24 h. In general, inactivation of L. monocytogenes was greatest at the highest level of lactocin 705 tested ( 1 6,80OAU/mL), with the fewest listeriae being recovered from a meat slurry and autoclaved ground beef. Inhibition of L. monocytogenes by starter cultures also has been assessed in minimally heat-treated vacuum-packaged beef cubes either with or without gravy and/or glucose. For these experiments, Lactobacillus bavaricus strain MN was inoculated into beef at 10sor 103CFU/g, along with 102CFU/g of L. monocytogenes, then vacuum sealed, and stored at 4 or 10°C [ 1861. Strain MN grew and produced bacteriocin during the early stages of growth, with inhibition of L. monocytogenes being most pronounced at the higher MN inoculum level and lower incubation temperature. Bacteriocin production was independent of the presence of glucose in the meat. Addition of sugar enhanced the antilisterial activity even though the pH was not greatly reduced in those meats with gravy and glucose.

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Greater antilisterial activity was seen in meats containing gravy with glucose, suggesting that the gravy may have enhanced diffusion of the bacteriocin. Bacteriocin-producing lactic acid bacteria also have been used to minimize growth of L. rnonocytogenes on frankfurters. In these experiments, either high ( 107CFU/g) or low ( l 03- 1O4 CFU/g) levels of a bacteriocin-producing strain of Pediococcus acidilactici, as well as its plasmid-cured, bacteriocin-negative derivative (bac- ), were inoculated separately onto frankfurters along with a five-strain cocktail of L. rnonocytogenes. Frankfurters were stored both aerobically and anaerobically at 4 and 15°C. The presence of P. acidilactici on the frankfurters inhibited listeriae to various degrees depending on the pediococci levels, storage temperature, and packaging atmosphere [32]. Yousef et al. [ 1913 reported that L. rnonocytogenes grew in two of five wiener exudates tested, with the best growth being observed in exudates containing the lowest concentration of phenols, the lowest indigenous levels of lactic acid bacteria, and the highest pH. Those exudates which did not support growth of the organism, including those at 4OC, proved to be listericidal. Glass and Doyle [85] previously showed that L. monocytogenes could grow on wieners stored at 4.4"C. These conflicting results could be explained by the loss of wiener exudate during manipulation, uneven distribution of exudate on the wiener surface, or variations in the intensity of smoking within and among different brands of wieners [ 19I]. Both P. acidilactici H and pure pediocin AcH inactivated listeriae that had been inoculated into one brand of wiener exudate and stored at 25°C for 8 days. In control samples, L. rnonocytogenes populations increased from about 104to nearly 107CFU/g within 4 days. In a follow-up study, the latter authors assessed the ability of the same lactic acid bacteria to limit growth of L. rnonocytogenes in temperature-abused vacuum-packaged wieners stored at 4 and 25°C. At 25OC, the presence of P. acidilactici strain LB42 inhibited but did not completely inactivate L. rnonocytogenes. However, P. acidilactici strain JBL 1095 was listericidal, decreasing counts of L. rnonocytogenes by 2.7 logs. This inactivation was not solely caused by pH, since pH values in wieners inoculated with both strains of pediococci were similar. The only difference between the two strains was that production of pediocin AcH was confined to JBL 1095, strongly suggesting that this bacteriocin enhanced the antilisterial activity of lactic acid bacteria in vacuum-packaged wieners.

Fermented Sausages Similar work also has been done to examine the effects of starter cultures and/or their bacteriocins on survival and growth of L. rnonocytogenes in sausages. Foegeding et al. [76] used pediocin-producing strains of P. acidilactici (along with an isogenic mutant as a control) to minimize growth of L. rnonocytogenes on dry fermented American-style sausages. Recovery of the organisms during fermentation was made easier by using antibiotic-resistant strains of pediococci and listeriae. The study was unique in showing that in combination with other fermentation endproducts, inhibition of L. rnonocytogenes was enhanced by bacteriocin production in situ during fermentation and drying. Berry et al. [31] had also previously shown the benefits of using P. acidilactici as a starter culture to minimize growth of listeriae during sausage fermentation. According to these authors, a commercial summer sausage mix was inoculated to contain - 106CFU/g of L. rnonocytogenes and fermented with either a bacteriocin-producing or non-bacteriocin-producing strain of P. acidilactici. Following a 12- to 14-h fermentation at 37.8"C, populations of listeriae decreased approximately 100-and 10-fold in summer sausage fermented with bacteriocin-producing and non-bacteriocin-producing strains of P. acidilactici, respectively. The pathogen also was inactivated in sausages with slower acid production (pH >5.5),

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which suggests that bacteriocin production occurred independent of carbohydrate fermentation. However, L. monocytogenes was still detected in 9 of 90 (10.0%)sausage samples that had been heated to an internal temperature of 64°C and then refrigerated for up to 2 weeks. Thus, although use of this bacteriocin-producing starter culture led to a dramatic decrease in numbers of viable listeriae in summer sausage, it did not completely inactivate the pathogen in finished product prepared from sausage mix containing 106CFU/g of L. monocytogenes. However, since the presence of > I O3 L. monocytogenes/g in commercially prepared sausage mix is highly unlikely, it appears that current heat treatments are adequate to produce Listeria-free semidry sausage. Another starter culture, Lactobacillus sake‘, was found to be capable of minimizing growth of L. monocytogenes in Germanstyle fresh sausages. Results varied depending on the initial pH of the “fresh Mettwurst,” with bacteriostatic and bacteriocidal ( 1-log reduction) effects on L. monocytogenes observed at initial pH values of 6.3 and 5.7, respectively. In another study [ 1051, a similar bacteriocin-producing (sakacin K) strain of L. sake‘ isolated from a naturally fermented sausage was able to reduce the numbers of L. monocytogenes in dry fermented sausage by 1.25 logs as compared with the isogenic bac- control strain. The fate of L. monocytogenes in naturally and artificially contaminated salami also has been assessed using Luctobacillus plantarurn as the starter culture [42]. In this study, the starter culture prevented growth of listeriae but was not listericidal. In inoculated samples, little difference was observed between samples containing bac+ or bac- strains of L. plantarurn. However, among naturally contaminated samples, only those inoculated with the bacteriocin-producing strain of L. plantarurn were free of listeriae. Bacteriocins also have been used in studies aimed at inactivating or preventing attachment of L. monocytogenes to muscle surfaces. El-Khateib et al. [65] investigated the ability of lactic acid, nisin, and pediocin to inactivate L. monocytogenes on beef muscle, as well as to prevent its attachment after short-term exposure and during 48 h of refrigerated storage. The latter experiments were done to simulate secondary contamination of meat during refrigerated storage. Lactic acid (2%), nisin (4 X 104IU/mL), and pediocin (3.2 X 103AU/mL) decreased the numbers of listeriae on the meat surface by 1.7, 1.1, and 0.6 log 1o CFU/6 cm2 (cubical piece), respectively. Interestingly, compared to the control, treatment with lactic acid caused a greater percentage of listeriae cells to attach to the meat surface. This was partially attributed to the fact that acidic conditions can sometimes enhance adhesion of bacteria to surfaces [65]. Using nisin and pediocin, the percentage of L. monocytogenes cells that attached to beef muscle in the presence of both bacteriocins either did riot change significantly or decreased slightly. However, after 1 h, 30 and 17% of the cells were attached to the nisin- and pediocin-treated samples, respectively, as compared with 6.3 and 12% for the controls. Other work done by Cutter and Siragusa [56,57] has shown nisin to be effective in reducing (by 2.0-2.83 log,,, CFU/cm2) the numbers of L. innocua on beef. In the latter study, the combination of vacuum-packaging and nisin spraying suppressed growth of L. innocua to the point where at the end of the 4-week incubation period at 4OC, counts were lower than in the corresponding nontreated controls. In summary, bacteriocins, especially pediocin, appear to be good candidates for controlling growth of listeriae in meat and poultry, with their effectiveness having been proven in wieners and wiener exudates, semidry and dry sausages, and fresh beef and pork. The main obstacles to their present use in foods are the development of bacteriocinresistant strains, activity loss over time, inactivation by proteases, and/or adsorption to meat and fat particles resulting in inactivation, limited diffusion, especially in minced meats, and poultry, and finally regulatory acceptance.

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REFERENCES Adesiyun, A.A. 1993. Prevalence of Listeria spp., Curnpylobacter spp., Salmonella spp., Yersinia spp. and toxigenic Escherichia coli on meat and seafoods in Trinidad. Food Microbiol. 10:395-403. 2. Adesiyun, A.A., and C. Krishnan. 1995. Occurrence of Yersinia enterocolitica 0:3, Listeria rnonocytogenes 0 :4 and thermophilic Curnpylobacter spp. in slaughter pigs and carcasses in Trinidad. Food Microbiol. 12:99- 107. 3. Amtsberg, G. von, A. Elsner, H.A. Gabbar, and W. Winkenwerder. 1969. Die epidemiologische und lebensmittelhygienische Bedeutung der Listerieninfektion des Rindes. Dtsch. tierarztl. Wochenshr. 76:497-536. 4. Anonymous. 1988. AM1 data show frankfurters most likely Listeria carrier in meat. Food Chem. News 30(16):37-39. 5. Anonymous. 1988. Hot dog processing “borderline” for Listeria destruction: ARS. Food Chem. News 30(41):46. 6. Anonymous. 1988. Listeria destruction in cooked meat products ineffective: Hormel. Food Chem. News 30( I5):32-34. 7. Anonymous. 1989. Listeria in raw meat restricted in Germany. Food Chem. News 31(31): 28-29. 8. Anonymous. 1989- 1994. Annual Reports. Agri-Food Safety Division, Food Production and Inspection Branch, Agriculture Canada, Ottawa, Canada. 9. Anonymous. 1989- 1996. Food recalls. Health Protection Branch, Health and Welfare Canada, Ottawa, Canada. 10. Anonymous. 1990. The microbiological safety of food. Part 1. Report of the Committee on the Microbiological Safety of Food. Her Majesty’s Stationery Office, p 137. I I . Anonymous. 1990. Data needed to change zero Listeria tolerance stance: FDA. Food Chem. News 32(40):9- 10. 12. Anonymous. 199 1. Listeria in food. Report of the West and North Yorkshire Joint Working Group on a two year survey of the presence of Listeria in food. Environ. Health 99:132137. 13. Anonymous. 1991. All L. rnonocytogenes said to be potentially pathogenic. Food Chem. News 32(45): 18- 19. 14. Anonymous. I99 1 . Skinless hot dogs recalled for Listeria. Food Chem. News, 33( 19):39. 15. Anonymous. 1991. Ham salad recalled due to Listeria. Food Chem. News, 33(40):38. 16. Anonymous. I99 1 - 1997. Food and Drug Administration Enforcement Reports, 1991 - 1997. Food and Drug Administration, Washington, DC. 17. Anonymous. 1992. Frankfurters recalled by Connecticut firm for Listeria. Food Chem. News 34(5):5 1-52. 18. Anonymous. 1992. Mrs. Drake, Good ‘n’ Fresh sandwiches recalled due to Listeria. Food Chem. News, 34(30):36. 19. Anonymous. 1992. Class I recalls involve botulinum, Listeria. Food Chem. News, 34(36): 42. 20. Anonymous. 1993. Listeria found in 20% of hot dogs in L.A. Times survey. Food Chem. News 35(2 1):45-46. 21. Anonymous. 1995. FSIS issues snapshot of raw meat, poultry microbiological profile. Food Chem. News 37(43): 19-20. 22. Anonymous. 1996. FDA targets high risk foods in pathogen monitoring program. Food Chem. News 38(8): 15-1 6. 23. Anonymous. 1997. Possible L. rnonocytogenes contamination prompts recall of ‘fresh’ sandwiches. Food Chem. News 38(49):41. 24. Anonymous. 1997. Food recalls. Canadian Food Inspection Agency, Ottawa, Canada. 25. Anonymous. 1997. Microbiological monitoring of ready-to-eat products, 1993- 1996. United States Department of Agriculture, Food Safety and Inspection Service, Washington, DC. 1.

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25a. Anonymous. 1998. Gould’s smoked breakfast ham recalled from New Hampshire for Listeria. IJSDA-FSIS press release, July 3 1. 26. Arumugaswamy, R.K., G.R.R. Ali, and S.N. Hamid. 1994. Prevalence of Listeria monocytogenes in foods in Malaysia. Int. J. Food Microbiol. 23: 1 17- 121. 27. Avery, S.M., J.A. Hudson, and N. Penney. 1994. Inhibition of Listeria monocytogenes on normal ultimate pH beef (pH 5.3-5.5) at abusive storage temperatures by saturated carbon dioxide controlled atmosphere packaging. J. Food Prot. 57:33 1-333. 28. Avery, A.M., A.R. Rogers, and R.G. Bell. 1995. Continued inhibitory effect of carbon dioxide packaging on Listeria monocytogenes and other microorganisms on normal pH beef during abusive retail display. Int. J. Sci. Technol. 30:725-735. 29. Barbosa, W.B., J.N. Sofos, G.R. Schmidt, and G.C. Smith. 1995. Growth potential of individual strains of Listeria monocytogenes in fresh vacuum-packaged refrigerated ground top rounds of beef. J. Food Prot. 58:398-403. 30. Benezet, A., J.M. De La Osa, M. Boras, N. Olmo, and F.P. Florea. 1993. Study of Listeria monocytogenes in meat products. Alimentaria 30: 19-23. 31. Berry, E.D., M.B. Liewen, R.W. Mandigo, and R.W. Hutkins. 1990. Inhibition of Listeria monocytogenes by bacteriocin-producing Pediococcus during manufacturing of fermented semitlry sausage. J. Food Prot. 53: 194- 197. 32. Berry, E.D., R.W. Hutkins, and R.W. Mandigo. 1991. The use of bacteriocin-producing Pediococcus acidilactici to control post processing Listeria monocytogenes contamination of frankfurters. J. Food Prot. 54:68 1-686. 33. Beuchat, L.R., R.E. Brackett, D.Y.-Y. Hao, and D.E. Conner. 1986. Growth and thermal inactivation of Listeria rnonocytogenes in cabbage juice. Can. J. Microbiol. 32:79 1795. 34. Beumer, R.R., M.C. te Giffel, E. de Boer, and F.M. Rombouts. 1996. Growth of Listeria monocytogenes on sliced cooked meat products. Food Microbiol. 13:333-340. 35. Bhaduri, S.P., W. Smith, S.A. Palumbo, C.O. Turner-Jones, J.L. Smith, B.S. Marmer, R.L. Buchanan, L.L. Zaika, and A.C. Williams. 1991. Thermal destruction of Listeria monocytogenes in liver sausage slurry. Food Microbiol. 8:75-78. 36. Boyle, D.L., J.N. Sofos, and G.R. Schmidt. 1989. Thermal destruction of Listeria monocytogenes in a meat slurry and in ground beef. J. Food Sci. 55:327-329. 37. Brahmbhatt, M.N., and J.M. Anjaria, 1993. Analysis of market meats for possible contamination with listeria. Ind. J. Anim. Sci. 63:687. 38. Breer, C., and K Schopfer. 1988. Listeria and food. Lancet ii: 1022. 39. Buchanan, R.L., and L.A. Klawitter. 1992. Effectiveness of Carnobacterium piscicola LK5 for controlling the growth of Listeria monocytogenes Scott A in refrigerated foods. J. Food Safety 12:219-236. 40. BunEid, S. 199 I . The incidence of Listeria monocytogenes in slaughtered animals, in meat, and in meat products in Yugoslavia. Int. J. Food Microbiol. 12:173- 180. 41. BunEid, S., L. Paunovic, and D. Radisic. 1991. The fate of Listeria monocytogenes in fermented sausages and in vacuum-packaged frankfurters. J. Food Prot. 54:413-4 17. 42. Campanini, M., I. Pedrazzoni, S. Barbuti, and P. Baldini. 1993. Behaviour of Listeria monocytogenes during the maturation of naturally and artificially contaminated salami: effect of lactic-acid bacteria starter cultures. Food Microbiol. 20: 169- 175. 43. Cantoni, C., S. d’Aubert, M. Valenti, and G. Comi. 1989. Listeria species in cheese and fresh sausage products. Indust. Aliment. 28: 1068- 1070. 44. Carlier, V., J.C. Augustin, and J. Rozier. 1996. Heat resistance of Listeria monocytogenes (phagovar 2389/2425/3274/267 1/47/ 108/340): D-and Z-vidues in ham. J. Food Prot. 59: 588-59 1 . 45. Carlier, V., C.A. Jean, and R. Jaques. 1996. Destruction of Listeria monocytogenes during a ham cooking process. J. Food Prot. 59592-595. 46. Carlin, F., C. Nguyen-the, and A.A. Da Silva. 1995. Factors affecting the growth of Listeria monocytogenes on minimally processed fresh endive. J. Appl. Bacteriol. 78:636-646.

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Unda, J.R.R., A. Molins, and H.W. Walker. 199 1. Clostridium sporogenes and Listeria monocytogenes: survival and inhibition in microwave-ready beef roasts containing selected antimicrobials. J. Food Sci. 56: 198-206. Van Laack, R.L.J.M., J.L. Johnson, C.J.N.M. Van der Palen, F.J.M. Smulders, and J.M.A. Snijders. 1993. Survival of pathogenic bacteria on pork loins as influenced by hot processing and packaging. J. Food Prot. 56:847-85 1. Varabioff, Y. 1992. Incidence of Listeria in smallgoods. Lett. Appl. Microbiol. 14:167- 169. Velani, S., and R.J. Gilbert. 1990. Listeria monocytogenes in prepacked ready-to-eat sliced meats. PHLS Microbiol. Dig. 756. Vignolo, G., S. Fadda, M.N. de Kairuz, A.A.P. de Ruiz Holgado, and G. Oliver. 1996. Control of Listeria monocytogenes in ground beef by ‘lactocin 705’, a bacteriocin produced by Lactobacillus casei CRL 705. Int. J. Food Microbiol. 29:397-402. Villari, P., M.M. D’Errico, E. Prospero, G.M. Grasso, and F. Romano. 1991. Isolation of Listeria spp. in fresh meats produced in Campania. L’Igiene Moderna 96:274-278. Vorster, S.M., R.P. Greebe, and G.L. Nortjk. 1993. The incidence of Listeria in processed meats in South Africa. J. Food Prot. 56:169-172. Wang, C., and P.M. Muriana. 1994. Incidence of Listeria monocytogenes in packages of retail franks. J. Food Prot. 57:382-386. Wang, G.-H., K.-T. Yan, X.-M. Feng, S.-M. Chen, A.-P. Lui, and Y. Kokubo. 1992. Isolation and identification of Listeria monocytogenes from retail meats in Beijing. J. Food Prot. 55: 56-58. Wendorff, W.L. 1989. Effect of smoke flavorings on Listeria monocytogenes in skinless franks. Seminar presentation, Department of Food Science, University of Wisconsin-Madison, Jan. 13. Wilson, I.G. 1995. Occurrence of Listeria species in ready to eat foods. Epidemiol. Infect. 115:519-526. Wimpfheimer, L., N.S. Altman, and J.H. Hotchkiss. 1990. Growth of Listeria monocytogenes Scott A, serotype 4, and competitive spoilage organisms in raw chicken packaged under modified atmosphere and in air. Int. J. Food Microbiol. 11:205-214. 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. Wong. H.-C., W.-L. Chao, and S.-J. Lee. 1990. Incidence and characterization of Listeria monocytogenes in foods available in Taiwan. Appl. Environ. Microbiol. 56:3 101-3 104. Yen, L.C., J.N. Sofos, and G.R. Schmidt. 199 1. Effect of meat curing ingredients on thermal destruction of Listeria monocytogenes in ground pork. J. Food Prot. 54:408-4 12. Yen, L.C., J.N. Sofos, and G.R. Schmidt. 1992. Destruction of Listeria monocytogenes by heat in ground pork formulated with kappa-carrageenan, sodium lactate and the algin/calcium meat binder. Food Microbiol. 9:223-230. Yen, L.C., J.N. Sofos, and G.R. Schmidt. 1992. Thermal destruction of Listeria monocytogenes in ground pork with water, sodium chloride and other curing ingredients. Lebensm. Wiss. Technol. 25:61-65. 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 or 25°C. Appl. Environ. Microbiol. 57:1461-1467. Yu, L.S.L., R.K. Prasai, and D.Y.C. Fung. 1995. Most probable number of Listeria species in raw meats detected by selective motility enrichment. J. Food Prot. 58:943-945. Zaika, L.L., S.A. Palumbo, J.L. Smith, F. Del Corral, S. Bhaduri, C.O. Jones, and A.H. Kim. 1990. Destruction of Listeria monocytogenes during frankfurter processing. J. Food Prot. 53: 18-21.

175. 176. 177. 178. 179. 180. 181. 182. 183. 184. 185. 186. 187. 188. 189. 190. 191. 192. 193.

Incidence and Behavior of Listeria monocytogenes in Poultry and Egg Products NELSON A. Cox AND J. STANLEY BAILEY Agricultural Research Service, U.S. Department of Agriculture, Athens, Georgia

ELLIOTT. RYSER Michigan State University, East Lansing, Michigan

INTRODUCTION Avian listeriosis was first recognized in 1932 when TenBroeck isolated Listeria monocytogenes (then Bacterium monocytogenes) from diseased chickens [ 1 1 1,1121. Chickens have remained a common avian host for L. monocytogenes since avian listeriosis was first recognized. Listeriosis also has been observed in at least 22 other avian species, including such frequently consumed fowl as turkeys [25,59,94], ducks [55,113], geese [55,96], and pheasants [55].Large outbreaks of listeriosis in domestic fowl are relatively rare [93]; however, sporadic cases occur much more frequently and are often accompanied by asymptomatic shedding of Listeria in feces. According to one report, 4.7% of cecal samples from Danish broiler chickens harbored L. monocytogenes [96]. Hence, as was true for beef, pork, and lamb, poultry meat destined for human consumption also is at risk of becoming contaminated with L. monocytogenes, particularly when birds are slaughtered, defeathered, and eviscerated. Several early studies suggested that poultry- and egg-processing workers can contract Listeria infections by handling contaminated birds 565

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[40,71,721. Additionally, several reports from England during the 1970s indicated that L. monocytogenes could be isolated with some frequency from raw chicken as well as turkey, duck, and pheasant. Nevertheless, consumption of poultry products has been only recently linked to listeriosis in humans. An added concern with poultry relates to eggs that might become contaminated with this pathogen during collecting and processing. The emergence of L. rnonocytogenes as a bonafide foodborne pathogen following the Mexican-style cheese outbreak of 1985 prompted immediate concern about presence of Listeria in dairy products and also generated a parallel interest in the incidence and behavior of L. rnonocytogenes in meat and poultry products; the latter is the topic of this chapter. Interest in this area has increased as a result of listeriosis cases that were directly linked to consumption of turkey frankfurters and ready-to-eat/cook-chill poultry products in the United States and England, respectively. A discussion of the incidence and behavior of L. monocytogenes in egg products appears toward the end of this chapter.

USDA-FSIS LISTERIA-MONITORING/VERIFICATION PROGRAM FOR COOKED/READY-TO-EAT POULTRY PRODUCTS Public health concerns about Listeria-contaminated raw and, particularly, processed readyto-eat poultry products sold in the United States also stem directly from the 1985 listeriosis outbreak in California associated with consumption of Mexican-style cheese. Soon thereafter U.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) officials announced their intentions to develop Listeria-monitoring/verification programs for cooked and ready-to-eat meat as well as poultry products. Since these monitoring/verification programs for meat and poultry products developed in parallel and were both similar in terms of sampling scheme, methodology, and action taken when L. rnonocytogenes is found in a product, the following discussion focuses on the various products tested and pertinent results rather than on an in-depth analysis of this program. A Listeria-monitoring/verification program for cooked/ready-to-eat poultry was first suggested in December 1985 and was to cover all such products prepared in federally inspected establishments as well as those produced by certified foreign manufacturers [60]. However, actual testing of poultry sausage, that is, the first category of ready-to-eat poultry products examined, did not begin until September 1988, 1 year after the Listeriamonitoring/verification program was first instituted for cooked beef, roast beef, and cooked corned beef. Before April 1989, the USDA’s Listeria policy, which gave firms the opportunity to clean up their facility before additional verification samples were analyzed under holdtest procedures, was consistent with the fact that listeriosis had not yet been linked to consumption of poultry products. However, the official USDA-FSIS position regarding the presence of L. monocytogenes in cooked and ready-to-eat poultry products changed radically on April 14, 1989, when Centers for Disease Control and Prevention (CDC) investigators directly linked consumption of contaminated turkey frankfurters to a case of listerial meningitis in a breast cancer patient in Oklahoma [ 141. After isolating L. monocytogenes serotype 1/2a of the same electrophoretic enzyme type from the woman and opened, as well as unopened, retail packages of turkey frankfurters, USDA-FSIS officials requested that the manufacturer issue an immediate Class I recall for approximately

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567

600,000 pounds of Texas-produced turkey frankfurters that were marketed by retail and institutional establishments in Alaska, Arizona, Arkansas, California, Florida, Georgia, Idaho, Illinois, Indiana, Kentucky, Louisiana, Mississippi, Missouri, New Jersey, New York, Ohio, Oklahoma, Pennsylvania, Tennessee, Texas, Utah, and Washington. As expected, this recall immediately prompted an intensified monitoring/verification program to determine the extent of Listeria contamination in a far wider range of cookedheadyto-eat poultry products marketed in the United States. Despite pleas by the National Turkey Association to adopt tolerance levels for L. rnonocytogenes in cooked and ready-to-eat poultry products [9], USDA-FSIS officials maintained that since an “acceptable level” of L. monocytogenes in such products could at that time not be determined, the only acceptable alternative was to adopt a policy of “zero tolerance” for this pathogen in cookedheady-to-eat poultry and meat products [ 101. Consequently, under the program [36] which is identical to that developed for cooked and ready-to-eat red meat products, USDA-FSIS officials request firms to issue a Class I recall for all lots of cooked and ready-to-eat poultry products in which L. rnonocytogenes is detected in monitoring samples taken from intact packages of product. However, Listeriapositive lots that are still under direct control of the manufacturer can be recalled internally, thus avoiding adverse publicity. If the pathogen is initially detected in monitoring samples from unpackaged products, USDA-FSIS officials do not request firms immediately to recall the sampled lot. Instead, government officials will analyze subsequent lots and, if necessary, initiate further steps (i.e., hold-test programs) to prevent distribution of contaminated products. Our knowledge concerning the incidence of Listeria in cookedh-eady-to-eat poultry products marketed in the United States has come primarily from the USDA-FSIS Listeriamonitoringherification programs with results indicating that 1.5-2.0% of all such products are contaminated with L. rnonocytogenes [7,18,31]. Poultry products in which L. rnonocytogenes has been found include chicken patties [7], chicken thighs [7], chicken salad [7], diced poultry [7], poultry salad [18], poultry spread [18], poultry frankfurters [7], poultry bologna [7], and turkey sausage [7]. In all likelihood, the pathogen entered these products during the later stages of manufacture and/or packaging. Since most manufactures now retain all sampled lots of product until results of Listeria testing become known, formal Class I recalls for such products are quite limited and include the aforementioned recall of turkey frankfurters [14], two recalls of chicken salad [4,6], and one additional incident involving 13,000 pounds of chicken spread produced by a Virginia-based firm [ 151. However, numerous Class I recalls have been issued for prepared delicatessentype sandwiches, with at least two of these recalls [ 11,171 involving items that also contained processed chicken and/or turkey.

-

INCIDENCE OF LISTERIA SPP. IN RAW POULTRY MARKETED IN THE UNITED STATES Immediately after the 1985 listeriosis outbreak in California was announced, public concerns were raised about safety of dairy products and other potentially contaminated foods, including meat and poultry products. Interest in the safety of raw poultry soon escalated following a nationwide telecast which informed the general public that -50% of all raw chicken marketed in the United States was contaminated with Salmonella. Consequently,

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several surveys were initiated to determine the extent to which raw chicken and turkey meat are contaminated with Listeria and Salmonella.

Chicken The aforementioned concerns prompted USDA-FSIS officials to initiate a poultry back/ neck testing program for L. monocytogenes, Salmonella, and Escherichia coli serotype 0157 :H7 in January of 1989. L. monocytogenes and Salmonella were detected in 508 of 2686 (18.9%) and 792 of 2739 (28.9%) samples, respectively, with E. coli serotype 0157 : H7 being absent from 2696 samples [ 181. Bailey et al. [20] determined the incidence of L. monocytogenes and other Listeria spp. on the surface of broiler carcasses processed in the southeastern United States. They also compared L. monocytogenes serotypes isolated from raw chicken with those that are commonly associated with human cases of listeriosis. Using an enrichment procedure together with three selective plating media, Listeria spp. were detected in rinse samples from 34 of 90 (37.8%) chicken carcasses; however, recovery of Listeria varied widely with three lots of 10 birds each being reported as negative. More important, 21 of 90 (23.3%) carcasses contained L. monocytogenes, with 64, 18, 6 and 12% of the isolates being identified as serotypes I /2b, 1/2c, 3b, and nontypable strains, respectively. Although only 7 of 1 15 (6.1%) L. monocytogenes isolates from listeriosis victims in the United States were of serotype 1/2b or 1/2c, the fact that most L. monocytogenes strains isolated from chickens were pathogenic to mice, suggests chicken meat as a possible vehicle in human cases of listeriosis. Between June 1988 and May 1989, Genigeorgis et al. [45,46] conducted two large surveys which examined the incidence of Listeria spp. on fresh and/or semifrozen, chicken and turkey parts obtained from retail and slaughterhouse sources. According to their results for chicken, 12.5% of fresh wings, 16.0% of fresh legs, and 15.0% of fresh livers purchased at three supermarkets in northern California contained detectable levels of L. monocytogenes (Table 1). Furthermore, with the exception of fresh chicken liver, L. innocua was generally two to three times more prevalent in the remaining samples than was L. monocytogenes. Overall, the highest incidence of Listeria spp. was observed for fresh legs (54.0%) followed by fresh wings (42.5%) and fresh livers (32.5%). In contrast to fresh products, only 10% of semifrozen chicken wings, legs, and livers contained Listeria spp. However, finding L. monocytogenes alone in 1 of 10 semifrozen legs and livers points to the ability of this pathogen to survive in semifrozen raw chicken and turkey, as also was observed by Palumbo and Williams [98]. In addition to these efforts, Genigeorgis et al. [45] also attempted to trace the route of Listeria contamination on fresh chicken wings, legs, and livers by examining samples at various stages of production and storage. Although all chicken parts from the beginning of the production line were free of L. monocytogenes, results in Table 2 indicate that most contamination occurred during the latter stages of production when carcasses came in direct contact with Listeria-laden fecal material, since at the time of packaging, 70, 30, and 33% of chicken wings, legs, and livers contained L. monocytogenes, respectively. Not surprisingly, L. innocua, which was virtually absent from chicken parts at the beginning of production, also was routinely isolated from wings, livers, and particularly legs at the end of production. Despite these relatively high contamination rates, both Listeria spp. failed to grow on all three packaged products during the first 4 days of refrigerated storage. Wimpfheimer et al. [ 1321 observed a 3- to 4-day lag phase for L. monocytogenes when inoculated samples of raw minced chicken were held at 4°C. Given this information, the

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TABLE 1 Incidence o f Listeria spp. on Fresh and/or Semi-Frozen Chicken a n d Turkey Parts Purchased f r o m Three California Supermarkets Between J u n e 1988 a n d M a y 1989 No. (%) of positive parts Type and part of poultry Chicken wings legs livers Turkey wings legs tails

No. of parts examined Fresh Semifrozen Fresh Semifrozen Fresh Semifrozen

40

Fresh Fresh Fresh

60 60 60

10

50

10

40 10

L. monocytogenes

L. innocua

(16.0) (10.0) (15.0) 1 (10.0)

14 1 19 0 8 0

(35.0) (1 0.0) (38.0)

12 (20.0) 8 (13.3) 7 (11.7)

3 0 0

(5.0)

5 0 8 1 6

(12.5)

a Some chicken parts contained both L. monocytogenes and L. innocua or L. innocua and L. welshimeri. Source: Adapted from Refs. 48 and 49.

(20.0)

L. welshimeri

Total Listeria spp.

I (2.5) 0 0 0 1 (2.5) 0

17a (42.5) 1 (10.0) 27 (54.0) 1 (10.0) 13a (32.5)

12 (20.0) 9 (15.0) 7 (11.7)

27 (45.0) 17 (28.3)

1 (10.0)

14 (23.2)

Cox et al.

570

TABLE 2 Incidence of Listeria spp. on Commercially Produced Fresh Chicken and Turkey Parts Before and After Being Packaged and/or Stored at 4°C

Production line

Type and part of poultry Chicken wings legs livers Turkey wings legs livers

Listeria sp.

beginning

L. L. L. L. L. L.

monocytogenes innocua monocytogenes innocua monocytogenes innocua

0/20a

L. L. L. L. L. L.

monocytogenes welshimeri monocytogenes welshimeri monocytogenes welshimeri

1/30 (3.3) 1/30 (3.3) 0/30 1/30 (3.3)

0/20

0/20 0/20 0/3 1 2/31 (6.5)b

0/30

1/30 (3.3)

end 21/30 (70) 6/30 (20) 11/30 (37) 19/30' (67) 5/15 (33) 4/15 (27)

0/30 4/30 (13.3) 2/30 (6.7) 1/30 (3.3) 0/30 5/30 (16.7)

4-day -old packaged product stored at 4°C 18/25 4/25 13/25 17/25 6/15 4/15

(72) (16) (52) (68) (40) (27)

NDd ND ND ND ND ND

ND, not determined. a Number of positive partdnumber of parts examined. Percentage positive. Strain of L. welshimeri also detected. Source: Adapted from Refs. 48 and 49.

failure of Genigeorgis et al. [45] to detect growth of L. rnonocytogenes and L. innocua on naturally contaminated packaged chicken parts is not surprising. In more recent surveys, 91% of raw poultry samples and 8% of cooked samples contained listeriae. Although L. rnonocytogenes was isolated from 59% of raw samples, all cooked samples were negative [76]. Among the chicken parts examined (i.e., drumsticks breasts, wings, livers), drumsticks were most frequently contaminated with listeriae and also harbored the highest populations. In a survey of seven Danish abattoirs [96], 0.318.7% of processing line and final product samples contained L. rnonocytogenes. Based on serotyping, phage typing, pulsed-field gel electrophoresis, and ribotyping, 62 distinct L. rnonocytogenes strains were identified from the 247 isolates tested, several of which predominated in poultry-processing samples. Subsequently, Ryser et al. [ 1081 identified Listeria spp. in 34 of 45 retail samples of chicken pieces, with 11 different L. rnonocytogenes ribotypes, including three ribotypes associated with previous foodborne listeriosis cases, being detected among the Listeria-positive samples. These findings suggest the presence of multiple incoming sources of contamination and/or heavily contaminated single sites within the poultry-processing environment. Hence, improved disinfection procedures may be necessary to effectively control Listeria spp. in the processing environment. In a study by Cox et al. 1351 to determine how often L. rnonocytogenes enters poultry processing plants on live chickens, L. rnonocytogenes was infrequently found in hatchery samples and on the exterior of fully grown birds. Although L. rnonocytogenes was not

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571

recovered from the intestinal tract of broiler chickens at the time of slaughter, 25% of postprocessing and retail-level carcasses contained L. monocytogenes. In another study involving perorally dosed chicks, Husu et al. [65] reported that L. monocytogenes was generally eliminated within 9 days, which again suggests that intestinal carriage of L. monocytogenes is most often transient. It is important to remember that like other meats, poultry products also can be used for purposes other than human consumption. Al-Sheddy and Richter [ 11 determined the incidence of L. monocytogenes in frozen ground meat that contained raw chicken together with chopped beef by-products. Although not conclusive, recovery of L. monocytogenes from all five samples examined and the fact that this pathogen is more commonly found in chicken rather than beef products leads one to conclude that raw chicken was the most likely source of contamination. Hence, considering the high incidence of L. monocytogenes in raw chicken, it may be prudent to eliminate raw poultry products from the diet of zoo animals to curb the number of listeriosis cases occurring in zoological parks. The scientific literature relating to pathogens commonly associated with processed poultry is extensive, and a review of this literature has been published [129]. In another review paper [69] covering the years 1971-1994, the prevalence of L. monocytogenes in meats appears to be highly variable, with approximately 16?6 of products being positive. In general, the highest numbers of L. monocytogenes have been found in processed meat and poultry products, with fresh meats generally containing much lower numbers. Although serotypes 1/2a, 1/2b, and 1/2c are most frequently isolated from meats, most human outbreaks have been traced to serotype 4b, thus suggesting that poultry products play a relatively minor role in foodborne listeriosis.

Turkey Since chickens and other types of domesticated fowl are similarly processed, one would expect various Listeria spp., including L. monocytogenes, superficially to contaminate other raw poultry products, including turkeys, ducks, and pheasants. After completing the aforementioned survey of California chicken parts for Lisleria [45], Genigeorgis et al. [46] initiated a similar study to determine the prevalence of various Listeria spp. on fresh turkey parts obtained from retail sources and slaughterhouses. Listeria contamination rates were generally similar to those previously observed for fresh chicken, with 45.0% of fresh turkey wings, 28.3% of fresh legs, and 23.3% of fresh tails obtained from three local northern California supermarkets harboring various Listeria spp. (see Table 1). Although their findings further demonstrate that L. monocytogenes is equally common on fresh chicken and turkey parts, with isolation rates of 10.0-16.0% and 11.7-20.0%, respectively, the same cannot be said for I,. innocua and L. welshimeri. In fact, L. innocua, the Listeria sp. most commonly detected on fresh chicken, was recovered from only 3 of 180 (1.7%) fresh turkey parts. Similarly, L. welshimeri, the dominant Listeria sp. on fresh turkey, was only rarely observed on fresh chicken. Although both surveys were confined to fresh chicken and poultry parts available from three local supermarkets, these findings still suggest the interesting possibility that chickens and turkeys may be preferential hosts for L. innocua and L. welshimeri, respectively. However, additional data need to be gathered from other parts of the country to prove or disprove this theory. In a subsequent survey [33], 9 of 42 turkey skin samples harbored L. monocytogenes with L. monocytogenes contamination rates apparently unrelated to the incidence in the

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flock before processing or after defeathering. As was true for fresh chicken, additional testing at a local slaughterhouse once again demonstrated that fresh turkey parts are most likely to become contaminated with Listeria during later stages of processing (i.e., evisceration, chilling) (see Table 2). This scheme, mentioned earlier as the route by which fresh poultry becomes contaminated, was further confirmed by identifying various Listeria spp., including L. monocytogenes, in 4 of 15 (26.7%) samples of mechanically deboned raw turkey meat obtained from the same slaughterhouse. Ryser et al. [ 1081 further stressed the importance of postprocessing contamination when they reported that 33 of 45 (73%) retail samples of ground turkey contained Listeria spp., including a diverse group of L. monocytogenes strains belonging to nine different ribotypes.

INCIDENCE OF LISTERIA SPP. IN POULTRY PRODUCTS MARKETED IN WESTERN EUROPE AND ELSEWHERE Raw Poultry European scientists have been aware of the possible relationship between listeriosis in fowl and humans for nearly 40 years, as evidenced by several reports in which infected poultry was found in the immediate vicinity of human cases. Considering the fecal carriage rate for L. monocytogenes in domestic and wild fowl as well as the mechanized slaughtering practices that result in heavy fecal contamination of carcasses during defeathering, evisceration, and subsequent chilling in “spin-chillers,” it is not surprising that interest in the incidence of Listeria in poultry has escalated. However, given that the first European case of avian listeriosis was diagnosed in England during 1936 [99], and that no additional cases were recorded in England over the next 22 years [55], one would probably not expect to learn that our first knowledge concerning the incidence of Listeria in European raw poultry products originates from surveys made in England and Wales during the 1970s. After identifying L. monocytogenes in human stool samples from 32 of 5 100 (0.6%) asymptomatic individuals who resided in an area of Wales in which clinical cases of listeriosis had not been identified for 15 years, Kwantes and Isaac [74] postulated that the fecal carriage rate may be related to food consumption and began surveying both fresh and frozen chicken for L. monocytogenes. Following a 1971 preliminary report [74], Kwantes and lsaac [75] published final results of their study in 1975 when they reported detecting L. monocytogenes on the internal/external surfaces of 27 of 51 (52.9%) raw chickens obtained from a local processor (Table 3), with 23 (85.2%) and 4 (14.8%) L. monocytogenes isolates being identified as serotype 1 and 4b, respectively. To determine the public’s actual exposure to contaminated poultry, these investigators went to homes of poultry consumers in Wales and swabbed the externalhnternal surfaces of locally purchased fresh and frozen chicken, turkey, duck, and pheasant carcasses. Overall, L. monocytogenes was isolated from 50% of fresh chickens sampled from home refrigerators, and also from 64% of frozen chickens stored in home freezers, thus demonstrating the ability of this pathogen to persist on frozen carcasses. However, unlike chickens obtained directly from processors, L. monocytogenes isolates of serotype 4b outnumbered those of serotype 1 on fresh and, particularly, on frozen chickens obtained from consumers’ homes. Although relatively few samples were examined, isolation of L. monocytogenes from the internal/external surface of one turkey, three ducks, and one wild pheasant (see Table 3)

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TABLE 3 Incidence of L. rnonocytogenes in or o n Raw Poultry Carcasses Marketed in Western Europe and Elsewhere Between 1971 and 1994

Origin Denmark England/ Wales

Italy Sweden Switzerland West Germany

Type of poultry Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh chicken Frozen chicken Frozen chicken Frozen chicken Turkey Fresh turkey Frozen turkey Duck Frozen duck Wild pheasant Fresh chicken Fresh chicken Fresh chicken Fresh chicken Fresh/frozen chicken Unspecified poultry Unspecified poultry

No. of carcasses examined

17 51 38 50 6 100 30 16 32 64 50 56 4 1 3 3 2 2 --200 50 45 24 100 30 11

No. (%) of positive carcasses

Ref.

8 27

120 75

(47.1) (52.9)

19 (50.0) 33 (66.0) 2 (33.3) 60 (60.0) 15 (50.0) 10 (62.5) 21 (65.6) 41 (64.0) 27 (54.0) 7 (12.5) 1 (25.0) 0 0 3 (100.0) 1 (50.0) 1 (50.0) 0 18 (36) 0 5 (20.8) 85 (85.0) 6 (20.0) 3 (27.3)

75 102, 103 51 104 63 88 83 75 102, 103 51 75 51 51 75 51 75 34 44 123 29 110, 122 97 122

indicates that improperly handled poultry products other than chicken also may pose a potential threat to consumers. One year later, Gitter [51] published results from a similar study which examined the incidence of L. monocytogenes on surfaces of various “oven-ready” poultry products purchased at 26 different shops and supermarkets in southern England. Using a combination of direct plating and cold enrichment, L. monocytogenes was identified on 7 of 56 (12.5%) frozen and 2 of 6 (33.3%) fresh chickens as well as on 1 of 2 frozen ducks (see Table 3). Although the incidence of L. monocytogenes on raw poultry products was markedly lower than that previously found by Kwantes and Isaac [75], L. monocytogenes isolates identified as serotype 4 again outnumbered those of serotype 1/2. Following emergence of L. monocytogenes as a serious foodborne pathogen in June 1985, Pini and Gilbert [ 1031 determined the prevalence of this pathogen in uncooked fresh and frozen chickens obtained from retail outlets throughout metropolitan London. Unlike previous studies, which relied on swab samples from carcasses, these researchers examined two different samples from each chicken carcass whenever possible-one sample

574

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consisting of edible offal (trimmings and/or viscera) and the other a composite sample of skin and carcass remnants. Using cold enrichment in conjunction with the Food and Drug Administration (FDA) procedure, L. rnonocytogenes was recovered from 33 of 50 (66%) fresh and 27 of 50 (54%) frozen chickens. These results are similar to those reported from other 1987- 1994 surveys [49,63,83,88,104] in which L. rnonocytogenes was detected on 10 of 16 (62.5%), 15 of 30 (50%), 60 of 100 (60%) and 21 of 32 (66%) fresh chicken carcasses marketed in England and Wales (see Table 3). According to a second report by Pini and Gilbert [102], other Listeria spp., including L. innocua, L. seeligeri, and L. welshirneri, also were detected either alone or together with L. rnonocytogenes in 26 and 30% of the fresh and frozen chicken samples tested, respectively. Overall, 74 L. rnonocytogenes strains representing serotypes 1/2, 3a, 3b, 3c, 4b, 4d, and two nontypable strains, with serotype 1/2 predominating, were isolated from 160 samples consisting of 60 edible offal and 100 composite samples. Composite samples yielded more isolates of L. rnonocytogenes (57%) than did edible offal samples (22%) and also a higher percentage of other Listeria spp. (23%) than did edible offal samples (15%). Despite differences in methodologies and types of samples analyzed in the above-mentioned studies, averaging the results in Table 3 indicates that 187 of 323 (58%) fresh chickens and 75 of 170 (44.1%) frozen chickens marketed in England and Wales between 1971 and 1994 contained L. monocytogenes. That the 1986 findings of Pini and Gilbert [103,104] are similar to those obtained in both American and European surveys as far back as the mid 1970s underscores the continuing need for proper kitchen hygiene, cooking of raw poultry products, and continuous inspection of carcasses (e.g., identification of liver and heart lesions), along with use of good manufacturing and sanitizing practices in poultry-processing facilities. Since 152 of 214 (71%) clinical L. rnonocytogenes isolates obtained from British patients between November 1986 and 1987 [87] were of serotype 4b, poultry products, in which L. rnonocytogenes serotype 1/2 predominates, may be a less common vehicle for listeriosis than other foods. However, pit&, which are in essence poultry spreads prepared from chicken or goose liver, may be an exception. European concern about the incidence of L. rnonocytogenes in raw poultry products consumed outside of England/Wales dates back to at least 1982 when two Swedish workers, Ternstrom and Molin [ 1231, examined 45 chickens obtained from two local slaughterhouses (see Table 3). Although these researchers failed to isolate L. rnonocytogenes from any of the chickens examined, it appears that the Listeria isolation/detection methods used in this study were primarily responsible for their lack of success, since listeriosis in Swedish poultry is relatively common, with 112 cases being diagnosed in the 10-year period between 1948 and 1957 [94]. In view of the high incidence of L. rnonocytogenes in raw poultry marketed in the United States and England, inadequate isolation/detection methods of the early 1980s also were likely responsible for the inability of Comi and Cantoni [34] to recover this pathogen from approximately 200 chicken samples (i.e., carcass, skin, entrails) obtained from slaughterhouses in northern Italy, with more recent Italian surveys [44,84] showing L. monocytogenes contamination rates of 10.6 and 36% for raw poultry. After the 1985 cheeseborne listeriosis outbreak in California, western European scientists began to determine the incidence of Listeria in a wide range of foods, including fresh poultry products. In the first of these studies, which was published in 1988, Skovgaard and Morgen [ 1201 visited two large Danish poultry slaughterhouses and examined chilled chicken carcasses for evidence of Listeria contamination. According to these authors, Listeria spp. were detected in neck-skin samples from 16 of 17 (94.1 %) chicken carcasses, with L. innocua being identified in all but two Listeria-positive samples. Al-

Listeria monocytogenes in Poultry and Egg Products

575

though most of the poultry processed at these two facilities was heavily contaminated with L. innocua, 8 of 17 (47.l%), 1 of 17 (5.9%), and 2 of 17 (1 1.8%) carcasses also contained detectable levels of L. monocytogenes (see Table 3), L. innocua, and other Listeria spp. (L. welshimeri, L. murrayi, and/or L. denitrificans), respectively. Thus the L. monocytogenes contamination rate for chickens processed in Denmark was only slightly lower than the average (56.7%) for fresh chicken carcasses marketed in England and Wales. These Danish researchers also identified Listeria spp., including L. monocytogenes, in chicken feces and transport cage material, further supporting the widespread belief that poultry carcasses most likely become contaminated with Listeria during evisceration and subsequent handling, as also was suggested by Genigeorgis et al. [45,46] and Cox [35] based on results of surveys of poultry-processing facilities in California and Georgia. Interest in the incidence of Listeria in European fresh poultry again intensified following reports that 34 individuals in Switzerland died after consuming contaminated Vacherin Mont d’Or soft-ripened cheese. Breer [29] isolated L. monocytogenes, L. innocua, and 1,.seeligeri from 5 of 24 (20.8%), 6 of 24 (25.0%), and 1 of 24 (4.2%) raw chickens purchased in Switzerland, (see Table 3), respectively. Using a modified version of the FDA procedure, German researchers [ 110,121], found Listeria spp. and L. monocytogenes in 94 of 100 (94%) and 85 of 100 (85%) chicken carcasses, respectively. However, results from two smaller surveys of German poultry [97,121] suggested far lower L. monocytogenes contamination rates, with only 20.0-27.3% of unspecified fresh poultry carcasses (presumably chicken) containing this pathogen (see Table 3). Similarly, Rijpens et al. [ 1051 more recently recovered Listeria spp. and L. monocytogenes from 35.5% and 15.5%, respectively, of poultry samples examined in Belgium, with the incidence of Listeria spp. markedly higher in unpackaged (41.7%) than in prepackaged (1 1.1%) poultry. L. monocytogenes also has been detected in French poultry since 1988 [26]. During a subsequent large-scale survey of poultry carcasses in France and Belgium, Uyttendaele et al. [ 1271 reported that the L. monocytogenes contamination rate decreased from 32.1% in 1992 to 9.2% in 1995, with 87%-98% of the carcasses tested yielding < I L. monocytogenes CFU/cm2. Contamination of poultry parts also decreased from 25.8% to 3% during this same 4-year period, with L. monocytogenes-positive samples primarily being confined to chicken legs and wings. In another survey similar to that conducted in the United States by Genigeorgis et al. [45], Franco et al. [43] identified Listeria spp. on 96, 84, 80, and 0% of the fresh chicken legs, wings, breasts, and livers, respectively, that were processed at a Spanish facility, with Listeria counts ranging from <2.0 to 2.8 log CFU/g on chicken wings. Follow-up testing of the processing environment again showed that most of the contamination occurred during the later stages of production as carcasses entered the quartering room and exited on conveyor belts. In a Danish survey [96], L. monocytogenes was not observed in cecal samples from over 2000 broilers representing 90 randomly selected broiler flocks. However, L. monocytogenes was isolated from 0.3 to 18.7% of all poultryprocessing environmental samples examined. Using several strain-specific typing methods, L. monocytogenes contamination was shown to be primarily localized in the processing facility, with the incidence reducible through improved hygiene. Elsewhere, 10 of 30 domestically grown fresh chickens from the United Arab Emirates [54] contained Listeria spp., with 1 of these carcasses being positive for L. monocytogenes. However, Listeria spp. were detected on 32 of 39 (82%) imported frozen chickens, 18 (46%) of which contained L. monocytogenes. Similarly, Rusul et al. [47] identified L. monocytogenes in 4 of 16 (25%) poultry samples collected from six local Malaysian markets, with one of these markets subsequently yielding L. monocytogenes in 15 of 24

576

Cox et al.

(62.5%) samples. In a South African study [38] of Listeria in poultry-processing facilities, L. innocua was recovered from the rubber fingers of a defeathering machine and on all neck skin samples after evisceration. L. monocytogenes strains were detected on the rubber fingers, packaging funnel, and all neck skin samples after chilling.

Cooked/Ready-to-Eat Poultry Increasing evidence indicates that contamination of ready-to-eat foods, including cooked chicken, is most likely to originate from the processing environment. With this premise in mind, Lawrence and Gilmour [76] examined the incidence of Listeria spp., including L. monocytogenes, in one poultry-processing facility, along with raw and cooked chicken processed at this same facility in Northern Ireland between March and August 1992. Within the raw and cooked poultry-processing environments, 36 of 79 (46%) and 51 of 173 (29%) samples harbored Listeria spp., whereas 21 of 79 (26%) and 27 of 173 (15%) yielded L. monocytogenes, respectively. Contamination rates were fairly uniform, with several environmental sites yielding L. monocytogenes throughout the study. Among raw and cooked products tested, 53 of 58 (91%) and 8 of 96 (8%) contained Listeria spp., whereas 34 of 58 (59%) and none of 96 cooked samples yielded L. monocytogenes, respectively. Although L. monocytogenes was absent from all cooked products tested, the presence of other Listeria spp. and a subsequent report by the same authors [77] attesting to isolation of identical L. monocytogenes strains from raw poultry, cooked poultry, and the processing environment (some environmental strains of which persisted for up to 1 year) confirms the importance of minimizing postprocessing contamination. An association between human listeriosis and consumption of cooked/cookedchilledheady-to-eat poultry products which are cooked, rapidly chilled, and frequently held refrigerated for at least 5 days before being consumed without further heating has been observed in both the United Kingdom and the United States. According to one survey conducted by the Public Health Laboratory Service in London [49,50,104], L. monocytogenes was present in 63 of 527 (1 2.0%) precooked ready-to-eat poultry products collected from London-area retail establishments between mid November 1988 and mid January 1989. Little information is available concerning actual numbers of L. monocytogenes present in cooked poultry products; however, 14 samples that were examined quantitatively contained < 100 CFU/g. In addition L. monocytogenes was isolated from 13 of 74 (18%) retail chilled meals, most of which were poultry products given to hospital patients. The pathogen was also discovered in 6 of 24 (25%) cook-chilled poultry products [13], 7 cook-chilled poultry dishes at levels up to 700 L. monocytogenes CFU/g, and 2 cooked chicken products labeled “ready-to-eat” which contained up to 400 L. monocytogenes CFU/g. Working at the Cardiff Public Health Laboratory Service in Wales, Morris and Ribeiro [90] determined the potential Listeria-related risks associated with consumption of pit&,an appetizer-type poultry spread that is typically prepared from chicken or goose liver. According to their report, 14 varieties of pit& (primarily imported from Belgium) were obtained in bulk from area delicatessen counters or in unopened packages from supermarket refrigerators and examined for L. monocytogenes using established methods. Overall, this pathogen was isolated from 37 of 73 (50.4%) pitis, with 28 (75.7%) and 9 (24.3%) of these positive samples originating from delicatessens and supermarket refrigerators, respectively. These pit& were subsequently withdrawn from the market after officials discovered dangerously high L. monocytogenes populations of l 0 4 - r 1O5 CFU/g in

Listeria monocytogenes in Poultry and Egg Products

577

seven samples [8]. L. monocytogenes strains of serotype 4b, the serotype responsible for -80% of all human listeriosis cases in England and Wales, were isolated from 36 of 37 positive samples, with one strain of L. monocytogenes serotype 4b matching clinical isolates from a 1987 cluster of listeriosis cases in which the exact origin of illness could never be determined. These findings prompted Public Health Laboratory officials greatly to expand their survey of pihi. By March 1990 [48,104], workers at 48 of 53 (90.6%) participating laboratories in England and Wales isolated L. monocytogenes from 187 of 1834 ( 10.2%) samples of imported and domestic pit& As in the previous survey, -10% of all positive samples contained 104-> 106L. monocytogenes CFU/g, with over half of all isolates belonging to serotype 4. Following this pitk-related outbreak discussed in Chapter 8, the number of listeriosis cases reported in England and Wales decreased to approximately half the level reported in 1988. Although some individuals expected to see a further decline [48,104], the incidence of human listeriosis in the United Kingdom has since stabilized, with about 100-125 cases, being reported annually over the last 5 years. In one other European survey of cookedheady-to-eat poultry products, Lieval et al. [80] isolated L. monocytogenes, L. seeligeri, and L. innocua from one of nine, two of nine, and one of nine chicken sandwiches obtained from cafes in and around Paris. Although identical efforts to recover Listeria from 20 fast-food fried chicken items ended in failure, the ability of such foods to harbor Listeria, including L. monocytogenes, has been well established by the previously discussed surveys in England.

BEHAVIOR OF L. MONOCYTOG€N€S IN RAW AND COOKED POULTRY PRODUCTS Although L. monocytogenes was first detected on European raw chicken nearly 20 years ago, behavior of Listeria in raw and processed poultry products received no attention until this organism was recognized as a bonafide foodborne pathogen in the mid 1980s. Subsequent research efforts have provided the poultry industry with valuable information concerning growth of L. monocytogenes in raw and cooked chicken products, including the levels of heat and microwave/gamma irradiation needed to destroy this pathogen in raw chicken. However, our present-day knowledge of Listeria behavior in raw and processed chicken products is still incomplete. Although results from these efforts will now be summarized, several listeriosis cases that were positively linked to consumption of processed poultry products in the United States and England have prompted additional work in thiq area. Results from these studies should add much to our knowledge about behavior of Listeria in poultry products and aid in development of processing methods that will decrease the incidence of this pathogen in raw and cooked-chilled poultry products.

Growth-Raw

Chicken

Wimpfheimer et al. [ 1321 examined the behavior of L. monocytogenes in raw chicken. In their study, raw minced chicken meat was inoculated to contain 10, L. monocytogenes CFU/g, packaged anaerobically (75% CO2:25% N2), microaerobically (72.5% CO,: 22.5% N 2 : S % O,), or aerobically (air) and examined for numbers of L. monocytogenes as well as aerobic spoilage organisms during storage at 4, 10, or 27°C. Neither L. monocytogenes nor aerobic spoilage organisms grew in anaerobically packaged raw chicken dur-

Cox et al.

578

ing extended storage at any of the three temperatures, with both populations decreasing to <10 CFU/g after 6 days of storage at 4°C (Fig. 1). When packaged microaerobically under conditions more closely simulating commercial practices, numbers of L. monocytogenes in raw chicken increased rapidly during extended storage at 4"C, whereas growth of aerobic spoilage organisms was strongly inhibited (see Fig. 1). Under these conditions, L. monocytogenes can rapidly proliferate in normal unspoiled raw chicken during refrigerated storage. Zeitoun and Debevere [133] later reported on the successful use of a 10% lactic acid/sodium acetate buffer (pH 3) in conjunction with modified atmosphere packaging (90% CO2, 10% 0,) to prevent growth of L. monocytogenes on uncooked chicken legs and extend the product's shelf life at 6°C to 17 days. According to Wimpheimer et al. [132], the ability of L. monocytogenes to grow in microaerobically packaged raw chicken was not affected by initial levels of Listeria (<10' or 102CFU/g) or aerobic spoilage organisms ( l O4 or 1O8 CFU/g). However, the ratio of Listeria to spoilage organisms was strongly temperature dependent, with both organisms reaching populations of 107-10Rand 109-10'" CFU/g in microaerobically packaged chicken following <2 and 8 days of storage at 10 and 27"C, respectively. Neither L. monocytogenes nor aerobic spoilage organisms were inhibited in aerobically packaged raw chicken, with Listeria and spoilage organisms attaining populations > l O7 and 1O9 CFU/g, respectively, in products stored at 4, 10, and 27°C. Thus with exception of the microaerobically packaged product, raw chicken would likely become overtly spoiled before L. monocytogenes could proliferate

L. monocytogenes

0

Aerobic Spoilage Organisms

98 1

7l

c.v

/

P'

/

6

0

2

4

6

8

10

12

14

16

18

20

22

Days

FIGURE1 Growth of L. rnonocytogenes and aerobic spoilage organisms in aerobically ( O), microaerobically (01 and , anaerobically ( 0 )packaged raw chicken during incubation at 4°C.(Adapted from Ref. 132)

Listeria monocytogenes in Poultry and Egg Products

579

to the point where the pathogen might be detectable in minimally cooked chicken. Nevertheless, it is important to remember that consumers must take special precautions to prevent cross contamination between raw chicken that may contain L. monocytogenes and/ or Salmonella spp. and ready-to-eat products including cooked chicken.

Growth-Cooked / Ready-to-Eat Poultry Products Confirmation of cooked-chilled chicken and turkey frankfurters as vehicles of Listeria infection in England and the United States during 1988 and 1989 prompted immediate international efforts to assess the potential hazards associated with growth of L. monocytogenes in a wide range of retail cookedheady-to-eat poultry products, six of which have been briefly summarized in Table 4. Since all of these studies differ in experimental design, sampling times and initial inoculum levels of L. monocytogenes, these findings cannot be compared directly. However, it is evident that numbers of Listeria increased 1-6 orders of magnitude in all six artificially contaminated products after 6-28 days of storage at 3-7"C, with higher populations being observed in aerobically packaged as opposed to vacuum-packaged or modified-atmosphere-packaged products. Equally important, sensory acceptability of these products was not altered by growth of this pathogen. Overall, L. monocytogenes grew most abundantly in vacuum-packaged sliced chicken and in one brand of sliced turkey, both of which were similar in pH (6.3, 6.4) and in contents of moisture (7 1.3, 74.0%) protein (18.9, 22.6%) carbohydrate (1.3, 0.9%) and salt (1.7, 1.4%). These authors attributed decreased growth of the pathogen in a second brand of sliced turkey to higher levels of salt (2.7%) and carbohydrate (1.7%); the latter was largely responsible for the eventual decrease in pH of this product to 4.97. In another study by Ingham et al. [68], cooked/sterilized chicken loaf was inoculated to contain 1 O3 L. rnonocytogenes and l O3 Pseudomonasfragi CFU/g, packaged aerobically (air), microaerobically (10% O,), or anaerobically and examined for both organisms during 6 days of incubation at 37 and 11°C. Regardless of incubation temperature, P. fragi attained maximum populations of approximately 4 X 109 CFU/g in all aerobic samples after 6 days of storage (Fig. 2). However, growth of Listeriu was totally or partially suppressed in similar samples stored at 3°C for up to 15 days [67]. Although both organisms attained lower maximum populations in cooked chicken loaf following 6 days of microaerobic or anaerobic incubation at all three temperatures, these conditions led to Listeria populations that were 1-2 orders of magnitude higher than those attained by P. fragi. Thus, as was true for raw poultry (see Fig. l), microaerobic and anaerobic refrigerated storage both appear to selectively favor growth of L. rnonocytogenes over P. fragi and possibly other spoilage organisms which, in turn, could potentially yield an organoleptically acceptable product with dangerously high numbers of Listeria. Identification of turkey frankfurters as the infectious vehicle in a 1988 case of listeriosis involving an Oklahoma breast cancer patient eventually led to a safety assessment of poultry sausage. According to McKellar et al. [86], L. monocytogenes grew on the surface of I3 of 27 (48%) artificially contaminated retail poultry weiners, with populations on some vacuum-packaged samples increasing nearly 4 orders of magnitude during 21 days of storage at 5°C. Wederquist et al. [131] reported sirnilar growth of L. monocytogenes when vacuum-packaged slices of turkey bologna were stored at 4°C. However, incorporating 0.5% sodium acetate, 2.0% sodium lactate, or 0.26% potassium sorbate into the product completely suppressed growth of the pathogen during the first 35 days of refrigerated storage, with populations in 3-month-old samples remaining 2-5 orders of

Cox et al.

580

TABLE 4 Populations of L. monocytogenes (log,, CFU/g) in Artificially Contaminated Cooked/Ready-to-Eat Poultry Products of Acceptable Organoleptic Quality During Extended Refrigerated Storage

Product Sliced chickena Sliced turkey (brand A)a Sliced turkey (brand B)a Chicken homogenate Breaded chicken fillets Chicken casserole Chicken nuggets Chicken nuggetsd

Incubation temp. ("C)

Initial inoculum

4.4 4.4 4.4 4.4 4.4 4.4 4 4 5 5 3 6 3 7 3 7

2.79 0' 3.04 - 1.30 2.87 - 1.70 6.7 2.7 2.7 1.7 2.7 2.7 4.8 4.8 4.7 4.7

Vacuum packaged. Not tested. 1 CFU/g. Modified atmosphere--80% CO,: 20% Nz

Length of incubation (days)

3

6

8

10

14 6.94 5.90 5.04 2.38 6.70 4.79

-b

-

2.9 3.6 4.8 5.3 4.8 4.9

3.6 3.6 3.5 5.3 5.2 6.0 4.8 5.1

-

-

3.3 6.3 5.7 6.1 5.0 5.3

3.6 7.5 6.1 7.4 5.2 5.3

-

6.3 -

6.0

20

28

Ref.

Listeria monocytogenes in Poultry and Egg Products

581

10

I

1

9 8

7

6 m

3

5

* I

4

U-

r cs, 0

J

.-a

5

3

*-.

0

a m

2 1 I

1

1

I

4

6

Days

FIGURE2 Growth of L. monocytogenes and Pseudomonas fragi in aerobically (O), microaerobically (01,and anaerobically ( 0 )packaged cooked chicken loaf during incubation at 7°C. (Adapted from Ref. 68.) magnitude lower than those in the controls. Two additional studies on fermented summer sausage prepared from ground chicken [ 191 and turkey [8 I] also demonstrated the importance of an active starter culture in limiting growth of L. rnonocytogenes during fermentation. When these sausages were prepared from a chicken or turkey batter (pH 6.6) containing a pediococcal starter culture and L. rnonocytogenes at a level of 1O4--1O7CFU/g, numbers of Listeria decreased 0.9- 1.8 orders of magnitude after an I 1-h fermentation (pH 5). Replacing this pediococcal starter culture with a pediocin-producing strain of Pediococcus acidilactici essentially doubled the inactivation rate of L. rnonocytogenes during fermentation. However, regardless of the starter culture used, all remaining listeriae were subsequently inactivated during 45 min of cooking to an internal temperature of 66.5"C. Thus, an active fermentation combined with normal thermal processing before packaging should result in a Listeria-free product. Finally, several investigations also have assessed behavior of Listeria in inoculated samples of chicken gravy and chicken broth during cooling and/or refrigerated storage. According to Huang et al. [62], L. monocytogenes populations in individual 1000-g samples of artificially contaminated chicken gravy (prepared from poultry stock, spices, waxy maize wash, and chicken base) increased by 2 orders of magnitude as the product cooled from 40 to 9°C during 24 h of storage in a refrigerator at 7°C (Fig. 3). Even though generation times for L. rnonocytogenes approximately doubled after the chicken gravy stabilized at 7"C, the pathogen still attained a maximum population of 109CFU/g in 8day-old gravy. Huang et al. [61] subsequently reported a reduction in L. rnonocytogenes

Cox et al.

582

+ L. monocvtoaenes

0- - Temperature

--

- -0- -- -0- - -e- -a-- - 0 - - -0

0

1

2

3

4

I

1

I

I

5

6

7

8

Days

FIGURE 3 Behavior of L. monocytogenes in chicken gravy during cooling to 7°C and extended storage. (Adapted from Ref. 62.)

when similar chicken gravy was held at 65°C for 1.3 min; however, such a heat treatment is clearly inadequate to inactivate higher Listeria populations that can develop in chicken gravy during prolonged refrigerated storage. Walker et al. [ 1301 also demonstrated the ability of three L. monocytogenes strains to multiply in artificially contaminated sterile chicken broth (pH E 6.4) held at heat-freezing temperatures, with Listeria populations in this product increasing 100-fold during extended incubation at 03°C (Fig. 4). In fact, growth of this pathogen was also evident in samples of chicken broth that were held at temperatures as low as -0.4"C, below which the broth froze and was no longer sampled. Results from these investigations stress the importance of cooling foods as rapidly as possible and show that hazardous situations can easily develop if refrigerated foods are subjected to mild temperature abuse, that is, holding at temperatures above 4°C. In an effort to increase the safety of cooked, cooked-chilled, and ready-to-eat poultry, USDA officials lowered the recommended long-term storage temperature for such products from 4.4 (40°F) to 1.7"C (35°F) and also have developed stricter guidelines that require faster (than previously recommended) cooling of warm products at the end of manufacture [3]. Continued attention to rapid cooling of finished products and avoidance of postprocessing contamination are both essential to decrease the incidence of this psychrotrophic pathogen in cookedh-eady-to-eat poultry products.

Listeria monocytogenes in Poultry and Egg Products

I

0

I

10

I

I

20

30

Days

I

40

583

1

50

FIGURE4 Growth of L. monocytogenes in sterile chicken broth during extended incubation at 8.7 (01,3.5 (m), 1.5 (01 and , 0.8”C (0).(Adapted from Ref. 130.)

Thermal Inactivation Heating is the most obvious means of destroying L. monocytogenes and other foodborne pathogens i n any raw food, including poultry. However, numerous reports attesting to unusual thermal tolerance of L. monocytogenes in various foods, coupled with discovery of L. monocytogenes in several cooked poultry products that were directly linked to cases of listeriosis, have raised questions about the exact thermal processing times and temperatures required to eliminate this pathogen completely from raw poultry products. In response to these concerns, Carpenter and Harrison conducted three studies in which raw chicken breasts were surface inoculated to contain 1OS- l O7 L. monocytogenes CFU/g and cooked to internal temperatures of 65.6, 71.1, 73.9, 76.7, or 82.2”C using dry heat [32], moist heat [57], and microwave radiation [58].All cooked chicken breasts were then vacuum-packaged or wrapped in oxygen-permeable film and analyzed for numbers of Listeria during 4 weeks of storage at 4 and 10°C. Overall, L. monocytogenes was recovered from chicken breasts cooked to all five internal temperatures, using dry heat, moist heat, and microwave radiation. As expected, the magnitude of lethality was directly related to cooking temperature. Since chicken breasts contained somewhat different levels of L. monocytogenes before heating, one cannot directly compare the effectiveness of the three cooking methods used in these studies. However, assuming that L. monocytogenes populations in these chicken breasts decreased linearly during heating (admittedly, some “tailing” of the survivor curve likely occurred at the three highest temperatures using dry heat, moist heat, and microwave irradiation), then the number of survivors in chicken breasts that contained any initial inoculum can be estimated. Thus, if Carpenter and Harrison had used an initial population of 1 .O X 106 L. monocytogenes CFU/g in all three studies, one would expect their results to have been similar to the estimated number of survivors shown in Table 5. Considering these approximations, it appears that L. monocytogenes was generally more tolerant of microwave radiation than dry or moist heat, with numbers of Listeria decreasing less than 4 orders of magnitude on chicken cooked to an internal temperature of 822°C. Of greater importance is the fact that Listeria populations decreased 1 2 orders of magnitude on chicken breasts cooked to an internal temperature of 7 1.1 “C, the minimum internal temperature to which poultry must be heated to designate the product as fully cooked in the United States [2]. Although numbers of Listeria decreased approximately 5 orders of magnitude when

-

Cox et al.

584

TABLE 5 Estimated Decrease in Numbers of L. rnonocytogenes o n the Surface of Chicken Breasts Inoculated to Contain 6.00 log,, CFU/g and Cooked t o Various Internal Temperatures Using Dry Heat, Moist Heat, or Microwave Radiation NO.^ of L. monocytogenes (log,, CFU/g) decrease after cooking to

internal temp. of

~

Cooking method Dry heat Moist heat Microwave radiation

~

65.6"C

71.1"C

73.9"C

76.7"C

82.2"C

2.42 2.08 0.82

2.00 1.83 1.95

5.05 3.46 2.50

5.24 5.08 3.77

5.04 4.96 3.26

Initial inoculum of 6 log,, L. rnonocyrogenes CFU/mL. Source: Adapted from Refs. 32, 57, and 58. a

chicken was cooked to higher internal temperatures using either dry or moist heat, these authors [56] later demonstrated that moist heating of surface-inoculated chicken breasts to an internal temperature of 73.9"C also failed to completely inactivate more realistic L. monocytogenes populations of 102-104CFU/g. Overall, microwave heating was less effective than either dry or moist heating, with Listeriu populations decreasing less than 4 orders of magnitude on chicken breasts cooked to an internal temperature of 82.2"C (see Table 5). In 1989, researchers in England [82] also reported that microwave heating was less effective than other forms of cooking for eliminating L. monocytogenes from the surface and interior (stuffing) of whole stuffed chickens (- 1.7 kg each) inoculated to contain 106and 107 Listeria CFU/g of skin and stuffing, respectively. Uneven heating, which is an inherent problem in microwave cooking, accounted for the 20 min of additional standing time that was required after 38 min of cooking (final skin temperature of 8099°C) to completely inactivate the pathogen on the surface of whole chickens. However, low levels of L. monocytogenes (<10 CFU/g) were still detected in stuffed samples from one of two similarly treated whole chickens that attained temperatures of 72-85°C after 20 min of standing. Thus, these findings serve as a warning to people who regularly cook large stuffed birds (particularly turkeys) in microwave ovens, and they also stress the importance of postcooking standing time for further inactivation of Listeriu and other foodborne pathogens in poultry products after microwave cooking. Not surprisingly, follow-up work by Carpenter and Harrison [32,57,58] demonstrated that L. monocytogenes survivors (likely sublethally injured during heating) can persist and multiply on both oxygen-permeable film-wrapped and vacuum-packaged cooked chicken during extended storage at 4 and 10°C. As shown in Figure 5, growth of Listeriu on chicken breasts packaged in oxygen-permeable film was most evident after 2 weeks of refrigerated storage with larger populations generally developing on products that were cooked using microwave radiation, followed by those given moist or dry heat. Most important, L. monocytogenes was recovered via direct plating from all 4-week-old aerobically packaged chicken breasts except those that were cooked to an internal temperature of 82.2"C using moist heat. In addition, higher numbers of Listeriu also developed on aerobically packaged chicken breasts that were exposed to less severe heat treatments. As expected, increasing the incubation temperature also led to much faster growth of Listeria, with the pathogen generally attaining populations of 106-107CFU/g on aerobically packaged chicken breasts after only 7 days at 10°C. Behavior of L. monocytogenes on chicken breasts was influenced by the heating

Listeria monocytogenes in Poultry and Egg Products

585

P

3

U.

0

0

I

I

1

I

1

2

3

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FIGURE5 Effect of three different heating methods on behavior of L. rnonocytogenes on inoculated chicken breasts that were cooked to internal temperatures of 71.1 (O), 76.7 (A),or 82.2"C (U), packaged in oxygen-permeable film, and stored at 4°C. (Adapted from Refs. 32, 57, and 58.) methodkreatment, temperature at which the cooked product was ultimately stored, and type of packaging material. According to these authors, Listeria populations were generally 1 to 2 orders of magnitude lower in vacuum-packaged than in aerobically wrapped product following 4 weeks of storage at 4°C; however, the pathogen was present in all 4-week-old samples except those that were originally cooked to an internal temperature of 82.2"C using moist or dry heat (Fig. 6). Although numbers of Listeria were again generally 1-2 orders of magnitude lower in vacuum-packaged than in aerobically wrapped chicken breasts following 1 week of storage at 10°C, populations were as much as 5 orders of magnitude lower in vacuum-packaged chicken breasts that were cooked to an internal temperature of 82.2"C using moist heat. Since raw chicken normally contains
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FIGURE6 Effect o f three different heating methods o n behavior o f L. monocytogenes o n inoculated chicken breasts cooked t o internal temperatures of 71.1 (O), 76.7 (A), or 82.2"C (W), vacuum-packaged, and stored at 4°C.(Adapted f r o m Refs. 32, 57, and 58.)

and Carpenter [56] reported that moist heating of inoculated chicken breasts to an internal temperature of 73.9"C failed to completely inactivate L. monocytogenes surface populations of 102-104CFU/g, with the pathogen reestablishing itself at levels equal to or greater than the original inoculum level after 4 weeks at 4°C. Nonetheless, the adequacy of current poultry-processing methods was maintained by another report [5] which indicated that a turkey meat emulsion (containing salt, sodium tripolyphosphate, carrageenan, and water) inoculated to contain 5.78 L. monocytogenes log,,, CFU/g was free of the pathogen after = 0.28 min). However, in view holding the product at 7 1.1 "C for 2 min (estimated D71,10C of at least three human listeriosis cases linked to cooked-chilled chicken meat and turkey frankfurters (the latter was reportedly warmed 45-60 s in a microwave oven before consumption), and that small numbers of L. monocytogenes survived a wide range of heat treatments given to chicken breasts and frequently grew in these products during refrigeration, it is prudent for poultry processors to cook their products to somewhat higher internal temperatures (76.7-82.2"C) than is now routinely practiced until the adequacy of the current minimum heat treatment can be firmly established. Future studies should address the effect of processed poultry ingredients (i.e., salt, preservatives) on thermal resistance of Listeria during conventional as well as microwave heating, since Harrison and Carpenter [58] found that the latter cooking method was less successful in eliminating L. monocytogenes from the surface of chicken breasts than either dry or moist heating.

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Chemical Treatments Various food additives have been evaluated for their ability to inactivate Listeria spp. on fresh poultry. Dipping inoculated chicken wings in 10% trisodium phosphate (TSP) at 10°C for 15 s and hot water (95°C) for 5 s resulted in a 79.49% reduction in numbers of L. monocytogenes, with minimal changes in subcutaneous temperature [ 1061. Hwang and Beuchat [66] also found that washing chicken skin in 1% TSP or 1% lactic acid significantly reduced viable populations of L. monocytogenes compared with washing in water. According to Shelef and Yang [ 1 161, growth of L. rnonocytogenes in sterile comminuted chicken was slowed during refrigerated storage by adding 4% sodium or potassium lactate. When used at levels 10.3%, sodium diacetate also was inhibitory to L. rnonocytogenes in poultry slurries [ 1091, with the antilisterial activity of sodium diacetate being further enhanced by supplementing the product with 0.25% ALTA, a commercially available microbially produced shelf life extender.

Susceptibility to Gamma Radiation Survey results indicate that up to -60% of all raw poultry products sold in retail stores may be contaminated with L. rnonocytogenes and Salmonella spp. These statistics have prompted development of various processes to eliminate such pathogens from raw poultry. Exposure to the bactericidal effects of gamma radiation appears to be among the most effective means of reducing populations of both pathogenic and spoilage organisms. Although presently allowable gamma radiation doses of 2.5-7.0 kGy in England [92] and <3.0 kGy in the United States [16] are generally regarded as sufficient for eliminating these organisms from raw poultry [ 1241, readers should be aware that exposing cooled poultry to levels >2.5 kGy may adversely affect product odor, color, and flavor [88]. In 1989, Patterson [ 1011 examined sensitivity of Listeria to gamma radiation using radiation-sterilized raw minced chicken meat that was inoculated to contain 1O6 L. rnonocytogenes CFU/g. After exposing the product to gamma radiation doses of 0, 0.5, 1.0, 1.5,2.0, and 2.5 kGy, L. monocytogenes exhibited a DIo-value(i.e., radiation dose required to decrease the population 10-fold) of 0.417-0.553 kGy, depending on the bacterial strain and the type of plating medium used to quantitate the pathogen in minced poultry. In support of these findings, Huhtanen et al. [64] also reported that a gamma radiation dose of 2 kGy was sufficient to inactivate an L. monocytogenes population of 104 CFU/g (average D-value of 0.45 kGy) in artificially contaminated, mechanically deboned chicken. Similar D-values also have been published for Salmonella spp. in fresh poultry [91]. Although all of the aforementioned studies support the use of 2.5 kGy of gamma radiation to eliminate < 1O4 L. monocytogenes CFU/g from raw poultry meat, researchers in England [ 881 recovered this pathogen from 1 of I2 (8.3%) fresh chicken carcasses that had been surface inoculated to contain approximately 102or 1O4 L. rnonocytogenes CFU/ cm2 and exposed to 2.5 kGy of gamma radiation. More important, after extended storage at 5-10°C, the pathogen was recovered from 1 of 18 (5.6%) and 7 of 18 (38.8%) irradiated carcasses that originally contained low and high inoculum levels, respectively. When poultry carcasses were inoculated to contain > 1O4 L. rnonocytogenes CFU/g, several investigators [79,128] also confirmed that small numbers of the pathogen survived irradiation at 2.5 kGy and eventually grew, particularly in the absence of air, in samples stored at 4°C. However, Shamsuzzaman et al. [ 1141 reported that combined use of 3.1 kGy irradiation and sous-vide cooking to an internal temperature of 71.1"C was sufficient to reduce L.

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monocytogenes populations in chicken breast meat from 1O6 CFU/g to undetectable levels, with no growth of the pathogen being observed during 5 weeks of storage at 8°C. Hence, in the absence of multiple treatments, these findings suggest that small numbers of Listeria may either escape sublethal injury during irradiation or undergo repair and grow on these carcasses during refrigerated storage. From this information, it appears that a gamma radiation dose of 2.5 kGy may be only marginally sufficient to inactivate levels of Listeria that one might reasonably expect to find on naturally contaminated raw poultry. Nevertheless, provided that irradiated poultry products are properly packaged to prevent recontamination (and subsequent growth) with Listeria and other foodborne pathogens, this procedure should markedly decrease the risk of contaminating ready-to-eat foods (e.g., salads, raw vegetables) when raw poultry is prepared by the consumer. Unfortunately, although the scientific community generally contends that foods exposed to such low levels of radiation are safe for human consumption, irradiated foods have not yet gained full acceptance by consumers. Perhaps the continued outpouring of scientific evidence will eventually curb the remaining unfounded fear of irradiated foods in the mind of the general public.

EGG PRODUCTS Listeria monocytogenes is most frequently isolated from heart, liver, and spleen tissue of poultry suffering from listeriosis; however, according to the early scientific literature, this pathogen also has been detected in necrotic oviduct lesions of several infected hens [55] and in follicles of one artificially infected chicken [72]. These observations prompted a large-scale survey in 1958 [70] in which 600 intact hen’s eggs were examined and found to be negative for L. monocytogenes. In keeping with these findings, consumption of eggs and egg products also has not yet been linked to a single case of listeriosis. However, the possible presence of L. monocytogenes on egg shells which may contain minute cracks along with the ability of this pathogen to survive 90 and > 14 days on eggs stored at 5 [22] and 10°C [24], respectively, persist on inoculated eggs treated with sodium hypochlorite containing 100 ppm available chlorine [24], and grow in artificially contaminated eggs stored at refrigeration as well as ambient temperatures [22] suggests that eggs cannot be ignored as a possible source of listerial infection. Hence, it is not surprising that recent concerns about contaminated poultry products also have prompted efforts to define both the incidence and behavior of L. monocytogenes in eggs and egg products, including pasteurized liquid and dried egg.

Incidence As mentioned in the preceding paragraph, isolation of L. monocytogenes from intact whole eggs has not yet been documented; however the same is not true for broken eggs. According to Leasor and Foegeding [78], Listeria spp. were isolated from 15 of 42 (36%) previously frozen samples of raw commercially broken solids-adjusted liquid whole egg (21 samples), natural-proportion liquid whole egg (20 samples), and yolk (1 sample) obtained on several occasions from 6 of 11 (54%) commercial manufacturers located throughout the United States. On closer examination of the data, L. innocua and L. monocytogenes were identified in 15 of 15 (100%) and 2 of 15 (13.3%) positive samples, respectively. Twelve of 15 (80%) and 3 of 15 (20%) positive samples, including one sample each with L. monocytogenes, were classified as solids-adjusted and natural-proportion

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liquid whole egg, respectively. Increased handling during manufacture and, as suggested by the authors, a higher solids content which may enhance growth and survival of Listeria are just two of many possible reasons why a higher incidence of Listeria was observed in solids-adjusted rather than natural-proportion liquid whole egg. Although results from direct plating indicated that the two L. monocytogenes-positive samples contained approximately 1 and 8 L. monocytogenes CFU/g at the time of analysis, the fact that these samples were held frozen for up to 4.5 months and subjected to two freeze-thaw cycles suggests that numbers of Listeria likely decreased by at least 50% during storage (see Chap. 6). Hence, both samples probably contained < 100 L. monocytogenes CFU/g before being frozen. Working in the United Kingdom, Moore and Madden [23] recovered Listeria from 125 of 173 (72%) in-line filters used to remove shell fragments from raw blended whole egg; 78 and 47 samples of which contained L. innocua and L. monocytogenes, respectively, generally at levels <400 CFU/mL. However, since 500 pasteurized egg samples yielded negative results for listeriae, pasteurization at 64.4"C for 2.5 min, as required in the United Kingdom, appears to afford a high degree of safety. During a subsequent survey of three Australian egg factories in New South Wales, Desmarchelier et al. [37] identified L. innocua in 9 of 13 (69%) samples of unpasteurized liquid whole egg, with no other Listeria spp. being observed. Floors, drains, and mobile equipment in raw processing areas of these factories were later confirmed as major sources of contamination through strainspecific typing of environmental Listeria isolates. Although 7 samples of raw sugared yolk and 26 peeled boiled eggs were Listeria free, L. innocua was recovered from 1 of 51 samples of pasteurized liquid whole egg, with this organism also being detected in 2 of 14 peeled boiled eggs following 3 weeks of modified-atmosphere storage at 4°C. Hence, under certain conditions, Listeria can multiply to detectable levels in egg products during extended cold storage.

Growth The ability of Listeria to grow in hen's eggs was first recognized in 1940 when Paterson [ 1001 inoculated a laboratory culture of L. monocytogenes into the chorioallantoic membrane of a chicken embryo. This procedure was traditionally used to determine virulence of L. rnonocytogenes isolates [ 1211. Information concerning growth of this pathogen in nonfertile eggs and egg products is limited. A search of the scientific literature has uncovered only two studies pertaining to growth of L. monocytogenes in raw whole eggs or egg components. According to results from the first such paper published in 1955, Urbach and Schabinski [ 1251 found that populations of L. monocytogenes in intact experimentally infected nonfertile eggs increased nearly 6 orders of magnitude during 10 days of storage at ambient temperature. Following this study, 20 years passed until viability of L. monocytogenes was again examined in artificially contaminated raw, as well as cooked (1 2 1"C/ 15 min) whole egg, albumen, and egg yolk during extended storage at 5 and 20°C [73]. Results for raw whole and separated egg showed that growth of L. monocytogenes was primarily confined to egg yolk (Fig. 7), with the pathogen exhibiting respective generation times of 1.7 days and 2.4 h at 5 and 20°C. Overall, Listeria populations in raw whole egg generally varied less than 1 order of magnitude from the original inoculum during extended storage at either temperature; however, numbers of Listeria in raw albumen (pH 8.9) decreased 3 and 5 orders of magnitude during prolonged incubation at 5 and 2OoC, respectively. Despite the reported ability of L. monocytogenes to grow in laboratory media

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FIGURE 7 Behavior of L. rnonocytogenes in raw and cooked whole egg (A),albumen (HI, and egg yolk ( 0 )during extended incubation at 5°C. (Adapted from Ref. 73.) having pH values as high as 10 [ 1131, loss of Listeria viability in raw albumen was pH related, with numbers of Listeria decreasing less than 2 orders of magnitude in samples that were preadjusted to pH 7 and held at 5°C. Unlike raw whole egg, albumen, and egg yolk, Listeria grew rapidly in corresponding cooked samples. Generation times for the pathogen in cooked whole egg, egg yolk, and albumen were 1.9,2.3, and 2.4 days, respectively, at 5"C, and 2.6, 2.6, and 3.5 h at 20°C. These authors speculated that loss of the aforementioned antilisterial properties of raw albumen resulted from inactivation of one or more binding proteins (i.e., ovotransferrin, ovoflavoprotein, avidin) during heating. Since L. monocytogenes can grow rapidly in cooked whole as well as separated eggs, investigators of foodborne outbreaks should not overlook these products as potential vehicles of infection. In 1990, Sionkowski and Shelef [ I171 provided the first information concerning growth of L. monocytogenes in pasteurized egg products. To simulate postpasteurization contamination, pasteurized (64.4OU2.5 min) samples of liquid egg and reconstituted dried egg were inoculated to contain 104- I O5 L. monocytogenes CFU/mL and examined for numbers of Listeria during 7 days of storage at 4°C. As shown in Fig. 8, the pathogen grew similarly in both products, reaching populations 1O7 CFU/mL after 7 days of refrigerated storage. Although transmission of L. monocytogenes by pasteurized egg products has not yet been documented, these findings suggest that a possible public health problem could develop if L. monocytogenes enters pasteurized liquid egg, particularly since the shelf life of some of these refrigerated products now has been extended to several months. Growth of L. monocytogenes in commercially processed, liquid whole egg was first assessed by Foegeding and Leasor [41]. In this study, commercially broken, liquid whole egg was ultrapasteurized (68"C/ I 18 s), homogenized, inoculated to contain one of five L. monocytogenes strains (Scott A [clinical isolate], F5069 [milk isolate], ATCC 191I 1 [poultry isolate], NCF-U2K3, and NCF-FI KKr [raw liquid whole egg isolate]) at a level of 5 X 102to 1 X 103 CPU/mL, overlaid with mineral oil to prevent oxygen transfer, and examined for numbers of Listeria during extended incubation at 4, 10, 20, and/or 30°C. Generation times and maximum populations were generally similar to those previ-

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ously observed in fluid milk products (see Chap. 11) with the exception that strain Scott A failed to grow in liquid whole egg during prolonged incubation at 4 and 10°C (Table 6). Although growth of strain Scott A in fluid milk, cheese, and cabbage juice during refrigeration is well documented, Buchanan et al. [30] recently reported that this strain failed to grow in meat and poultry products incubated at 4°C. As shown in Table 6, generation times for the five L. monocytogenes strains ranged from 24.0 to 51.0, 8.0 to 31.0, 7.5 to 26.0 and 4.3 to 15.0 h at 4, 10, 20, and 30"C, respectively. Maximum populations ranged from 5.0 to 7.0, 5.48 to 8.48, 6.85 to 8.0, and 7.0 to 8.0 L. monocytogenes log,, CFU/g in liquid whole eggs incubated at 4, 10,20, and 3OoC,respectively. However, Sheldon and Schuman [ 1151 reported that when stored at 4"C, L. monocytogenes populations in an inoculated commercially available reduced-cholesterol liquid whole egg product could be reduced as much as 3.9 orders of magnitude by adjusting the pH of the product to 6.6 with citric acid and adding nisin at a level of 1000 IU/mL. Considering current distribution and marketing practices, it is likely that perishable products such as liquid whole egg will occasionally encounter periods of temperature abuse. Hence, from these data, it follows that even brief exposure to temperatures 210°C can lead to a dramatic increase in both growth rate (i.e., decreased generation time) and maximum popula-

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TABLE 6 Generation Times and Maximum Populations of L. rnonocytogenes in Ultrapasteurized Liquid Whole Egg Incubated at 4, 10, 20, and 30°C Strain of L. rnonocytogenes

Incubation temperature ("C) 4

F5069 Scott A ATCC 19111 NCF-U2K3 NCF-F1KK4

24 NGa 51 26 25

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Generation time (h) 12 7.5 NG 26 31 22 7.8 NDb 8.0 ND Maximum population (log 1o CFU/g) 7.OO 8.00 ND 7.00 5.48 6.85 8.48 ND 8.30 ND

4.3 7.1 15 ND ND 8.00 7.85 7.00 ND ND

ND, not determined. a No growth. Source: Adapted from Ref. 41.

tions of L. monocytogenes attained in ultrapasteurized liquid whole egg. Since most L. monocytogenes strains can proliferate in ultrapasteurized liquid egg products at refrigeration temperatures, postpasteurization contamination should be avoided and the product should be stored at temperatures close to, or preferably below, 0°C. The behavior of L. monocytogenes has been assessed in several additional egg and egg-related products. Brackett and Beuchat [27] showed that L. monocytogenes can survive throughout the normal shelf life of powdered and frozen egg products. The survival characteristics of L. monocytogenes on shell eggs and after cooking raw whole and scrambled eggs by frying also were determined by Brackett and Beuchat [28]. On the surface of egg shells, L. monocytogenes popultions decreased from 104to <10 CFU/egg after 6 days of storage at 5 and 20°C. Frying scrambled eggs reduced L. monocytogenes populations >3 orders of magnitude, whereas frying eggs sunny side up did not significantly reduce levels of L. monocytogenes. In commercial mayonnaise products, Erickson and Jenkins [39] showed that L. monocytogenes inactivation rates were directly correlated with aqueous phase acetic acid concentration as follows: sandwich spread 2 real mayonnaise > cholesterol-free reduced calorie mayonnaise dressing > reduced calorie mayonnaise dressing. The higher antilisterial activity in the cholesterol-free formulation was attributed to egg white lysozyme. Additionally, Glass and Doyle [53] reported that L. monocytogenes populations in two types of commercially produced low-calorie mayonnaise containing 0.7% acetic acid in the aqueous phase decreased from an initial inoculum of -106 CFU/ g to nondetectable levels following 10-14 days of ambient storage. These studies document that commercial mayonnaise products represent a negligible consumer safety risk. In the only other egg-related growth study thus far reported, Notermans et al. [95] examined the viability of several foodborne pathogens, including L. monocytogenes, in an eggnog-like product prepared from raw whole egg and sugar (25%, w/v) with/without ethanol (7%, v/v). When samples of ethanol-free product were inoculated to contain 104-105 L. monocytogenes CFU/g, numbers of Listeria generally decreased 10-fold during the first

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2 days of incubation at 4°C and then slowly increased to levels near or slightly above the original inoculum level after 5 additional days of refrigerated storage. Although L. monocytogenes generally exhibited similar behavior patterns in nonalcoholic samples incubated at 22OC, initial population decreases were far more abrupt, with the pathogen then increasing to populations 1 to 3 orders of magnitude lower than the original inoculum after 7 days of incubation. Unlike alcohol-free samples, L. monocytogenes was slowly inactivated in product containing 7% ethanol, with populations typically 1-2 and more than 4 orders of magnitude lower in 7-day-old samples held at 4 and 22"C, respectively, than were present initially. Hence, given the normal 2-week refrigerated shelf life of similar commercially available nonalcoholic eggnog-like products, recontamination of these beverages during packaging could lead to potential public health problems involving Listeria and other foodborne pathogens, with Salmonella enteritidis and S. typhimurium reportedly also remaining viable in artificially contaminated samples during 63 days of refrigerated storage.

Thermal Inactivation Interest in possible heat resistance of L. monocytogenes in eggs is of recent origin; however, concerns by European scientists regarding potential transmission of Listeria through eggs prompted a 1955 study by Urbach and Schabinski [ 1251, which examined the ability of this pathogen to survive in artificially infected eggs that were fried. According to these authors, L. monocytogenes was isolated from fried eggs (congealed white, soft yolk) prepared from inoculated raw eggs in which the pathogen had previously grown to levels >5 X 105CFU/g. Whereas the aforementioned work appears to be fairly crude by current standards and is now primarily only of historical interest, Foegeding and Leasor [41] conducted a more sophisticated study in which D-values were determined for five strains of L. monocytogenes (Scott A [clinical isolate], F5069 [milk isolate], ATCC 19111 [poultry isolate], NCF-U2K3 and NCF-FlKK4 [raw liquid whole egg isolates]) in sterile raw egg. Inoculated samples of raw liquid whole egg were added to glass capillary tubes which were heat-sealed and immersed in a water or oil bath at 51.0, 55.5, 60.0, and 66.0°C. After various times, tubes were removed and contents examined for survivors. Numbers of Listeria decreased linearly in raw egg during all four heat treatments, with D-values for the five L. monocytogenes strains ranging from 14.3 to 22.6, 5.3 to 8.2, 1.3 to 1.7, and 0.06 to 0.20 min at 51.0,55.5,60.0, and 66.OoC,respectively. Strain Scott A was generally less heat resistant than were the other four strains, particularly at the two lower temperatures; however, strain F5069 and the two isolates from raw egg exhibited moderate thermal tolerance at all four temperatures. Muriana et al. [92] subsequently reported similar D-values at 60°C for L. monocytogenes strain Scott A when inoculated samples of liquid whole egg were tested using either capillary tubes (D-value of 1.8 min) or a flow injection system (D-value of 1.95 min). Although this pathogen appears to exhibit a similar degree of heat resistance in both raw whole milk (see Chap. 6) and raw liquid whole egg, survival of L. monocytogenes is enhanced by supplementing liquid whole egg or egg yolk with 10% NaCl [89]. At 64"C, L. monocytogenes exhibited D-values of 10 and 10.5 min in salted liquid whole egg and egg yolk, respectively, as compared with 1 .O and 1.8 min for unsalted samples, with increased thermal tolerance attributed to a decrease in water activity. In contrast, adding 10% sucrose to unsalted samples neither increased thermal resistance of listeriae nor altered the product's water activity. In more practical terms, USDA officials currently require that liquid whole egg be

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pasteurized at a minimum of 60°C for 3.5 min to effect a 9-order of magnitude (9-D) kill of Salmonella spp. [ 1261. Although results from the study just discussed indicate that minimum pasteurization of liquid whole egg would yield only a 2.1- to 2.7-D kill of L. monocytogenes, one must remember that current estimates place L. monocytogenes populations in liquid whole egg at < 100 CFU/g [4 11. Hence, as is true for milk pasteurization, current minimum pasteurization requirements for raw liquid whole egg appear adequate to inactivate normal levels of Listeria that might be present in the product. However, it is important to stress that current minimum pasteurization requirements for liquid whole egg, as specified in the USDA Egg Pasteurization Manual, indicate that the margin of safety is approximately 6 orders of magnitude lower for L. monocytogenes than for most Salmonella spp. Furthermore, such pasteurization treatments appear to be inadequate for salted liquid whole egg and egg yolk. In 1987, Ball et al. [2 11 documented that ultrapasteurization (i.e., pasteurization at>60"C for <3.5 min) in combination with aseptic processing and packaging can be used to produce liquid whole egg with a refrigerated shelf life of 3-6 months. After results from this study were published, FDA officials issued a temporary permit allowing a North Carolina firm to market ultrapasteurized liquid whole egg [ 12,1071. Although two of the four objectives of the process were to render the product free of Salmonella and L. monocytogenes, the exact time/temperature requirements to completely inactivate L. monocytogenes in liquid whole egg were not specified in the FDA temporary permit. Based on extrapolations from the aforementioned survivor curves which showed no evidence of tailing, Foegeding and Leasor [4 11 predicted that the ultrapasteurization processes used by Ball et al. [21] would effect a 1- to 34-D (average of 14-D) kill of L. monocytogenes in liquid whole egg. Assuming that the ultrapasteurization times and temperatures used in conjunction with the temporary FDA permit are those values that were previously determined by Ball et al. [21], Foegeding and Leasor [41] went on to speculate that four of the 10 thermal treatments used by Ball and coworkers may not conform to the definition of ultrapasteurization in the temporary permit, depending on how one views the necessity for a 9-D reduction in numbers of Listeria. However, it appears that Listeria-free ultrapasteurized liquid whole egg having a refrigerated shelf life of 1 to several months can be produced, provided that the raw product is processed using one of the six more severe timehemperatwe treatments proposed by Ball et al. [21] and then is aseptically packaged to eliminate postpasteurization contamination. Foegeding and Stanley [42] verified the previous predictions concerning heat resistance of Listeria by determining the thermal death time (F-value) for L. monocytogenes strain F5069 in liquid whole egg. Using their previously described submerged capillary tube method [39], they found L. monocytogenes was eliminated from inoculated samples (1.0 X 10' to 4.0 X 108CFU/mL) of sterile liquid whole egg after processing at 62, 64, 66, 69, and 72°C for 16.0, 8.0, 4.5, 1.6, and 0.6 min, respectively. Although results from this study confirm that minimum pasteurization (60°C/3.5 min) will not result in a Listeria-free product if initial populations are large, the need for a 9-D kill as currently required by the USDA may not be appropriate for L. monocytogenes, since present estimates place the population of this pathogen at < 100 CFU/g in raw liquid whole egg. Hence, based on maximum expected L. monocytogenes levels in raw liquid whole egg, pasteurization by current standards should render such products free of Listeria. The situation regarding ultrapasteurization appears to be somewhat different since the thermal death-time data obtained by Foegeding and Stanley [42] indicate that 4 of the 10 ultrapasteurization processes proposed by Ball et al. [21] (63.7OU26.2 s, 63.8*C/

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92.0 s, 67.7OU9.2 s, and 71.5OC12.7 s) would likely fail to produce a 9-D decrease in numbers of Listeria in raw liquid whole egg. Nonetheless, the >9-D kill effected by the remaining six ultrapasteurization processes proposed by Ball et al. [2 I ] indicates that ultrapasteurization processes can be designed to produce Listeria-free liquid whole egg with an anticipated refrigerated shelf life of 3-6 months.

REFERENCES 1. Al-Sheddy, and E.R. Richter., 1989. Microbiological quality/safety of zoo food. Annual Meeting of the Institute of Food Technologists, Chicago, June 26-29, Abstr. 476. 2. Anonymous. 1988. Code of Federal Regulations, Title 9, Section 381.150. 3. Anonymous. 1988. FSIS recommends 35°F for long-term storage of meat, poultry, Food Chem. News 30( 12):25-28. 4. Anonymous. 1989. Chicken salad recalled in New England due to Listeria. Food Chem. News 3 1(42):65-66. 5. Anonymous. 1989. Current meat processing may not kill Listeria, study shows. Food Chem. News 30(52):57-58. 6. Anonymous. 1989. Listeria-contaminated chicken salad recalled from 3 states. Food Chem. News 3 1(35):5 1. 7. Anonymous. 1989. Listeria found by FSIS in small number but wide range of products. Food Chem. News 3 1 (30):47-48. 8. Anonymous. 1989. Listeria rnonocytogenes: P&i. Common. Dis. Rep. 89(27): 1. 9. Anonymous. 1989. Listeria tolerances asked by meat, poultry group. Food Chem. News 3 1( 1 4):46-48. 10. Anonymous. 1989. Listeria zero tolerance is warranted, USDA says. Food Chem. News 3 I (19):41-48. 11. Anonymous. 1989. Refrigerated fresh and frozen sandwiches recalled. FDA Enforcement Report, Dec. 20. 12. Anonymous. 1989. Temporary permit granted antimicrobial liquid eggs. Food Chem. News 30(47):49. 13. Anonymous. 1989. U K establishes committee to investigate food safety. Food Chem. News 30(5 1):39-40. 14. Anonymous. 1989. USDA to toughen regulatory policy on Listeria in meat, poultry. Food Chem. News 3 1(8):52-53. 15. Anonymous. 1990. Chicken, potato salad recalled by Campbell unit due to Listeria. Food Chem. News 32(9):61-63. 16. Anonymous. 1990. Irradiation in the production, processing and handling of food. Fed. Reg. 55: 18538. 17. Anonymous. 1990. Prepared sandwiches recalled. FDA Enforcement Report, Jan. 3 1. 18. Anonymous. 1990. USDA monitoring finds Listeria in ready-to-eat products at 78 plants. Food Chem. News 32(7):71-73. 19. Baccus-Taylor, G., K.A. Glass, J.B. Luchansky, and A.J. Maurer. Fate of Listeria rnonocytogenes and pediococcal starter cultures during the manufacture of chicken summer sausage. Poultry Sci. 72: 1772-1778. 20. Bailey, J.S., D.L. Fletcher, and N.A. Cox. 1989. Recovery and serotype distribution of Listeria rnonocytogenes from broiler chickens in the southeastern United States. J. Food Prot. 52: 148--150. 21. Ball, H.R., Jr., M. Hamid-Samimi, P.M. Foegeding, and K.R. Swartzel. 1987. Functionality and microbial stability of ultrapasteurized, aseptically packaged, refrigerated whole egg. J. Food Sci. 52:1212-1218.

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22. Baranenkov, M.A. 1969. Survival rate of Listeria on the surface of eggs and the development of methods for disinfecting them. Tr. Vses. Nauch.-Issled. Inst. Vet. Sanit. 32:453-458. 23. Bartlett, F.M., and A.E. Hawke. 1995. Heat resistance of Listeria monocytogenes Scott A and HAL 957E1 in various liquid egg products. J. Food Prot. 58:1211-1214. 24. Bartlett, F.M. 1993. Listeria rnonocytogenes survival on shell eggs and resistance to sodium hypochlorite. J. Food Safety 13:253-261. 25. Belding, R.C., and M.L. Mayer. 1957. Listeriosis in the turkey-two case reports. J. Am. Vet. Med. Assoc. 131:296-297. 26. Bind, I.-L. 1988. Review of latest information concerning data about repartition of Listeria in France. WHO Working Group on Foodborne Listeriosis, Geneva, Feb. 15-19. 27. Brackett, R.E. and L.R. Beuchat. 1991. Survival of Listeria monocytogenes in whole egg and egg yolk powders and in liquid whole eggs. Food Microbiol. 8:331-337. 28. Brackett, R.E. and L.R. Beuchat. 1992. Survival of Listeria monocytogenes on the surface of egg shells and during frying of whole and scrambled eggs. J. Food Prot. 55:862865. 29. Breer, C. 1988. Occurrence of Listeria spp. in different foods. WHO Working Group on Foodborne Listeriosis, Geneva, Feb. 15- 19. 30. 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. 31. Carosella, J. 1989. Personal communication. 32. Carpenter, S.L., and M.A. Harrison. 1989. Survival of Listeria monocytogenes on processed poultry. J. Food Sci. 54556-557. 33. Clouser, C.S., S. Doores, M.G. Mast, and S.J. Knabel. 1995. The role of defeathering in the contamination of turkey skin by Salmonella species and Listeria monocytogenes. Poultry Sci. 74:723-73 1. 34. Comi, G., and C. Cantoni. 1985. Listeria spp. in poultry from slaughterhouses of Lombardia. Indust. Aliment. 24521-525. 35. Cox, N.A., J.S. Bailey, and M.E. Berrang 1997. The presence of Listeria monocytogenes in the integrated poultry industry. J. Appl. Poultry Res. 6: 116-1 19. 36. Crawford, L.M. 1989. Food Safety and Inspection Service-Revised policy for controlling Listeria monocytogenes. Fed. Reg. 54:22345-22346. 37. Desmarchelier, P., J. Cox, and R. Esteban. 1995. Study of Listeria spp. contamination in the egg industry. In Proceedings of XI1 International Symposium on Problems of Listeriosis, Perth, Western Australia, Oct. 2-6, Promaco Conventions Pty. Ltd., Canning Bridge, Western Australia, pp. 257-260. 38. Dykes, G.A., I. Geornaras, M.A. Papathanasopoulos, and A. von Holy. 1984. Plasmid profiles of Listeria species associated with poultry processing. Food Microbiol. 11: 5 19-523. 39. 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. 40. Felsenfeld, 0. 1951. Diseases of poultry transmissible to man. Iowa State College Vet. 13: 89-92. 41. Foegeding, P.M., and S.B. Leasor. 1990. Heat resistance and growth of Listeria monocytogenes in liquid whole egg. J. Food Prot. 53:9-14. 42. Foegeding, P.M. and N.W. Stanley. 1990. Listeria monocytogenes F5069 thermal death times in liquid whole egg. J. Food Prot. 53:6-8, 25. 43. Franco, C.M., E.J. Quinto, C. Fente, J. L. Rodriguez Otero, L. Dominguez, and A. Cepeda. 1995. Determination of the principal sources of Listeria spp. contamination in poultry meat and a poultry processing plant. J. Food Prot. 58: 1320-1325. 44. Galli, R., C.G.T. Sannipoli, and C. Valente. 1992. Listeria monocytogenes as broiler carcasses contaminant. Indust. Aliment. 3 1:21-23.

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15 Incidence and Behavior of Listeria monocytogenes in Fish and Seafood* KARENC. JINNEMAN, AND MARLEEN M. WEKELL Seafood Products Research Center, U.S. Food and Drug Administration, Bothell, Washington

MELW. EKLUND**

U.S.National Marine Fisheries Service, Northwest Fisheries Science Center, Seattle, Washington

INTRODUCTION Listeria monocytogenes is ubiquitous in nature. Many aquatic creatures, including fin fish, oysters, shrimp, crabs, lobsters, squid and scallops, are harvested from natural environments; therefore, fish and seafood have been targeted as potential sources of Listeria in the human diet. Many of these products also undergo various processing procedures, some of which can inactivate Listeria present on the raw product. Listeria also can enter the

* The views expressed here are those of the authors and are not necessarily endorsed by the U.S. Food and Drug Administration, National Marine Fisheries, or the Government of the United States. ** Retired. 60 1

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product both during and after processing by poor sanitation conditions or manufacturing practices. The psychrotrophic nature of L. monocytogenes allows survival or even multiplication of this potential pathogen during refrigerated storage or temperature abuse situations. This is a special concern for those products which receive minimal or no heat treatment before consumption. Since the first time L. monocytogenes was isolated from imported cooked crabmeat in 1987, at least 112 Class I recalls (i.e., a situation where reasonable probability exists that the use of or exposure to a violative product will cause serious adverse health consequences or death) have been issued by the U S . Food and Drug Administration (FDA) for more than 250,000 pounds of ready-to-eat domestidimported fish and seafood, with this pathogen routinely being found in 8.7% of all such products marketed in the United States. The first of several cases of listeriosis positively linked to consumption of fish or seafood was not reported until 1989 when a 54-year-old woman in Italy contracted listerial meningitis 4 days after consuming steamed fish from which L. monocytogenes was later isolated [36]. This case and the potential hazard associated with consumption of other Listeria-contaminated ready-to-eat food such as cooked crabmeat, cooked shrimp, and smoked salmon has prompted studies to determine the incidence and control of Listeria in various seafoods. The incidence and behavior of L. monocytogenes in fish and seafood have been addressed in several review papers [ 13,26, 32,391. In this chapter, data reviewed are from a series of FDA surveys from I987 to 1996. These were designed to determine the incidence of L. monocytogenes in domestic and imported shrimp, crab, and various other fish and seafood products. As in previous chapters, Class I recalls that have been issued for Listeria-contaminated fish and seafoods also will be mentioned. Surveys of fish and seafood products for Listeria conducted by many other international groups will be reviewed. The behavior of L. monncytogenes in these foods, data concerning growth and thermal resistance of L. monocytogenes in seafoods, as well as measures used such as the application of lactic acid for controlling growth of Listeria in seafood also will be covered.

FDA SURVEYS OF L. MONOCYTOGENES IN DOMESTIC AND IMPORTED SEAFOOD Immediately after the June 1985 outbreak of cheeseborne listeriosis in California, FDA officials focused their attention on urgent problems that confronted the dairy industry. Despite a lack of evidence linking consumption of meat and poultry products to cases of human listeriosis before 1988, as early as December 1985 U.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) officials began taking an active interest in determining the incidence of L. monocytogenes in meat and poultry products. Increased concern about the potential hazard of Listeria-contaminated seafood to public health began in the spring of 1987 after a private testing laboratory in the United States isolated L. monocytogenes from frozen cooked crabmeat obtained from a Mexican supplier [4]. L. monocytogenes was confirmed in this product by the FDA in Baltimore, Maryland, in May of 1987. In maintaining FDA’s “zero-tolerance” policy for L. monocytogenes in ready-to-eat foods, the first in a series of Class I recalls was issued for nearly 4 tons of tainted crabmeat that was marketed in four states. These events also prompted an import alert on June 17, 1987 [ 5 ] ,which called for automatic detention and testing for

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Listeria and Escherichia coli in all frozen crabmeat shipped to the United States from Mexico. Less than 1 month after this product was recalled, the FDA in Seattle, Washington, detected L. monocytogenes in samples of imported frozen raw shrimp [3] and lobster tails [15]. Although no recalls were issued for these products, which are almost invariably cooked before consumption, confirmation of Listeria in these seafoods together with the finding in cooked crabmeat noted previously prompted the FDA to initiate two surveys in July of 1987. In the first of these surveys, six imported samples of frozen raw shrimp were collected monthly and examined for Listeria at each district office. The samples represented as many different countries as possible (Table 1) [3]. Additionally, each district also was requested to collect three domestic samples of frozen raw shrimp per month at the wholesale or retail level. Using the original FDA method [69], Listeria spp. were detected in 18 of 74 (24.3%) samples of frozen raw shrimp imported from 10 different countries between July and October of 1987 (see Table 1). L. monucytugenes also was isolated from 4 of 74 imported samples of frozen raw shrimp, with all positive samples originating from

TABLE1 Results from an FDA Survey of Imported Frozen Raw Shrimp, July-October, 1987. No. of positive samples (%)

Country of origin Brazil Ecuador Guyana Honduras Hong Kong India Indonesia Macau Mexico Nigeria Norway Pakistan Panama People’s Republic of China Peru Philippines Taiwan Thailand Venezuela Total

No. of samples analyzed

L. monocytogenes

3

1 (25) 1 (12.5) 1 (100) l a (20) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

74

4 (5.4)

4 8 1 5 1

4 1 1 10 3 1 4 7 4

1 3

9

4

a One sample contained L. monocyotgenes and other Listeria spp. Source: Adapted from Ref. 40.

Other Listeria SPP.

Jinneman et al.

604

Central or South American countries. Subsequently, three lots of raw shrimp, imported from Ecuador and Honduras, were found to contain 103-105L. monocytogenes or L. innocua CFU/g [76]. However, since shrimp are normally not consumed raw in the United States, FDA officials did not request the recall of any of these contaminated lots. In the second FDA survey, domestic and imported samples of cooked, frozen, and refrigerated crabmeat (i.e., picked or extracted) were examined for the presence of L. monocytogenes, Staphylococcus aureus, Vibrio cholera, V. parahaemolyticus, V. vulniJcus, and Yersinia enterocolitica and numbers of E. coli [3]. Again, samples of imported crabmeat from as many different countries as possible were collected. As of January 1988, 6 of 98 (6.1 %) domestic samples of cooked crabmeat contained Listeria, with L. monocytogenes and L. innocua being recovered from 4 and 2 samples, respectively (Table 2). Similarly, Listeria spp. were detected in 3 of 24 (12.5%) imported samples of cooked crabmeat, with L. monocytogenes being discovered in 2 of 24 (8.3%) samples of product marketed in the United States. Weagant et al. [106] formally published the first results of a survey dealing with the incidence of Listeria spp. in imported/domestic frozen seafood products analyzed at the FDA District Laboratory in Seattle during the second half of 1987; 31 of 50 (62%) imported and 4 of 7 (57%) domestic samples of frozen seafood tested positive for Listeria spp. using the FDA method [69]. The only Listeria spp. detected were L. monocytogenes (15 of 57,26.3%) and L innocua (26 of 57,45.6%); both L. monocytogenes and L. innocua were isolated from several of the samples. Although the number of samples examined from the various product categories was limited, results suggested that frozen seafood more frequently contains L. innocua than L. monocytogenes. Hence, as was true for raw milk, meat, and poultry products, both organisms also likely occupy similar niches in seafood-processing environments. Therefore, the presence of L. innocua in raw and particularly in cooked seafood should not be ignored but rather should be viewed as an indicator of possible contamination with L. monocytogenes.

TABLE 2 Results from an FDA Survey of Domestic/lmported

Refrigerated or Frozen Cooked Crabmeat, July 1987 to January 1988

No. of positive samples (%)

Country of origin

No. of samples analyzed

L. monocytogenes

Other Listeria SPP. ~~

United States Canada Chile Korea Japan Mexico Venezuela

98 3 2 11 2 3 3

4 (4.1) 0 0 2 (18.2) 0

Total (imported)

24

2 (8.3)

0

0 ~

One sample contained L. monocytogenes and L. innocua. Source: Adapted from Ref. 15.

a

~~~~~~~

2 (2.0) 1 (33.3) 0 2a (18.2) 0 0 0 3 (12.5)

Listeria monocytogenes in Fish and Seafood

605

Discovery of Listeria in raw shrimp, crabmeat, and other seafood products, coupled with an increased concern about the general safety of seafood, prompted FDA officials in October 1987 to include analysis for L. monocytogenes in a compliance program for domestic/imported shrimp [7] and to increase testing of many other domestically produced seafoods for Listeria spp. under the program for pathogen monitoring of select high-risk foods (CPGM 7303.030) [ 11,15,40]. This increased sampling effort sought to determine the geographical distribution of Listeria in domestic/imported seafood and to identify the incidence of Listeria spp. in such products. In March 1988, a processed seafood assignment was issued [42]. The purpose of the processed seafood compliance program (CPGM 7303.036) was to test for several bacterial pathogens, including Listeria, in imported and domestic processed seafood that receives minimal to no processing by the consumer. Products selected for Listeria analyses under the processed seafood compliance program included crabmeat (cooked or pasteurized), crayfish/crawfish, lobster, langostinos (cooked, parboiled), molluscan shellfish, processed imitation seafood (surimi), seafood salads, shrimp (cooked), smoked or salted fish, and other processed seafood. In addition, the National Advisory Committee on Microbiological Criteria for Foods (NACMCF) in April 1988 began the laborious task of developing microbiological criteria for cooked shrimp and crabmeat [8]. During the FDA surveys from October 1988 through September 1990, L. monocytogenes was recovered from domestic samples of crabmeat, lobster, shrimp, smoked salmon, and surimi. Imported fish, lobster, shellfish, shrimp, smoked fish, squid, and surimi also tested positive for L. monocytogenes during the same time period [42]. Three FDA compliance programs in effect since 1991 have surveyed the incidence of Listeria in fish and seafood products and reported analytical data into the FDA Microbiological Information System. The Domestic Fish and Fisheries Products Compliance Program (CPGM 7303.842) covers investigations and sampling of domestic fish and fishery products [45]. Imported seafood and seafood products are surveyed for presence of Listeria under the Import Seafood Products Compliance Program (CPGM 7303.844) [44]. The Processed Seafood Compliance Program (CPGM 7303.036) was in existence through 1994 and covered both domestic and imported processed seafood and seafood products [42,43]. Since 1994 the items covered by this program have been incorporated into the CPGM 7303.842 and CPGM 7303.844 programs for domestic and imported products, respectively. Ready-to-eat food products that require no further or minimal processing by the consumer or products collected as a follow-up to suspected cases of foodborne illness are identified for Listeria analyses in each of these compliance programs. The domestic fish and fisheries product compliance program also includes analyses for Listeria of in-line and swab samples collected during processor establishment investigation reviews. Among these three compliance programs a total of 7158 samples of fish, seafood products or seafood processing in-line samples were analyzed by the FDA between 1991 and 1996. Listeria monocytogenes was detected in 622 of these samples for an overall incidence of 8.7%. The breakdown of samples analyzed and those in which L. monocytogenes was detected is given by year and origin (domestic or import) in Table 3. There is no significant difference based on a heteroscedastic T-test ( P = .OS) between the incidence of L. monocytogenes in imported compared with domestic products. The 8.7% incidence found by the FDA is comparable to the 4-12% incidence of L. monocytogenes in seafood and seafood products from temperate areas as reported by Embarek [32]. Surveys of other food products have indicated a 4-60% incidence in raw meat, 23-60% in fresh poultry, and 2.2% in raw milk [32,39,64].

Jinneman et al.

606

TABLE 3 Seafood Product and Seafood Processor In-line Samples Analyzed for Listeria monocytogenes by the FDA from 1991 through 1996 ~

Samples

1991

1992

1993

1994

1995

1996

Domestic product (CPGM) positive negative total % positive Import product (CPGM) positive negative total % positive Overall positive negative total % positive

(1,3) 20 403 423 4.7% (3) 32 362 394 8.1%

( 193)

63 50 1 564 1 1.2% (273) 51 643 694 7.3%

(1,3) 89 558 647 13.8% (293) 65 80 1 866 7.5%

(193)

94 623 717 13.1% (273) 42 737 779 5.4%

(1) 67 48 I 548 12.2% (2) 41 589 630 6.5%

(1) 41 382 423 9.7% (2) 17 456 473 3.6%

52 765 8 17 6.4%

114 1144 1258 9.1%

154 1359 1513 10.2%

136 1360 1496 9.1%

108 1070 1178 9.2%

58 838 896 6.5%

Overall

374 2948 3322 11.3% 248 3588 3836 6.5% 622 6536 7158 8.7%

Source: Data compiled from the FDA Microbiological Information System. Samples collected from the following Compliance Programs (CPGM) [42-451: 1 . CPGM 7303.842 Domestic Fish and Fisheries Products (19911996); 2. CPGM 7303.844 Import Seafood Products (1992-1996); 3. CPGM 7303.036 Processed Seafood (1991- 1994).

Samples found positive for L. monocytogenes in the FDA compliance programs represent a wide range of fish and seafood products (Table 4). Among the crustacean products, (crab, shrimp/prawns, lobster, and crawfish), 2 18 samples were positive, with crab accounting for 142 positive samples. Fifteen samples were positive for L. monocytogenes from the shellfish category which includes mussels, oysters, clams, scallops, and snails. The fin fish category had 231 samples positive for L. monocytogenes, with 164 of these from a smoked seafood product. The remaining positive L. monocytogenes samples represent a diverse group of products, including squid/calamari, 3 samples; eel, 9 samples; roe/caviar, 19 samples; imitation seafood, 17 samples; seafood salad/spread/p$tb or mousse, 13 samples; or processor in-line or swab samples, 94 samples. Overall, the two fish or seafood products identified in the FDA compliance programs which account for the highest incidence of L. monocytogenes are crab and smoked fin fish. Together these two product categories represent nearly half (306 of 622 positive samples) of all the fish or seafood product samples in which L. monocytogenes was detected between 1991 and 1996 by the FDA. A closer look at the FDA Microbiological Information System data for these two products appears in Table 5. For crab products, 1886 samples were analyzed for Listeria. Of these, 142 (7.5%) were positive for L. monocytogenes. This is within the 0-29.2% L. monocytogenes incidence rate reported in several other surveys [20,30,37,84,94,106]. It is presumed that the presence of L. monocytogenes in ready-to-eat crab is the result of postprocess contamination of the product. In the FDA studies, a total of 1210 smoked fin fish products were analyzed for Listeria, with 164 (13.6%) samples being positive for L. monocytogenes. Between 1991 and 1995 for those smoked seafood samples in which the smoking process was known, the incidence of L. monocytogenes was higher in cold smoked 21.3% (5 1 of 240) compared

Listeria monocytogenes in Fish and Seafood

607

TABLE4 Fish and Seafood Products from which L. monocytogenes Was Isolated by FDA from 1991 to 1996.

Product/Sample

1991

1992

1993

1994

1995

12 7 8 1

34 1 9 0

37 7 9 2

28 11 7 1

25 2 4 2

0 0

1 1

1 0 0

0 0 0

0 0 3 0 1

1 0 1 0 0

16 2

32 12

38 10

1 0 1 0 0

0

3 0

Crustacean crab shrimplprawns lobster crawfish Shelljish mussels oysters clams scallops snails Fin Fish smoked other Other seafood products squid/calamari eel roelcaviar imitation seafood seafood (salad, spread, pit& mousse) processor in-line or swabs not specified

1996

Total

6

1 0

142 32 38 6

0 0 3 1 0

2 0 0 0 0

4 1 8 1 1

33 25

23 11

22 7

164 67

1 2 4

1 4 9 7 3

0 0 2 2 4

1 0 5 2 1

0 0

3 9 19 17 13

12 0

22 0

19

28 0

10

94 1

5

1

4

1 4 1

0

Source: Data compiled from the FDA Microbiological Information System. Samples collected from the following Compliance Programs (CPGM) [42-451: CPGM 7303.842 Domestic Fish and Fisheries Products (1 99 I - 1996); CPGM 7303.843 Import Seafood Products (1992- 1996); CPGM 7303.036 Processed Seafood (1991-1994).

TABLE 5 Crab and Smoked Fin Fish Samples Analyzed for L. monocytogenes by FDA from 1991 Through 1996

Crab positive negative total % positive Smoked fin fish positive negative total % positive ~

1991

1992

1993

1994

1995

1996

Total

12 248 260 4.6%

34 324 358 9.5%

37 363 400 9.3%

28 320 348 8.0%

25 272 297 8.4%

6 217 223 2.7%

142 1744 1886 7.5%

16 117 133 12.0%

32 175 207 15.5%

38 193 233 16.3%

33 23 1 264 12.5%

23 154 177 13.0%

22 176 198 11.1%

164 1046 1210 13.6%

~~

Source: Data compiled from the FDA Microbiological Information System. Samples collected from the following Compliance Programs (CPGM) [42-451: CPGM 7303.842 Domestic Fish and Fisheries Products (1 99 I - 1996); CPGM 7303.844 Import Seafood Products (1 992- 1996); CPGM 7303.036 Processed Seafood ( I 99 1- 1994).

Jinneman et al.

608

TABLE 6 Class I Recalls Issued i n the United States for Domestic and Domestic/

Import Ready-to-Eat Seafood Products Contaminated with L. monocytogenes Since 1987

Product Crustacean crab

shrimp lobster ShellBsh mussels (marinated) mussels (smoked) snails

Fin Fish

No. of Class I recalls since 1987

lb affected

46

>141197

7 2

>3 1332 >264

1 1 1

Unknown Unknown 1455

MA New Zealand CO, FL, GA, IL, KS, LA, NH, NJ, NY, OR, PA, TX, WA KY, MD, ME, NY, WA CA, MA, ME, NJ, NY, OR, WA, United Kingdom CA, FL, IL, MD, ME, NC, NY, SC, TN, VA, WA Canada

hot smoked cold smoked

6 22

>253 >93722

smokeda

16

>9292

salted Other imitation seafood seafood salad or spread

1

Unknown

5

> 1773

3

>42

Location of manufacturer AL, FL, GA, ME, NC, OR, TX, VA, WA, Chile, Mexico FL, GA, MA, NY, WA Canada

ID, NV, OR, UT, VA, WY, Japan, Korea FL, ME, WA

Hot or cold smoking process not identified. Source: Data compiled from FDA Enforcement reports, Refs. 14 and 41.

a

with hot smoked 8.8% (19 of 215) samples [57]. Several other investigations have evaluated the prevalence of L. monocytogenes in smoked fin fish products, with incidence rates ranging from 0 to 75% [32]. In surveys with over 100 samples, the incidence of L. monocytogenes also tended to be greater in cold smoked fin fish products (1 1.3-24.0%) [60-621 compared with hot-smoked fin fish products (8.4-8.9%) [61,62]. Similar to ready-to-eat crab products, postprocess contamination likely accounts for the presence of L. rnonocytogenes when it occurs in hot-smoked fin fish products. However, with cold-smoked fin fish, the process may not eliminate L. monocytogenes present on the raw product; nevertheless, it is also possible that contamination could occur during or after processing of the product [29]. Overall, there have been 112 Class I recalls for domestic and domestichmported ready-to-eat seafood products resulting from presence of L. monocytogenes during 1987 through August 1998 in the United States [14,41]. Recalls only have been issued when L. monocytogenes was found in seafood or seafood products which are readyto-eat and would therefore receive no subsequent or minimal heat treatment by the consumer before consumption. The number of recalls by product category is shown in Table 6. Products which resulted in the greatest number of recalls reflect the types of products which were most frequently identified as being positive for L. monocytogenes

Listeria monocytogenes in Fish and Seafood

609

in compliance program surveys. Crab accounted for 47 and smoked fin fish for 16 of the 112 recalls.

OTHER SURVEYS FOR L. MONOCYTOG€N€S IN FISH AND SEAFOOD PRODUCTS The documented presence of Listeria in fish and seafood products and subsequent product recalls prompted several surveys to determine the incidence of Listeria spp. in various products from many geographical locations (Table 7). Results from these studies have been extensively reviewed [ 13,26,32,62]. Sampling strategies, number of samples analyzed, and detection methods varied, so that although it is useful to note the results from these studies, the data from them cannot always be directly compared.

Crustaceans Listeria spp., including L. monocytogenes, have been recovered from cooked and picked, ready-to-eat crabmeat, and, as noted earlier, this product has been the subject of several recalls. Since crab meat is heat processed to eliminate or reduce microorganisms, the presence of L. monocytogenes on the finished product most likely represents postprocessing contamination. Several surveys have included small numbers of crab samples. L. monocytogenes was detected in 7 and L. innocua in 12 of 24 cooked imported crab products [ 1061. Although L. monocytogenes was not detected in another study, L. welshirneri was found in one of two cooked crab samples [20]. Listeria spp. were recovered in two of five crab samples collected as part of a survey in Alexandria, Egypt [30], and L. monocytogenes was isolated from one of seven crab samples from the United States and China [37]. Two larger U.S. studies of cooked and processed crab also have been published. In the first, 3 1 of 138 (22.5%) processed crab samples contained Listeria spp., identified only to the genus level [84]. In the second study which examined 126 cooked and picked blue crab samples, 10 samples (7.9%) were positive for L. monocytogenes and 3 samples (2.4%)

TABLE 7 Incidence o f Listeria spp. in Fresh, Frozen, and Processed Seafood % positive for

Product (country) Crab crab (c) (multiple countries) crab (c) (USA) crab (r) (China) crab (c or p) (USA) crab (QY Pt 1 blue crab (c) (USA) Shrimp/prawn s shrimp (f), (multiple countries) shrimp (f and fr) (USA) shrimp (f) (Japan) shrimp (f) (Trinidad) shrimp (raw, fr) (France) shrimp (c and p) (multiple countries)

No. of samples 24 2 7 138 5 126 7 4 70 41

17 8

Listeria spp.

50 22.5 40 10.3 25 8.6 5 23.5

L. monocytogenes

29.2 0 14 0 7.9

28.6 0 1.4 11.8 25

Ref. 106 20 37 84 30 94 106 20 75 1 93 106

Jinneman et al.

610

TABLE 7 Continued % positive for

Product (country) shrimp (r) (multiple countries) shrimp (f and p) (India) shrimp (Canada) shrimp in brine (r) (Norway) shrimp (c and f ) (Iceland) prawn (r) (Japan) shrimp (Egypt) prawns/shrimp/cockles (c) (UK) Lobster lobster tail (fr) (multiple countries) Mussels mussels (sm) (New Zealand) mussels (f) (Spain) mussels (f) (Australia) Oysters oysters (fr) (multiple countries) oysters (p) (USA) oysters (f) (Japan) oysters (f) (Egypt) oysters (f) (Australia) Clams clam (f) (India) clam (f) (USA) Scallops scallops (fr) (multiple countries) scallops (raw) (USA) Other shellfish and invertabrates shellfish (c) (Iceland) non-oyster shellfish (f) (Japan) shellfisha Donax spp. (coquina) (f) (Egypt) Ruditapes spp. (clam) (f) (Egypt) Fin Fish fish (f) (USA) catfish (f) (USA) fish (f) (Trinidad) minced fish (f) (Norway) fish (f) (Japan) fish (f) (India) fish (f) (Egypt) fish (fr) (Egypt) fish (f) (India) fish (fr) (India) fish (r) (New Zealand) fish minced (raw, r) (Japan) fish (trout) (f) (Iceland)

No. of samples 49 19 20 16 11 38 5 40

Listeria spp.

10.5 9 15.8 40

2

L. monocytogenes

Ref.

8.2 0 20 18 9 2.6 20 0

37 73 37 97 56 99 30 95

50

106

35.7 7.5 15.4

59 101 102

14 40

22.5

1 2 84 2

0 0 0 0

0 0 0 0 15.4

106 20 75 30 102

1 1

0 0

0 0

49 20

2 1

50 0

0 0

106 20

0

0 1.4 25 16.7 25

56 75 59 30 30

50 100 14.8

50 0 2 12 2.4 0 12.8 5.9

20 20 1 97 75 73 30 30 49 49 59 99 56

11 147 25 6 4

4 1 61 8 382 51 39 17 4 10 25 37 2

11.6 44 16.7 50

12.6 3.9 25.6 17.6 25 20 52 43.2 0

32 8.1 0

Listeria monocytogenes in Fish and Seafood

611

TABLE7 Continued % positive for

Product (coutitry) fish (dried haddock) (Iceland) fish (fr) (multiple countries) fish (ceviche) (Peru) fish (lightly pickled) (Switzerland) fish (gravad) (Iceland) fish (cold-sm, salmon) (Switzerland) fish (cold-sm, salmon) (Switzerland) fish (cold-sm, fish) (Switzerland) fish (cold-sin, fish) (Switzerland) fish (cold-sin, salmon) (Norway) fish (sm,-salmon) (Iceland) fish (cold-sin, salmon) (Canada) fish (cold-srn, salmon) (Italy) fish (cold-srn, salmon) (New Zealand) fish (cold-sm, salmon) (USA) fish (sm)-(New Zealand)” fish (sm fish) (Canada) fish (sm fish) (Canada) fish (hot-sm fish) (Switzerland) fish (hot-sm fish) (Switzerland) fish (sm and/or salted) (Egypt) fish (f) (Denmark) fish (cold-sm, cured) (Denmark) Other fish and seafood products seafood (squid, langostinos) (multiple countries) seafood (f arid fr) (Taiwan) seafood (f and p) (Iceland) seafood (India) seafood ( f ) (USA) seafood (p) (USA) seafood (other) (Iceland) seafood raw, (r) (Japan) seafood (r) (New Zealand) seafood (c) (Japan) seafood (other) (Japan) seafood salad (p) (USA) fish salads (r) (Iceland) seafood (past ) (pasta with minced fish) (Iceland) seafood (surirni) (multple countries) seafood (surirni) (USA) seafood (surirni) (Canada)

No. of samples 5 4 32 89 22 100 64 324 434 33 31 32 37 12 61 12 71 496 69 1 11

232 335 2 57 26 200 59 14 5 28 50 5 6 2 37 3 7 1 46

Listeria spp.

L. monocytogenes

0 25 9 25.8 22.7 24 6.3 13.6 11.3 9 3.2 31.2 0 75 78.7 66.7

0

75 63.6

29 0-80

11.3 20.4

4.4 8.9 8.4 5.6 14.2 10.8

18.2

I00

0 10.5 3.9 0

3.9 8 49.2 0 20 7.1 48 0 0 0 32 0

20 10.7 26 0 0 0 16 0 28.6 0 2

0 ~

c, cooked; f, fresh: fr, frozen; p, processed; r, ready-to-eat; sm, smoked. “ Includes the 14 smoked mussel samples listed in mussles catagory. Included in the 25 ready-to-eat fish samples in the New Zealand study listed above. Source: Adapted in part from Ref. 32.

Ref.

56 106 48 61 56 60 52 61 62 97 56 37 104 59 29 59 27 25 61 62 30 2 2 106 105

56 65 84 84 56 99 59 99 99 20 56 56 106 20 37

~-

672

Jinneman et al.

positive for L. innocua [94]. Very few studies have enumerated L. monocytogenes in naturally contaminated cooked and processed crab. Among the 10 samples positive for L. monocytogenes in one study [94], one sample contained 1100 Listerialg, but in the remaining samples, <100 Listeridg were detected, indicating a generally low level of contamination. Listeria spp. have been detected in fresh or raw shrimp. Two of seven samples contained L. monocytogenes [ 1061; one of four samples contained Listeria innocua 1201 in two U.S. studies. In a survey of fresh shrimp in Japan, Listeria spp. were recovered from 6 of 70 samples (8.6%), with one (1.4%) being positive for L. monocytogenes [70]. In France, L. monocytogenes was isolated from 2 of 17 (1 13%) uncooked shrimp samples and Listeria spp. from 4 samples (23.5%) [93]. In Trinidad where shrimp are consumed in a nearly raw state, Listeria spp. were recovered from 2 of 41 ( 5 % ) fresh, uncooked shrimp samples [I]. Despite cooking and other heat-processing steps which should eliminate Listeria spp. present on raw product, several investigators have recovered Listeria spp. from cooked and ready-to-eat shrimp/prawns. L. monocytogenes was detected in 2 of 8 (25%) cooked and processed shrimp in a U.S. study [106], 4 of 49 (8.2%) ready-to-eat shrimp in Canada [37], and 1 of 38 (2.6%) ready-to-eat shrimp products in Japan 1993. No L. monocytogenes was recovered from 40 retail samples of cooked prawns, shrimp, and cockles sold in England and Wales between 1987 and 1989 [95]. Ready-to-eat shrimp in brine had L. monocytogenes in 3 of 18 (16.7%) samples, tested in Norway [97]. As with the surveys of cooked and processed crab, there have been few studies where numbers of L. monocytogenes were determined in cooked shrimp products. However, low levels of L. monocytogenes in three lots of naturally contaminated ready-to-eat shrimp (0.54,5.5, and 0.04 most probable number (MPN)/g)and lobster (2.0,0.23, and 0.4 MPN/ g) were reported in a Canadian study [37].

She1Ifish Smoked mussels have been associated with several listeriosis cases in Australia and New Zealand [35,77] and with recalls in the United States. L. monocytogenes was isolated from 5 of 14 smoked mussel samples in a survey [59] in New Zealand, whereas fresh Spanish mussels yielded L. monocytogenes from 3 and other Listeria spp. from 9 of 40 samples [loll. The incidence of Listeria in other shellfish products, including oysters, clams, and scallops has been remarkably low. No Listeria spp. were isolated from oysters in two studies in the United States and one in Egypt which included one or two samples [20,30,106]. In a study in Japan, no Listeria spp. were recovered from 84 oyster samples [75]. No Listeria spp. were found in a single clam sample in each of two studies [20,49]; however, in a study in Egypt, Listeria spp. were found in two of four Ruditapes spp. (clam) samples, with L. monocytogenes being present in one of these samples [30]. Only a limited number of samples of scallops have been tested; however L. innocua was isolated from one of two frozen U.S. samples [ 1061, but no Listeria spp. were recovered from a single sample in a later study [20]. No Listeria were found in 11 Icelandic cooked shellfish samples [56]. In a survey of fresh seafood samples purchased from markets in Alexandria, Egypt, one of six Donax spp. (coquina) was positive for L. monocytogenes [28]. In Japan, L. monocytogenes was isolated from 2 samples and Listeria spp. were recovered from 17 samples of a total of 147 non-oyster shellfish samples [75,81].

Listeria monocytogenes in Fish and Seafood

613

Fin Fish In the United States, raw fresh or frozen fish are generally not consumed without further processing; therefore, surveys for Listeria spp. have included very few fresh or frozen fish samples. In surveys conducted in the United States, L. monocytogenes was isolated from two of four fresh fish samples, L. innocua from one catfish sample [20] and L. monocytogenes from one of four frozen fish samples [106]. In India, Fuchs and Surrendan [49] reported Listeria spp. in 1 of 4 fresh and 2 of 10 frozen fish samples. In a larger survey of fresh fish in India, Listeria spp. were isolated from 2 of 51 (3.9%) samples, but L. monocytogenes was not recovered [73]. In Alexandria, Egypt, Listeria spp. were present in 10 of 39 (25.6%) fresh and 3 of 17 (17.6%) frozen fish samples, with L. monocytogenes being isolated from 5 of 39 (12.8%) and 1 of 17 (5.9%) of these fresh and frozen fish samples, respectively [30]. In Norway, L. monocytogenes was isolated from one of eight (12.5%) minced fresh fish samples [97]. The practice of consuming fresh seafood in an almost raw state is common in Trinidad where Listeria spp. were detected in 9 of 61 (14.8%) fresh fish samples [I]. In Japan, 3 of 37 (8.1%) raw ready-to-eat minced fish samples were positive for L monocytogenes and 16 of 37 (43.2%) for Listeria spp. [99]. In a similar Japanese survey, L. monocytogenes was found in 9 (2.4%) and Listeria spp. in 48 of 382 (12.6%) samples [75]. However, it is not clear if all these samples were ready-to-eat.

Smoked Fish Products The presence of Listeria in smoked and lightly processed fish products is often a concern, because many of these products are commonly eaten without further heating. The coldsmokmg process does not generate sufficient heat to inactive Listeria organisms which may be present on fish [29,53,71]. In Switzerland, L. monocytogenes was recovered from 24 and 6% of cold smoked salmon [52,60] and 13.5 and 11.3% of cold smoked fish samples [61,62]. In studies in Norway [96], Canada [37], and New Zealand 1591, the organism was isolated from 9, 3 1, and 66%, respectively, of cold smoked salmon samples (see Table 7). A detailed study on the incidence and sources of L. monocytogenes in several processing facilities producing cold-smoked salmon showed the primary sources of L. monocytogenes were surface areas of frozen or fresh raw fish coming into the plant. As the processing of fish progressed, this pathogen was transferred to other processing areas and these became secondary sources of the bacterium [29]. In those studies which specifically identified hot smoked fish samples, L. monocytogenes also was recovered from 8.9 and 8.4% of samples despite the heat processing these products received [61,62].

Lightly Processed Fish Products Other lightly processed ready-to-eat fish products also can harbor L. monocytogenes. These include lightly pickled fish from which L. monocytogenes was isolated in 25.8% of samples surveyed in Switzerland [61]. Cerviche is a lightly acidified ready-to-eat fish product, which is popular in several South American countries. Listeria spp. were recovered from 75% and L. rnonocytogenes was recovered from 9.4% of cerviche samples examined in Peru [45]. In Iceland, Listeria spp. were identified in 32.4% and L. rnonocytogenes in 16.2% of ready-to-eat fish and seafood salads tested [56]. No Listeria spp. were recovered from two seafood salad samples included in a U.S. survey [20] or three pasta salads with minced fish in the Icelandic study [56].Imitation seafood made from surimi is another

614

Jinneman et al.

processed fish-based product from which L. monocytogenes was found, with an incidence of 28.6% in the United States [I061 and 2.2% in Canada. No Listeria were recovered from a single sample in a second U.S. survey [20].

HUMAN LlSTERlOSlS ASSOCIATED WITH FISH AND SEAFOOD PRODUCTS Despite the high prevalence of L. monocytogenes, there have been few reported human listeriosis outbreaks associated with consumption of fish and seafoods. The first case of listeriosis positively linked to consumption of fish or seafood was not reported until 1989 when a 54-year-old woman in Italy contracted listerial meningitis 4 days after consuming steamed fish from which L. monocytogenes was later isolated [36]. The fact that the two L. monocytogenes isolates from the patient’s cerebrospinal fluid and leftover portion of fish both were of serotype 4 and were identical in terms of phage type and DNA restriction analysis, confirms fish as the vehicle of infection in this case of listerial meningitis. However, the mode by which this fish became contaminated remains unknown. In 1991, three previously healthy people, aged 83, 37, and 10 years, became ill in two separate incidents in the State of Tasmania, Australia, after consuming smoked mussels [77]. Symptoms included malaise, chills, fever, and headache followed by diarrhea. Samples of implicated mussels from both incidents contained over I million L. monocytogeneslg. In addition, L. monocytogenes also was isolated from feces of the patients. Mussels had been imported from New Zealand, repackaged illegally in Australia by a retail outlet, and labeled with a code date that overestimated their shelf life by 3 months or more. Newborn twins died from a L. monocytogenes infection in Auckland, New Zealand, in 1992. Their deaths were attributed to consumption by their mother of smoked mussels contaminated with L. monocytogenes. Reports from New Zealand have indicated that the company producing the smoked mussels had detected Listeria in their product several months before the deaths of the infants. In 1993, the owner of the company and a consultant to the company were charged with manslaughter by New Zealand police [ 19,351. Since these outbreaks, one report indicating a contamination rate of 15.4% for Australian oysters and mussels [ 1021, and concerns by the Australian Quarantine and Inspection Service [74], the Australian National Health and Medical Research Council [82,83] has issued two bulletins providing special dietary advice for pregnant women, transplant patients, and other immunocompromised patients and information for medical practitioners on diagnosis, treatment, and advice to patients.

REGULATORY ASPECTS OF L. MONOCYTOGENES IN FISH AND SEAFOODS Although discussion of proposed criteria for L. monocytogenes and other pathogens in foods appears in Chapter 17, the reader should be aware that the NACMCF has recommended a “zero tolerance’’ for L. monocytogenes in cooked shrimp and crabmeat [ 10,121. The regulatory policies of different countries vary concerning allowable levels of L. monocytogenes in food products. In 1994, Madden stated, “The policy of the [U.S.] FDA remains what has been commonly referred to as the ‘zero tolerance’ policy, which is very conservative. No L. monocytogenes organisms are permitted in a food which was not intended for further heat treatment.” [72]. Canadian regulations for presence of L. monocy-

Listeria monocytogenes in Fish and Seafood

615

togenes in ready-to-eat foods have been based on the ability of L. monocytogenes to grow in a given food product [38]. There still is not complete agreement among the countries which compose the European Economic Community (EEC) for criteria regarding L. monocytogenes in various foods [ 1031. Criteria for L. monocytogenes developed by the EEC are only within the Milk Hygiene Directive. Several public health approaches to food safety including addressing industry groups about HACCP-based hygiene plans and educating the most susceptible groups, (e.g., pregnant women and immunocompromised individuals) have been used by individual European countries. A quantitative approach setting limits at the point of sale or at the end of product shelf life, also has been explored by several countries. For example, German regulations categorize foods into four risk levels and set specific L. monocytogenes action levels based on the risk category. Group I foods have the most restrictive limit (absence of L. monocytogenes in 25 g or 25 mL of food). Within the German approach, seafood products like heat-treated shrimp or prawns would be categorized as Group 111. For products in this category, low-level contamination (
BEHAVIOR OF L/ST€R/A IN FISH AND SEAFOOD Before 1987, very little was known about the incidence and behavior of Listeria spp. in fish and seafoods. Since this time, a number of studies have focused on the growth, inhibition, and thermal resistance of L. monocytogenes in different products. The results from these studies and the means by which this pathogen may be transmitted to various forms of aquatic life are discussed in this section.

Modes of Transmission Current data point to cross contamination as the major source of Listeria in cooked or otherwise processed seafood, as evidenced by the recovery of healthy, noninjured cells of L. monocytogenes from the surface of many heat-processed/ready-to-eat seafood products. However, a small percentage of aquatic creatures may become contaminated through direct/indirect contact with Listeria in their natural environment. This appears even more plausible when one recalls the salt-tolerant nature of L. monocytogenes and that this pathogen has been isolated from sewage effluent entering the North Sea [24] and also from crustaceans that were harvested from stream water in which L. monocytogenes was previously identified [86]. In 1989, Fuad et al. [47] evaluated the ability of L. monocytogenes to survive in the estuarine environment. Since there appears to be a higher incidence of Listeria in chitinous seafood (i.e., shrimp, crab, lobster), samples of filtered and unfiltered seawater with and without chitin and chitin-free filtered and unfiltered stream water were inoculated with various strains of L. monocytogenes, many of which possessed chitinase activity. Although Listeria populations decreased in chitin-free filtered and unfiltered seawater, adding chitin to both types of seawater stimulated growth of Listeria. Moreover, the pathogen grew in filtered stream water. These findings, along with those from a report in which L. monocytogenes was found on the exoskeleton but not in the digestive tract of shrimp that were exposed to high levels of L. monocytogenes in aquaculture tanks [ 1051, suggest that this pathogen may be

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ecologically adapted to chitin. If this is true, then it is imperative that holding tanks for chitinous marine animals be properly set up and maintained to avoid potential microbiological problems involving L. monocytogenes and other foodborne pathogens including Vibrio spp. and Aeromonas hydrophilia. It is well established that Listeria spp. are often associated with wild animals and birds which can serve as reservoir hosts. Fenlon et al. [46] demonstrated the role that scavenging birds can play in the Listeria cycle. In that study, a definite association between gulls feeding on sewage and fecal carriage of Listeria (26.3% positive) was shown, which compared with a carriage rate of only 8% for gulls feeding in less polluted areas. This higher carrier rate suggests that L. monocytogenes is a part of the normal microflora of the near estuarine environment [ 131. Two studies of estuarine waters, shrimp, and oysters along the northern Gulf of Mexico [80] and of freshwater tributaries, sediments, bay water, and oysters in the Humboldt-Arcata Bay of Northern, California 1221, reached similar conclusions. In one study [79], 5% of 78 saltwater samples were positive for Listeria spp. In comparison, 11% of 74 shrimp samples were positive for L. monocytogenes and no Listeria were isolated from oysters. Listeria species and L. monocytogenes were found in 8 1 and 62% (37 samples), respectively, of freshwater or low-salinity waters in tributaries draining into Humboldt-Arcata Bay. The incidence of Listeria spp. and L. monocytogenes in sediment (46 samples) from the same tributaries was 30.4 and 17.4%, respectively. One of three bay water samples contained Listeria spp. (including L. monocytogenes), whereas L. innocua was recovered from only 1 of 35 oyster samples [22].Both of these studies indicate that the estuarine environments are continuously subjected to potential contamination with Listeria spp. from, for example, processing effluents, agricultural runoff, and sewage effluents. Listeria spp. can be recovered from nonpolluted environments, and the source of these bacteria may very well be from avian species, especially sea-gulls [79].

Growth and Survival Raw and processed seafoods have been long regarded as excellent substrates for growth of most common agents of foodborne disease, particularly if seafoods are held at improper temperatures; however, interest in behavior of L. monocytogenes in these products is of recent origin [32]. According to data gathered by Lovett et al. [70) in 1988, L. monocytogenes grew readily (generation time G 12 h) in inoculated samples of raw shrimp, crab, surimi and whitefish, with the pathogen attaining maximum populations of > 10' CFU/g in all four products following 14 days of storage at 7°C. Two years later, Brackett and Beuchat [16] also reported that L. monocytogenes grew and retained similar levels of pathogenicity on artificially contaminated crabmeat during 14 days of storage at 5 to 10°C. In similar studies, Rawles et al. 1941 examined both the incidence and growth of L. monocytogenes in blue crab meat held at refrigeration temperatures. Of the 126 samples analyzed, 10 were positive for L. monocytogenes and 3 were positive for L. innocua. Populations of Listeria found in fresh-picked blue crabmeat were usually < 100 CFU/g. Based on these data, an inoculum level of 50 CFU/g was added to commercially pasteurized crabmeat and the growth rate determined at 1.1, 2.2, and 5°C. Calculated generation times were 68.7 h at I.1"C; 31.4 h at 2.2OC, and 21.8 h at 5°C. At 5"C, there was a 7 log 10 increase in L. monocytogenes population but only a 2.5 log,,, at I . 1"C after 2 I days. The authors therefore concluded that blue crab meat needs to be stored at 5 1.1"C. However, in contrast to what Lovett et al. [70] observed for shrimp and whitefish, Harrison et al. [54] and Shineman and Harrison [ 1001 found that L. monocytogenes failed to grow in overwrapped/vacuum-packaged raw shrimp and fin fish, with numbers of Listeria gen-

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erally decreasing by approximately I log after 2 I days of storage in an ice chest. When catfish were stored at 4°C the L. monocytogenes population increased slowly ( 1 .O- 1.5 log,,,) during the first 12 days and then decreased I .5 log,,, by day 16 [68]. During storage, psychrotrophic populations increased from 1O3 to > 1 O7 CFU/g, thus reinforcing the notion that L. monocytogenes can readily survive in refrigerated raw foods even when greatly outnumbered by other natural contaminants. Since L. monocytogenes was recovered from laboratory-contaminated shrimp (initial inoculum 2 10' CFU/g) after 90 days at -20°C [76], it is evident that this pathogen also is fairly resistant to subfreezing temperatures. Unlike the aforementioned products, preliminary results from Kaysner et al. [66] suggest that L. monocytogenes was unable to grow in artificially contaminated oysters, with Listerill populations remaining constant in shucked oysters after 21 days at 4°C. Apparent inability of Listeria to grow in raw oysters may be related to difficulties in isolating Listeria from retail raw oysters. According to Farber [37], L. monocytogenes (inoculum level of 2 X 103CFU/mL) grew fairly well on cooked lobster, shrimp, crab, and smoked fish and in most instances increased about 2-3 log,,, within 7 days at 4°C. When these same products were temperature abused for a short time (6 h) at room temperature, levels of L. monocytogenes increased by 1 log on shrimp. crab, and lobster, and only 0.2 log on smoked salmon. In a survey for the incidence of this pathogen on shrimp and lobster meat at the wholesale level, 13 of 113 samples were positive and were contaminated at a level of < 10 MPN/g. Storage of these naturally contaminated products at 4°C resulted in L. monocytogenes populations of
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CFU/g inoculum. In these same studies, the growth rate of L. monocytogenes also was determined in products with different initial bacterial counts. When the inoculum level was 6 CFU/g, L. monocytogenes populations increased faster in samples with the lower rather than higher initial total bacterial populations.

Inhibition Of the processes used to prepare smoked fishery products, the cold-smoking operation has been of special interest, because the temperatures used are not lethal to L. monocytogenes. Hence, the following interventions have been recommended to reduce the risks associated with L. monocytogenes in these products: (a) eliminate or reduce numbers of L. monocytogenes on the outside surfaces of frozen or fresh fish before filleting, (b) prevent recontamination and growth of L. monocytogenes during all stages of processing, and (c) inhibit growth of any possible survivors or recontaminants during processing and distribution 1291. Several papers have been published on inhibition of L. monocytogenes in coldsmoked fish processed with sodium chloride, sodium nitrite and sodium lactate. In these studies, smoke was not applied to the products, so that the efficacy of the different forms of inhibition could be addressed. Peterson et al. [91] studied behavior of L. monocytogenes (150 CFUI15 g) in cold-processed salmon containing 3.5 or 6.0% water phase sodium chloride. The products were packaged in either oxygen-permeable film or vacuum sealed in impermeable film and stored at 5 and 10°C. After the second week at 1OOC, L. monocytugenes populations increased to the range of 106-108 CFU/g, with no difference being attributed to the sodium chloride concentration. Vacuum packaging suppressed growth of L. monocytogenes by 10- to 100-fold in samples with 3 or 5% sodium chloride. Inhibition related to salt concentration was most apparent at 5"C, with L. rnonocytogenes populations being held below 10' CFU/g by 6% water phase salt, but increased to 104CFU/g in products with 5% water phase salt and to 10' CFU/g with 3% water phase salt. Brown sugar is often used in processing of cold-smoked salmon; use of the sugar in the product, however, did not influence growth of L. monocytogenes. In these same studies, growth of the clinical isolate Scott A and two L. monocytogenes strains isolated from salmon were comparable in cold-smoke salmon stored at 5 and 10°C. Given the salt tolerance of L. monocytogenes and consumer unacceptability of smoked fish products with water phase NaCl concentrations much above 3 or 4%, it was concluded that other inhibitors, in addition to NaCl, were needed to control growth of this bacterium. Pelroy et al. [90] therefore studied the behavior of L. monocytogenes (150 or 4.9 X 103CFU/15-g sample) in relation to sodium nitrite (190-200 ppm) combined with sodium chloride in cold-processed salmon stored at 5 and 10°C. The combination of NaCl and NaN0, was most effective at 5°C. With an initial inoculum of 150 CFU/l5 g, L. monocytogenes was held below IS CFU/g by a combination of 190-200 ppm NaN02 and 3% water phase salt and below 20 CFU/g with NaNO, and 5% NaC1. Packaging in oxygen-permeable or oxygen-impermeable (vacuum-sealed) films had little effect on growth of L. monocytogenes when NaN02 was included in the process. Increasing the storage temperature to 10°C markedly reduced the efficacy of both NaCl and NaCl NaN0, treatments. There was little difference in inhibition between 3 or 5% water phase NaCl at 10°C, and the combined effect of NaCl and NaNO, was only slightly greater than than that of NaCl alone. However, the packaging method had the most pronounced effect on growth of L. monocytogenes at 10°C. Growth was consistently greater in samples packaged in oxygen-permeable film than in the vacuum-sealed impermeable film pack-

+

Listeria monocytogenes in Fish and Seafood

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ages. L. monocytogenes populations had increased from 10 CFU/g to about 108 CFU/g in samples packaged in oxygen-permeable film and 106 CFU/g in impermeable film. Differences in inhibition attributable to inoculum level and NaCl + NaN02concentrations were obvious in products stored at 5°C. In comparison, when the initial inoculum level was increased to 327 CFU/g, L. monocytogenes reached a population of l 06- 107CFU/g after 20 days of storage. Of the different inhibitors studied by Pelroy et al. [89], sodium lactate used in combination with salt or salt plus sodium nitrite was most effective in controlling growth of L. monocytogenes (150 CFU/l5 g) on vacuum packaged cold-smoked salmon stored at 5 and 10°C [89]. A concentration of 3% lactate in combination with 3% salt or 3% salt + 125 ppm nitrite prevented any increase in L. monocytogenes populations during 40-50 days of storage at 5 or 10°C (Fig. 1). Addition of 2% sodium lactate prevented growth of L. rnonocytogenes in all samples stored at 5°C. At 10°C, however, a combination of sodium lactate (2%) and NaCl (3%) inhibited growth for 14 days, but then the pathogen reached populations about 1-2 logs less than those of the control samples with NaCl only. When the products contained NaN02 (125 ppm), sodium lactate (2%), and NaCl (3%), growth of L. monocytogenes was totally suppressed at 10°C except in one of the four samples where populations reached 9.3 X 102 CFU/g. Certain spices and herbs also can be used to minimize growth of Listeria, as previously discussed in Chapter 6. According to Rorvik et al. [98], L. monocytogenes populations remained unchanged on vacuum-packaged gravad salmon (i.e., an unsmoked salmon product containing dill with a similar pH, salt content, and a, to that of smoked salmon)

10°C 7

E

5°C 3% NaCl

c

$ 7 0 No Lactate

A

"0

10 20 30 40 500

2% Lactate

10 20 -30 40 50

Days stored

FIGURE1 Growth of L. monocytogenes (150 Scott A/15-g sample) in comminuted salmon containing 0, 2, or 3% water phase NaCI, with or without 125 ppm NaN02, during storage at 10" or 5°C in vacuum-sealed impermeable film packages. Symbols on baseline indicate L. monocytogenes was detectable by enrichment only. (Adapted from ref. 89.)

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during 4 weeks of storage at 4°C. Since addition of as little as 0.5% dill prevented growth of Listeria in laboratory media, dill was most likely responsible for inhibiting L. monocytogenes in gravad salmon during extended cold storage. Fermentation products from lactic acid bacteria have been studied to determine their efficacy in controlling L. monocytogenes growth in blue crab meat [23]. In these studies, steam-sterilized crab meat was inoculated to contain 5.5 log,, CFU/g of a three strain mixture of L. monocytogenes and then washed with various lactic acid bacteria fermentation product at levels of 2000-20,000 arbitrary units (AU) per milliliter of wash water. L. monocytogenes populations remained relatively constant in control samples stored for 6 days at 4°C. In comparison, numbers of L. monocytogenes on crabmeat washed with Perlac 1911 or Micro-Gard (10,000-20,000 AU) initially decreased (0.5-1 .O log,, unit/g) and then recovered to original levels within 6 days. When crab meat was washed with 10,000-20,000 AU of Alta 234 1, enterocin 1083, or nisin per milliliter, L. monocytogenes populations initially decreased by I .5-2.7 log units/g. Thereafter, counts increased by 0.5- 1.6 log 1o units within 6 days. Degnan et al. [23] also showed that when the crabmeat was washed with food-grade sodium acetate (4 M), sodium diacetate (0.5 to 1 M), and sodium lactate ( I M) or sodium nitrate (1.5 M), there was only a modest reduction in L. monocytogenes population (0.40.8 log,, unit/g). However, Listeria counts decreased 2.6 logl0/gwithin 6 days when the sodium diacetate concentration was increased to 2 M. Trisodium phosphate (1 M) also reduced L. monocytogenes counts from 1.7 to >4.6 log 10/gwithin 6 days. Hence, L. monocytogenes populations on crabmeat can be reduced by washing with selected antimicrobial agents. Potential use of certain strains of Enterococcus faecium to control L. monocytogenes was suggested by Embarek et al. [34]. E. faecium isolates from sous-vide cooked fish fillets were tested on different strains of L. monocytogenes and other pathogenic bacteria using Brain Heart Infusion broth with added CaC03 to avoid decreases in pH during growth of E. faecuim. Of the 19 isolates tested, 14 produced proteinaceous substances inhibitory to different strains of L. monocytogenes. An inoculum of 107CFU E. faecium/ mL reduced L. monocytogenes populations of 102CFU/mL to lO/mL after 14 days at 3°C and to I CFU/mL after 35 days. With a lower inoculum of 1 O4 CFU/mL, L. monocytogenes was only slightly inhibited at 15°C and not at 3 or 5°C. However, after 11 days at 15"C, spontaneous resistance was observed and L. monocytogenes reached I X I O8 CFU/ mL. All of these L. monocytogenes isolates were resistant to E. faecium. The antibacterial effects of 209 psychrotrophic Pseudoinonas strains isolated from spoiled iced fish and newly caught fish were assessed by screening L. monocytogenes and other organisms using agar diffusion assays [51]. Only eight strains inhibited growth of L. monocytogenes. Inhibitory action was most pronounced among Pseudomonas strains producing siderophores which chelate iron; addition of iron sometimes eliminated the antibacterial effect. Some strains of Pseudomonas enhanced growth of L. monocytogenes, with dense growth being observed around wells containing these Pseudomonas strains. These strains may have created a more advantageous nutritional environment for L. monocytogenes by increasing the supply of iron or the availability of low molecular weight nutrients.

Inactivation As was true for dairy, meat, and poultry products, the rash of Class I recalls during the past decade involving ready-to-eat seafoods has prompted concerns about the adequacy

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of thermal processing treatments used for raw seafood. Hence, in early 1988, Pace et al. [88] reported results from a study in which freshly shucked oysters were exposed to 150 ppm chlorine for 30 min, pasteurized at an internal temperature of 72-74°C for 8 min, and then periodically examined for major bacterial groups during 5 months of refrigerated storage. According to these authors, chlorination reduced initial aerobic plate counts of 4.5 X 1O5 C'FU/g by 40-90%, with pasteurization reducing the population by an additional 99.9%. Despite these large reductions in microbial flora, survivors classified in eight different genera of aerobic or facultatively anaerobic bacteria were present in the product. However, at this point, the oysters were unfit for consumption, as evidenced by profuse gas production and swelling of plastic pouches in which the product was pasteurized. In response to public and industry concerns, the FDA 1761 also examined the thermal resistance of Listeria in raw shrimp tails that were inoculated internally to contain approximately 104--1OS L. rnonocytogenes CFU/g. Using a combination of cold (with/without broth) enrichment and warm (selective) enrichment, these investigators failed to recover the pathogen from shrimp tails that were boiled longer than 5 min. Although appreciable numbers of heat-stressed cells were detected in inoculated shrimp tails that were boiled for 3 min, frozen storage of the product at -20°C eventually led to complete inactivation of the pathogen. More important, when this study was repeated using naturally contaminated frozen shrimp from Ecuador and Honduras in which 1 03-1OS L. monocytogenes or L. innocua CFU/g were presumably present only on the chitinous exoskeleton, all Listeria were eliminated after 1 min of boiling. Hence, since shrimp are more likely to be contaminated externally than internally with relatively low levels of Listeria, these findings suggest that present cooking methods are adequate to eliminate these organisms from raw shrimp. Since these initial studies, the thermal death time of L. monocytogenes has been determined in different seafoods and fish. The results of these studies are summarized in Table 8. In an effort to determine if the presence of L. rnonocytogenes in processed lobster could be the result of undercooking or postcooking contamination, Budu-Amaoko et al. [21] determined the thermal death time of 107cells of L. monocytogeneslg of product using 25-g samples. The observed D-values at 51.6, 54.4, 57.2, 60.0, and 62.7"C were 97.0, 55.0, 8.3, 2.39, and 1.06 min, respectively, with a z-value of 5.0"C (Table 8). After isolating this pathogen from plants using good manufacturing practices, the authors speculated that the presence of L. monocytogenes in the final product was probably the result of underprocessing . Blue crab is a popular seafood item, with more than 50% of picked meat being pasteurized to offset seasonal fluctuations in some regions. The lack of information about the growth and survival of this pathogen in blue crabmeat prompted Harrison and Huang [55] to determine the heat resistance of the Scott A strain in this product. The crabmeat was inoculated with 107 cells/g before distributing 7.5 g into sausage casings (1.6 X 4 cm) for thermal processing. D-values were 40.43, 12.0, and 2.6 I min at 50, 55, and 6OoC, respectively, with a z-value of 8.40"C as determined by using Trypticase Soy Agar (see Table 8). Use of Vogel Johnson agar, a selective plating medium that is less able to support repair and subsequent growth of sublethally injured Listericz, yielded lower D-values (34.48, 9.18, and 1.31 min) and a lower z-value (6.99"C) at the same temperatures. Heightened interest in foodborne listeriosis has also led to intense efforts toward determining whether current industry practices for cooking crawfish are adequate to inactivate L. rnonocytogenes. In 1993, Dorsa et al. [28] first determined the growth rate of L. monocytogenes in crawfish tail meat stored at 0.6 and 12°C. Exponential growth began with no apparent lag phase and 109CFU/g were observed after 10 and 4 days at 6 and

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TABLE 8 Thermal Death times of L. rnonocytogenes in Different Seafood and Fish Product Temperature

("C)

Lobster meat (21)

Blue crab meat (55)

Crawfish tail meat (28)

Mussels brine soaked (18)

Salmon (33)

Cod (33)

10.73

7.28

4.48 2.07

1.98 0.87

0.87 0.15 0.07

0.28 0.15 0.03

D-value (min)

50.0 51.6 54.4 55.0 56.0 57.2 58.0 59.0 60.0 62.0 62.7 65.0 68.0 70.0

97.0 55.0

40.43 12.00

10.23

8.3 2.39

1.06

2.6 1

1.98 0.19

48.09 16.25 9.45 5.49 1.85

z-values, lobster meat 5.0"C; blue crabmeat 8.40"C in Trypticase Soy Agar; crawfish tail meat 5.5"C; mussels 4.25"C, salmon 5.6"C; cod 5.7"C.

12"C, respectively. Rapid growth of L. monocytogenes at these temperatures further emphasized the need for information on thermal resistance of the bacterium in crawfish tail meat. In precooked crawfish meat, D-values for L. monocytogenes were 10.23, 1.98, and 0.19 min at 55, 60, and 65"C, respectively, with a z-value of 5.5"C. Since commercially processed crawfish are normally boiled for 5-10 min before hand peeling, most Listeria contamination occurs postboiling during peeling and packaging. Therefore, in addition to strict in-plant sanitation programs, postpicking or postpackaging heat treatments, as described by Dorsa et al. [28], can be used to produce Listeria-free product. Based on the psychrotrophic nature of L. monocytogenes and the lack of information on the minimum oral infective dose, the New Zealand Department of Health has taken a conservative approach and recommended a policy of "zero tolerance" for L. monocytugenes in a 25-g sample of ready-to-eat seafood. Hot water blanching at 68-72°C has been used by several New Zealand mussel harvesters to inactivate L. monocytogenes in greenshell mussels [87]. Bremer and Osborne [ 181 also determined the thermal death time of seven strains of L. monocytogenes in green shell mussels that were brined in preparation for hot smoking. Brined mussels were blended, inoculated to contain 106CFU/g, and heat resistance was determined in plastic pouches. The D-values at 56, 58, 59, 60, and 62°C were 48.09, 16.25, 9.45, 5.49, and 1.85 min, respectively, with a z-value of 4.25"C (see Table 8). When the D-values for different seafoods at 60°C are compared, values for lobster meat, crawfish tail meat, and crabmeat are similar. The D-value for brined mussels, however, were two to three times higher (see Table 8). The authors indicated that addition of salt and brown sugar (used in the smoke products) may have enhanced the thermal resistance of L. monocytogenes in mussels during hot smoking. These differences suggest that product form and composition should be considered when determining the heat resis-

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tance of a pathogen. Furthermore, these data emphasize the importance of determining the D-value for each product experimentally. Minimally processed foods, also called “refrigerated pasteurized” foods, of extended durability [78] that can be prepared using the sous-vide technology have raised new concerns regarding survival of Listeria. Embarek and Huss [33] studied the heat resistance of two strains of L. monocytogenes (062 and 057) in fatty fish (salmon) and nonfatty fish (cod), with the former strain being slightly more heat resistant. For sous-vide processing, the fish were vacuum packaged and then heated at 58, 60,62, 65, 68, and 70°C. For cod, D-values were 7.28, 1.98, 0.87, 0.28, 0.15, and 0.03 min, respectively, with a z-value of 5.7”C for L. monocytogenes strain 062 (see Table 8). Both strains of L. monocytogenes were one to four times more heat resistant in salmon than in cod. The D-values for salmon were 10.73, 4.48, 2.07, 0.87, 0.2, and 0.07 min, respectively, with a z-value of 5.6”C for L. monocytogenes strain 062 (see Table 8). The authors attributed the protective effect in salmon to its higher fat content. They also emphasized the importance of product form and ingredients in determining the heat resistance of L. monocytogenes. Embarek [311 subsequently assessed the thermal resistance of L. monocytogenes in sous-vide fish fillets containing various levels of NaCl and also the potential growth of this pathogen in the product during cold storage. When cod fish fillets were heated at 5868OC, destruction of Listeria took up to five times longer in fillets containing 5% added NaCl as compared with product prepared without salt. Thus, although the present time/ temperature treatments recommended by the NACMCF will easily inactivate expected numbers of L. monocytogenes in sous-vide fish prepared without salt, identical processing will likely produce some survivers in product containing added salt. Growth experiments also showed that L. monocytogenes populations increased 2 and 4 log,, CFU/g in saltfree sous-vide fish after 14 days of storage at 3 and 5-10°C, respectively. These findings stress the importance of eliminating L. monocytogenes from the product during sous-vide processing. If the aforementioned timekemperature treatments for the different seafoods had been followed, then the recent Class I recalls likely resulted from postprocessing contamination, as has already been implied by Kvenburg [9,67] and other FDA officials [6,105]. However, considering the possibility for errors during thermal processing, members of the seafood working group of NACMCF agreed it would be prudent to consider certifying or licensing persons who are directly involved in thermally processing the different seafoods, as has been done for many years in the canned food industry [9]. Recognizing that L. monocytogenes is more likely to contaminate the surface rather than the interior of most fishery products, several studies have concentrated on using different treatments to inhibit or inactivate L. monocytogenes on the surface of processed seafoods. Noel et al. [85]investigated the possibility of using various lactic acid treatments to inactivate L. monocytogenes on processed peeled and unpeeled shrimp. The shrimp were immersed in a broth culture of L. monocytogenes, drained, and then immersed in an aqueous solution of 1.5,3.0, or 6.0% lactic acid for 1, 10, or 120 min. The untreated and treated shrimp were examined for survivors during 28 days of storage at 20°C. Although all lactic acid treatments decreased the numbers of Listeria, exposure to 1.5% lactic acid for 10 min was deemed most appropriate, since the treatment did not adversely affect the product’s organoleptic quality. Overall, initial L. monocytogenes populations of 2.6 X 103 CFU/g on inoculated shrimp decreased to <3.2 X 102CFU/g following 10 min of exposure to 1.5% lactic acid. Although Listeria populations continued to decrease in all samples, fewer Listeria were observed in lactic acid-treated (-10 CFU/g) than in untreated shrimp (-40 CFU/g) after 28 days of frozen storage.

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In a similar investigation, decimal reduction times (D-values) were determined for a mixture of seven strains of L. monocytogenes exposed to marinades in the presence and absence of greenshell mussels [ 171. When an acetic acid (1.5% wt/vol) marinade was used, the calculated D-values in the presence and absence of mussels were 77.3 and 33.3 h, respectively. When a combination of acetic acid (1.5%) and glucono-delta-lactone (0.2%)-based marinades were used, the D-values increased to 86.3 h in the presence of mussels and decreased to 19.3 h without the mussels. Likewise, for acetic acid (0.75%) and lactic acid (0.75%) marinades the D-values increased to 125.5 h in the presence of mussels and 26.9 h in the absence of mussels. Through enrichment experiments, L. monocytogenes was detected after 26 days for acetic acid ( I .5%) and after 53 days for the acetic acid, lactic acid (0.75% each) combination, but no survivors were found after 29 days with the acetic acid (1 S % ) and the glucono-delta-lactone (0.2%) combination. Their results prompted the authors to emphasize that care must be taken in extrapolating from results of a model broth system to a "real food." Thus despite the inability of organic acids to eliminate completely L. monocytogenes from seafoods, storage of marinated products before release is an effective method for further decreasing the possibility of L. monocytogenes being associated with these products. This also was demonstrated with inoculated gravad salmon processed with dill and stored at 4°C. Dill at 0.5 and 5.0% prevented growth of L. monocytogenes during storage of the salmon. These studies also indicate that dipping products such as shrimp, mussels, lobster, crab, and scallops in organic acids could prove useful in decreasing the levels of Listeria before and during frozen storage. During the past decade, there have been several recalls of smoked fish products because of L. monocytogenes contamination. To estimate the potential health hazard for the consumer eating such products, Jemmi and Keusch [63] determined the heat resistance and growth of L. monocytogenes in artificially inoculated heat-smoked trout. In these experiments, two strains of L. monocytogenes were surface inoculated ( 106MPN/g) onto raw trout which then were kept in a salt and spice marinade (10% NaCl and 0.7% spices, pH 6.4) for 12 h before smoking in a kiln. The trout were surface dried at 60°C for 30 min, the kiln was heated to 1lO"C, and fish were held at this temperature for 20 min after an internal temperature of 65°C was reached. Then the product was smoked for 45 min, cooled, and stored at 8-10°C for up to 20 days. L. monocytogenes decreased from an initial population of 106MPN/g to nondetectable levels after hot smoking and throughout 20 days of storage at 4-10°C. However, when the smoked trout were inoculated with 30 or 45 MPN/g after processing and stored at 4 and 8-10°C, L. monocytogenes increased to 107 MPN/g. The presence of L. monocytogenes on hot-smoked fishery products typically suggests postprocessing contamination. Thus Poysky et al. [92] assessed various processing parameters required to inactivate L. monocytogenes during the hot-smoking process. Since L. monocytogenes contamination is most likely to occur on the surface of raw fish fillets or steaks [29], the effectiveness of using a combination of heat and smoke to inactivate L. monocytogenes on the surface of brined salmon steaks was investigated. When brined salmon steaks were heat processed without smoke, L. monocytogenes survived on steaks processed to an internal temperature of 181°F (823°C) for 30 min. Application of generated smoke reduced the minimum lethal temperature to 153°F (67.2"C). In contrast, when smoke was applied during the last half of the process after surface drying and formation of the surface pellicle, L. monocytogenes was recovered from steaks heated to an internal temperature of 176°F (8O.OOC) for 30 min. Poysky et al. [92] also reported that by using undiluted liquid smoke at the beginning

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of the process, the inactivation temperature could be decreased to as low as 138°F (58.9"C). Diluting liquid smoke to 50% reduced the effectiveness and increased the lethal temperature to 150°F (65.5"C). The oil soluble fraction of CharSol C-10, CharOil, was less effective and L. monocytogenes survived in samples processed to an internal temperature of 166°F (74.4"C), the highest temperature tested with this liquid smoke fraction. In these studies, preenrichment was used to enhance repair and recovery of injured cells. The processed product was stored at 5°C for 4 days and then preenriched in Trypticase Soy Broth for 6 h at 30°C before adding selective agents to UVM enrichment broth. Length of enrichment also played an important role in detection of survivors. Of the 245 samples tested, 57 that were negative for L. rnonocytogenes after 1 day at 30°C turned positive after 6-9 days of incubation. These studies of Poysky et al. [92] demonstrated that inactivation of L. monocytogenes is dependent on the interaction between heat and smoke. For best results, smoke should be applied before pellicle formation, since this pellicle on the fish surface serves to protect L. monocytogenes by limiting absorption of smoke.

ACKNOWLEDGMENTS The authors thank Cecilia Wolyniak (FDA, CFSAN), Pat Pinkerton (FDA, Seattle District), Stephanie Dalgliesh (FDA, Seattle District), and Daryl Thompson (FDA, Atlanta Regional Office) for retrieval and assistance with the FDA recall information. Maxine Heinitz (FDA, Midwest Laboratory for Microbiological Investigations, FDA Microbiological Information Systems Manager) and Jan Johnson (FDA, Seattle District) provided valuable assistance with accessing data from the FDA Microbiological Information System. Literature searches were conducted by Walter E. Hill and Jim Hungerford (FDA, Seattle District) for which we are grateful. We would also like to thank Walter E. Hill and Nancy Hill (FDA, Seattle District) for assistance with building and finalizing a data base for the reference list.

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Jinneman et al. trol. Proceedings of XI1 International Symposium on Problems of Listeriosis. Perth, Australia. Oct. 2-6. pp 397-401. Pace, J., C.Y. Wu, and T. Chai. 1988. Bacterial flora in pasteurized oysters after refrigerated storage. J. Food Sci. 53:325-327, 348. Pelroy, G.A., M.E. Peterson, P.J. Holland, andM.W. Eklund. 1994.Inhibition of Listeria monocytogenes in cold-process (smoked) salmon by sodium lactate. J. Food Prot. 57:108-113. Pelroy, G., M. Peterson, R. Paranjpye, J. Almond, and M. Eklund. 1994. Inhibition of Listeria rnonocytogenes in cold-process (smoked) salmon by sodium nitrite and packaging method. J. Food Prot. 57: 1 14- I 19. Peterson, M.E., G.A. Pelroy, R.N. Paranjpye, J.S. Almond, and M.W. Eklund. 1993. Parameters for control of Listeria rnonocytogenes in smoked fishery products: sodium chloride and packaging method. J. Food Prot. 56:938-943. Poysky, F.T., R.N. Paranjpye, M.E. Peterson, G.A. Pelroy, A.E. Guttman, and M.W. Eklund. 1997. Inactivation of Listeria rnonocytogenes on hot-smoked salmon by the interaction of heat and smoke or liquid smoke. J. Food Prot. 60:649-654. Ravomanana, D., N. Richard, and J.P. Rosec. 1993. Listeria spp. dans ded produits alimentaires-etude comparative de differents protocoles de recherche et d’une methode rapide par hybridation nucleique (Listeriu spp. in food products-a comparative study of some analytical methods including a rapid procedure by nucleic hybridization). Microbiol. Alim. Nutr. 11: 57-70. Rawles, D., G. Flick, M. Pierson, A. Diallo, R. Wittman, and R. Croonenberghs. 1995. Listeria rnonocytogenes occurrence and growth at refrigeration temperatures in fresh blue crab (Callinectes supidus) meat. J. Food Prot. 58:1219- 1221. Richmond, M. 1990. Report of the Committee on the Microbiological Safety of Food, HMSO pp. 133-137. Rorvik, L., and M. Yndestad. 1991. Listeria rnonocytogenes in foods in Norway. Int. J. Food Microbiol. 13:97-104. Rorvik, L.V., M. Yndestad, and E. Skjerve. 1991. Growth of Listeria monocytogenes in vacuum-packed, smoked salmon, during storage at 4°C. Int. J. Food Microbiol. 14: I 1 1-I 18. Rorvik, L.M., E. Skjerve, E.K. Nekstad, and M. Yndesat. 1995. Growth of Listeria rnonocytogenes on vacuum-packaged gravad salmon and in the presence of dill at 4°C. Proceedings of XI1 International Symposium on Problems of Listeriosis, Perth, Australia, Oct. 1-6, pp. 484. Ryu, C.-H., S. Igimi, S. Inoue, and S. Kumagai. 1992. The incidence of Listeria species in retail foods in Japan. Int. J. Food Microbiol. 16:157-160. Shineman, T.L., and M.A. Harrison. 1994. Growth of Listeria rnonocytogenes on different muscle tissues. J. Food Prot. 57: 1057- 1062. Simon, M.C., D.I. Gray, and N. Cook. 1996. DNA extraction and PCR methods for the detection of Listeria monocytogenes in cold-smoked salmon. Appl. Environ. Microbiol. 62322-824. Soontharanont, S., and C.D. Garland, 1995. The occurrence of Listeria in temperate aquatic habitats. Proceedings of XI1 International Symposium on Problems of Listeriosis. Perth, Australia, Oct. 2-6, pp. 145-146. Teufel, P. 1994. European perspectives on Listeria monocytogenes. Dairy Food Environ. Sanit. 14:212-214. Valenti, M., S. d’Aubert, L. Bucellati, and C. Cantoni. 1991. Listerie spp. in salmoni (Listeria spp. in salmons). Indust. Aliment. 30:74 1-743. Van Wagner, L.R. 1989. FDA takes action to combat seafood contamination. Food Proc. 5018- 12. Weagant, S.D., P.A. Sado, K.G. Colburn, J.D. Torkelson, F.A. Stanley, M.H. Krane, S.C. Shields, and C.F. Thayer. 1988. The incidence of Listeria species in frozen seafood products. J. Food Prot. 51:655-657. Wong, H.-C., W.-L. Chao, and S.-J. Lee. 1990. Incidence and characterization of Listeria rnonocytogenes in foods available in Taiwan. Appl. Environ. Microbiol. 56:3 101-3104.

16 Incidence and Behavior of Listeria monocytogenes in Products of Plant Origin ROBERTE. BRACKETT The University of Georgia, Griffin, Georgia

INTRODUCTION Use of adequate isolation procedures, enough time, and a little perseverance by investigators makes it possible to isolate Listeria spp., including L. rnonocytogenes, from most forms of animal life. A similar situation also exists with products of plant origin. Chapter 3 describes the apparent association between consumption of silage and occurrence of an illness resembling listeriosis in ruminants, which was observed as early as 1922; however, this link between silage consumption and listeriosis in domestic livestock was not confirmed until 1960 [43]. Although several papers published during the next 15-year period documented the presence of L. rnonocytogenes in vegetation grown primarily for consumption by animals [75-771 (see Chap. 2), scientists at the time were generally unconcerned about the incidence of listeriae in produce destined for human consumption, primarily since such products had not been positively linked to human listeriosis. In fact, the only instance in which listeriae were recovered from raw retail produce before the 1981 listeriosis outbreak in Canada involving coleslaw occurred in 1975 when successful isolation of three untypable Listeria strains from lettuce marketed in Brazil was reported [50]. Although 18 of 41 Canadians died of listeriosis in 1981 after consuming coleslaw from which L. rnonocytogenes was isolated and positively identified [69], it was not until 631

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632

the 1985 cheese-related listeriosis outbreak in California that researchers began to develop more than a passive interest in the public health significance of L. monocytogenes in vegetables, fruits, and other products of plant origin. Nevertheless, with the exception of one isolated listeriosis case in Finland involving homemade salted mushrooms [53] and a recent cluster of five cases traced to frozen broccoli and cauliflower in Texas [70], no additional cases of listeriosis have been positively linked to consumption of plant products produced in North America or elsewhere. This chapter is devoted to food products and it will specifically address the incidence of Listeria spp. in raw retail vegetables and fruits. As in earlier chapters, information concerning behavior of L. monocytogenes in fresh produce and related products (orange juice/serum, soy milk, pasta, beet pigment) will also be presented along with some possible means by which listeriae can be inactivated in some of these products.

INCIDENCE OF LISTERIA O N RAW VEGETABLES Unless fertilized with human and/or animal waste or irrigated with water containing such waste, raw vegetables (and fruits) normally should be free of most human and animal enteric pathogens. Although the presence of soilborne spore-forming organisms such as Clostridiurn perfringens and Bacillus cereus are of little consequence on raw vegetables, these organisms can pose potential health problems in cooked vegetables that have been held at inappropriate temperatures. Although fewer than 1 % of documented outbreaks of foodborne illness during the last 10 years have been associated with consumption of vegetables, such outbreaks are now beginning to occur with greater frequency. L. monocytogenes is among the foodborne pathogens most often associated with these foods. This may result, in part, from the variety of ways that L. monocytogenes can contaminate fresh vegetables, as was depicted [10,11] (Fig. 1 ) by Beuchat.

\

plants-

silage, feed

-

soil

(cross contamination)

meat, milk, eggs ’

FIGURE 1 Mechanism b y which fresh produce can become contaminated with pathogenic microorganisms and serve as vehicles of human disease. (Adapted from Ref. 11.)

Listeria monocytogenes in Products of Plant Origin

633

When one considers the enormous variety and quantity of produce being marketed annually, routine microbiological examination of raw vegetables (and fruits) seems highly impractical and probably unnecessary if good agricultural practices are used in growing crops along with acceptable hygienic practices while harvesting, packaging, and transporting raw produce to market. Although consumption of coleslaw prepared from contaminated cabbage was linked to a large Canadian outbreak of listeriosis in 1981 [68], in retrospect, it appears that this outbreak could have been easily avoided if the coleslaw manufacturer had realized that the cabbage farmer had fertilized the cabbage with sheep manure from a flock that was previously diagnosed as having listeriosis. However, van Renterghem et al. [74] suggested that L. rnonocytogenes dies quickly in fecal matter, and therefore animal manure may not be as important in the spread of L. monocytogenes as once thought. (The role of manure and sewage as vehicles for listeriae is discussed in Chapter 2. ) However, they were able to demonstrate that L. rnonocytogenes could be transferred from contaminated soil to vegetables. In these experiments, carrots and radishes were planted in soil which had been inoculated with L. rnonocytogenes (10’ CFU/g soil). They found that three of six radishes but none of the carrots grown in the inoculated soil contained L. monocytogenes. In spite of the Canadian outbreak, which resulted in 18 fatalities, it is not surprising that, unlike dairy, meat, poultry, and seafood, no surveillance and/or regulatory programs have been initiated until relatively recently to assess the incidence of listeriae in raw vegetables (or fruits) marketed in the United States, Canada, or elsewhere. Nevertheless, inadvertent isolation of L. rnonocytogenes from potato salad in April 1990 prompted a Virginia-based manufacturer to recall 5700 pounds of product that had been distributed in the southeastern United States [2]. During 1997 and the first half of 1998, six Class 1 recalls were issued for fresh frozen coconut [2c], hummus with red peppers and vegetables [2a,2d,2e], sprouts [2f], and potato salad [2b], the last of which involved over 5.5 million pounds of product. Although the risk of contracting listeriosis from such products is generally thought to be quite low, lack of routine microbiological analysis of raw produce should not be interpreted to mean that fresh vegetables and fruits will always be free of listeriae, including L. monocytogenes.

United States In response to heightened concern about foodborne listeriosis, several small surveys were conducted after the 1985 outbreak in California to determine the extent of Listeria contamination in raw fresh and frozen vegetables destined for human consumption. During 1986 and 1987, Petran et al. [65] used the U.S. Food and Drug Adniinistration (FDA) procedure in an attempt to isolate Listeria spp., including L. rnonocytogenes, from 23 retail samples of vegetables, including fresh beet peels, broccoli, cabbage (outer leaves), carrot peels, cauliflower sterns, corn husks, head lettuce, leaf lettuce, mushroom stems, potato peels, and spinach as well as frozen green beans, pea pods, green peas, and spinach. Using FDA and Centers for Disease Control and Prevention (CDC) procedures along with direct plating, officials at the CDC [3] tried to isolate listeriae from 22 samples of broccoli, carrots, celery, lettuce, green peppers, and potatoes in conjunction with several clusters of listeriosis cases in Los Angeles County, California, and Philadelphia, Pennsylvania. Finally, as part of a much larger survey dealing with the incidence of Listeria spp. in retail meat, poultry, and seafood products, Buchanan et al. 1231 used an MPN (most probable number) method to examine two samples of potato salad for listeriae. As already

634

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implied, no Listeria spp. were recovered from any samples examined in the three surveys just described. However, when one considers the small number of samples examined, these findings cannot assure consumers that these products will always be free of listeriae. In the first truly definitive survey reported, Heisick et al. [48] used the FDA procedure to determine the incidence of various Listeria spp., including L. monocytogenes, in 10 different varieties of raw unwashed vegetables (total of 1000 samples) obtained from two Minneapolis-area supermarkets between October 1987 and August 1988. As shown in Table 1, Listeria spp. were detected in one or more samples of cabbage, cucumbers, lettuce, mushrooms, potatoes, and radishes, but were never found in broccoli, carrots, cauliflower, or tomatoes. Although L. monocytogenes, L. innocua, L. welshimeri, and L. seeligeri were recovered from 5.0,2.6,0.8, and 1.3% of all raw produce examined, respectively, with 41 of 50 (82%) and 9 of 50 (18%) L. monocytogenes strains classified as serotypes l a and 4a/4ab, respectively, the overall incidence of Listeria spp. as well as L. monocytogenes was markedly higher in radishes and potatoes than in other types of vegetables. Given that carrots recently were shown to possess some inherent antilisterial activity [6,14,19,59,60], it appears that root crops such as potatoes and radishes more frequently carry viable listeriae than other vegetables because of their close association with soil. Interestingly, contamination rates for most raw vegetables were fairly consistent throughout the year, and this reinforces the belief that listeriae populations remain relatively constant in soil. These findings also are supported by those from a similar study in which Heisick et al. [48] used four procedures to ultimately identify Listeria spp. in 19 of 70 (27.1%) and 25 of 68 (36.8%) potato and radish samples, respectively, with no listeriae being detected in mushrooms, carrots, cabbage, broccoli, cauliflower, lettuce, tomatoes, or cucumbers obtained from the same two Minneapolis supermarkets. Use of an adequate isolation procedure and sufficient time to examine large numbers of samples, has made it clear that a small percentage of raw vegetables marketed in the United States is likely to harbor Listeria spp., including L. monocytogenes, with the incidence of this pathogen being highest in root crops. Hence, the inability to detect listeriae in raw vegetables examined in the three aforementioned surveys probably resulted because insufficient numbers of samples were examined. In support of this observation, Steinbruegge et al. [72] isolated L. monocytogenes from only 2 of 43 retail samples of head lettuce purchased in Nebraska. Although the occasional presence of listeriae in retail raw vegetables should not be viewed with alarm, careful handling and washing of all produce to be consumed raw is recommended, particularly for pregnant women, the elderly, and other individuals at greater than normal risk of developing listeriosis. Others in the United States have similarly found L. monocytogenes to be an infrequent contaminant of fresh vegetables. Lin et al. [57] determined occurrence of L. monocytogenes and other foodborne pathogens in vegetable salads served in 3 1 food service establishments in Florida. Of the 63 vegetable salad samples tested, only one was contaminated with L. monocytogenes. The vegetable salad from which the bacterium was isolated consisted of iceberg lettuce, red cabbage, carrots, cucumbers, and tomatoes. Interestingly, this salad and others yielding potentially pathogenic bacteria other than Listeria were purchased from only 5 of the 31 establishments. Moreover, several of these implicated facilities were apparently guilty of selling contaminated salads on more than one occasion, with contamination most likely a result of product mishandling by workers. The importance of proper sanitation and handling in minimizing L. monocytogenes contamination of salads and vegetables was mentioned by Harvey and Gilmour [47]. They suggested that systematic contamination of vegetable salads by L. monocytogenes was

Listeria monocytogenes in Products of Plant Origin

635

TABLE1 incidence of Various Listeria spp. in Unwashed Raw Retail Vegetables Marketed in the Minneapoiis, Minnesota, Area Between October 1987 and August 1988

Type of vegetable Broccoli Cabbage carrots Cauliflower Cucumbers Lettuce Mushrooms Potatoes Radishes Tomatoes Total Source: Adapted from Ref. 47

No. of samples analy zed

92 92 92 92 92 92 92 132 132 92 1,000

No. of positive samples (%)

L. rnonocytogenes 0

L. innocua

L. welshimeri

L. seeligeri

Total

0 2 (2.2) 0 0 28 (21.2) 19 (14.4) 0

0 0 0 0 5 (5.4) 1 (1.1) 11 (12.0) 5 (3.8) 4 (3.0) 0

0 0 0 0 2 (2.2) 0 0 1 (0.8) 5 (3.8) 0

0 1 (1.1) 0 0 0 0 0 0 12 (9.1) 0

0 2 (2.2) 0 0 9 (9.8) 1 (1.1) 1 1 (12.0) 34 (25.8) 40 (30.3) 0

50 (5.0)

26 (2.6)

8 (0.8)

13 (1.3)

97 (9.7)

1 (1.1) 0

636

Brackett

more likely a result of improper handling by food service workers rather than from natural contamination of the raw product.

Canada Despite the Canadian coleslaw outbreak of 1981, the incidence of listeriae in fresh produce has received relatively little attention in Canada, with only one formal Canadian publication on the subject presently recorded in the scientific literature. Using the original FDA procedure, Farber et al. [37] failed to recover any Listeria spp. from lettuce (50 samples), celery (30 samples), or tomatoes (20 samples) purchased in Ottawa during 1988. However, L. innocua was detected in 1 of 10 radish samples, which again suggests that the incidence of listeriae may be somewhat higher in root crops than in other vegetables.

Western Europe When the first edition of this book appeared in 1991, knowledge concerning the incidence of listeriae in raw vegetables marketed in western Europe was confined to a few scattered reports. Since then much more information has been published. An increase in the number of listeriosis cases in England, along with the possibility that some of these cases may have been food-related, prompted several surveys to determine the incidence of listeriae in various foods including dairy, meat, poultry and seafood products, raw vegetables, and prepackaged salads. Working at Cambridge, Sizmur and Walker [71] examined 10 different varieties of prepackaged salads obtained from two leading area supermarkets. Overall, L. monocytogenes serotype 1/2 was isolated from 4 of 60 (6.7%) samples, with L. monocytogenes serotype 4b also being present in one of these positive samples. Prepackaged salads from which the pathogen was recovered consisted of two varieties that contained either (a) cabbage, celery, sultanas, onions, and carrots or (b) lettuce, cucumbers, radishes, fennel, watercress, and leeks. Both of these salad varieties contained cabbage, cucumbers, and/or radishes-three of four raw vegetables from which L. monocytogenes (predominantly serotype la) was isolated in the United States (see Table 1). Although no Listeria spp. were recovered from plain bean sprout salads or those that contained nuts, possibly because of a low pH, L. innocua was detected in 13 of 60 (21.7%) samples representing five different varieties of mixed vegetables and/ or fruit salad. In addition to these findings, English investigators [41] also have isolated L. monocytogenes from coleslaw. Thus, raw salad vegetables can serve as a potential source of L. monocytogenes in the human diet. Bending and Strangeways 171 proposed that a 74-year-old postoperative patient in a London hospital may have acquired listerial septicemia and meningitis from consuming contaminated lettuce. Although different serotypes of L. monocytogenes (1/2a and 1/2c) were isolated from the patient and 1 of 11 (9.1 %) samples of washed English round lettuce, recovery of the pathogen from washed lettuce prepared in the hospital’s kitchen, but not from 44 other food samples examined, suggests that consumption of washed raw vegetables may pose a potential health threat to hospital patients, many of whom are debilitated and/or immunocompromised. Consumption of homemade uncooked salted mushrooms containing 1O6 L. monocytogenes serotype 4b CFU/g has been positively linked to a nonfatal case of listerial septicemia in an 80-year-old apparently healthy Finnish man [53] (see Chap. 10). (Working in the Netherlands, van Netten et al. [73] also isolated L. monocytogenes from 2 of 20 raw mushroom samples obtained from area markets.) During this investigation, low levels

Listeria monocytogenes in Products of Plant Origin

637

(<102CFU/g) of an unrelated L. monocytogenes strain belonging to serotype 1/2a also were detected in carrots that were stored in the same cow barn with the tainted mushrooms, thus making this the first account in which this pathogen has been recovered from raw carrots, a vegetable that reportedly possesses listericidal properties [6,14,19,59,60]. As was true for surveys in England, preliminary data from Switzerland indicated the presence of Listeria spp. in a small percentage of raw vegetable salads [21]. Although 27 raw vegetable samples obtained from a group of retail markets in Switzerland were free of listeriae, L. monocytogenes was detected in 3 of 64 (4.7%) raw salad (2 mixed salads and 1 parsley salad) obtained from the same group of markets. Similarly, L. innocua was recovered from 4 of 64 (6.3%) raw salads. These values are essentially the same as those reported 4 years later by Breer and Baumgarner [22] for German salads. Earlier reports documenting the presence of L. monocytogenes in fresh produce and the fact that this bacterium is ubiquitous in the environment has prompted Harvey and Gilmour [47] to conduct an extensive survey of foods in Northern Ireland. Their results were similar to previous reports in that low incidences of L. monocytogenes occurred in fresh salad vegetables (8.9%) and prepared salads (7.5%). However, the rates of contamination were not consistent. Vegetables and prepared salads obtained from one source never yielded Listeria, whereas one third of products obtained from a different source were contaminated. The authors used these results to suggest that, despite previous reports of L. monocytogenes contamination of vegetables, producing a Listeria-free product was possible. Several studies have been published reporting the incidence of L. rnonocytogenes in Spanish fresh produce. De S i m h et al. [31] surveyed 103 samples of raw vegetables and reported an overall listeriae contamination rate of 18%. Listeria monocytogenes was the most common Listeria species found (7.8%), followed by L. innocua (5.8%), L. welshimeri (2.9%), and L. seeligeri (1.9%). The most common serovars of L. monocytogenes isolated were 1/2a (62.5%), 1/2c (25%), and 4b (12.5%). More recently, Garcia-Gimeno et al. [39] also examined the prevalence and growth of L. monocytogenes in vegetable salads produced in Spain. Unlike other market sample surveys, these authors followed the presence and development of L. monocytogenes in vegetable salad containing lettuce (75%),carrot (15%), and red cabbage (10%) produced at a specific Spanish food processing plant. During processing, the individual ingredients were cut, washed in potable water containing 100 mg/L active chlorine, placed in barrier bags, and then stored or distributed. L. monocytogenes was isolated from 21 of the 70 samples examined. Isolates primarily belonged to serotype 3a, although 3b also was found. The authors pointed out that the number of positive samples was much greater than previously published [37,49,65,7I] but offered no explanations for the high percentages found. Most European surveys for the presence of listeriae in vegetables yielded similar results. Except for samples from Spain [31,39], the incidence of listeriae in European vegetables generally appears to be less than 10%. Moreover, the levels of L. monocytogenes are usually even lower.

Asia and the Middle East Although the presence of L. monocytogenes in food has received most attention in Europe and North America, scientists in the Middle East and in Asia also have conducted surveys for the bacterium. Wong et al. [79) were among the first to publish results of an extensive survey of foods, including a variety of vegetables, in Taiwan. They analyzed 49 different

Bracketf

638

vegetables and found L. monocytogenes present in 12.2% of vegetables sampled; the organism was only recovered from lettuce, Chinese cabbage, and green onions. On further characterization, all isolates obtained from vegetables were of serotypes other than 1 and 4. In contrast, more than 90% of isolates from turkey and beef were serotype 1, with these strains and those from seafood having greater hemolytic activity than isolates obtained from vegetables. Consequently, these authors hypothesized that characteristics of L. monocytogenes might be related to their food origins. A similar survey of occurrence of L. monocytogenes in foods sold in Tokyo was published by Ryu et al. in 1992 [66]. Their survey also included a variety of plant products, including fresh vegetables, potato salad, and pickled vegetables. L. monocytogenes was frequently isolated from meat (34%) and fish products (6.1%); however, no listeriae were recovered from vegetable products or from ready-to-eat vegetable foods such as fermented soybeans, cooked bamboo shoots, or coleslaw. Arumugaswamy et al. [4] surveyed various fresh and ready-to-eat foods in Malaysia and found the highest incidence of L. monocytogenes contamination yet reported. About 22% of leafy vegetables analyzed contained the bacterium, with similar proportions of bean cakes and peanut sauces also reported as being positive. Although these values are higher than those reported for other countries, they were lower when compared with other Malaysian vegetable products tested. Eighty percent of ready-to-eat cucumber slices and 80% of bean sprouts tested positive for L. monocytogenes. The authors suggested that a high percentage of positive samples may have resulted because many of the samples came from street vendors or small processors, which often employed less than desirable sanitation practices. They also suggested that the results indicate a need for health agencies to put greater priority on street-vended foods. Salamah [67] conducted an extensive survey for the presence of L. rnonocytogenes in various fresh market vegetables sold in Riyadh, Saudi Arabia. A summary of their results is given in Table 2. In general, all samples analyzed contained L. monocytogenes, with incidence rates varying from 1.3 to 16.3%. Salamah confirmed the observations of Heisick et al. [46] that root crops were more frequently contaminated with L. monocytogenes than vegetables grown above ground. Finally, Gohil et al. [42] examined 183 imported and locally grown vegetables in the United Arab Emirates for the presence of L. monocytogenes. Unlike other surveys,

TABLE 2 Presence of L. rnonocytogenes in Market Vegetables in Saudi Arabia No. of positive samples

Incidence

Vegetables

No. of samples examined

Cabbage Carrot Cucumber Lettuce Potato Radish

70 120 110 80 80 75

2 16 4 I 13 8

2.8 13.3 3.6 1.3 16.3 10.7

Source: Adapted from Ref. 67

(%)

Listeria monocytogenes in Products of Plant Origin

639

however, they were unable to detect any L. monocytogenes in vegetables. However, they isolated L. innocua from two samples each of imported and locally grown vegetables.

BEHAVIOR OF L. MONOCYTOG€N€S ON VEGETABLES Despite the 1981 listeriosis outbreak in Canada directly linked to consumption of contaminated coleslaw [69] and an earlier cluster of listeriosis cases in Massachusetts that appears to have been epidemiologically linked to raw celery, lettuce, and tomatoes, until recently scientists were generally unconcerned about behavior of L. monocytogenes in raw produce. This lack of concern probably existed because vegetables grown in modern industrialized nations only rarely have been associated with any type of bacterial foodborne illness. Furthermore, the source of contamination in the Canadian coleslaw outbreak was readily traced to a farmer who fertilized cabbage with untreated manure obtained from a flock of sheep that was previously diagnosed as having listeriosis. Consequently, most individuals in the scientific community probably viewed this outbreak as an isolated incident which could have been easily prevented if better agricultural practices had been followed in growing cabbage. The fast changing global market and increase in consumption of fresh produce has challenged the above assumptions. Many consumers do not realize that vegetables are frequently imported from other countries. Consequently, such products can sometimes be plagued with the same microbiological hazards as the countries in which they were grown. Hence, a more global perspective on potential microbiological hazards, including Listeria is warranted. Interest in L. monocytogenes as a serious foodborne pathogen increased in 1985 following reports from California that at least 40 individuals died after consuming contaminated Mexican-style cheese. This outbreak sparked a renewed interest in the Canadian coleslaw outbreak and also prompted a series of investigations to determine both the incidence and behavior of L. monocytogenes on raw vegetables including cabbage. Results from these studies will now be reviewed along with some data concerning thermal inactivation of L. rnonocytogenes on cabbage and the effect of modified atmospheric storage, chlorine, and lysozyme on growth and survival of this pathogen on various raw vegetables. As was true for meat, poultry, and seafood, behavior of listeriae on raw produce has become an active area of research. Hence, additional information regarding various means to inactivate Listeria (e.g., chlorine dioxide, ozone) on raw vegetables and particularly prepackaged refrigerated salads will probably be available in the near future.

Growth and Survival Coleslaw was the first vegetable that was directly linked to an actual outbreak of listeriosis in humans. Consequently, it is not surprising that growth and survival of L. monocytogenes in cabbage was initially investigated. In the first such study published in 1986, Beuchat et al. [ 181 determined behavior of L. monocytogenes strains Scott A (clinical isolate) and LCDC 81-861 (Canadian cabbage isolate) on inoculated samples of shredded raw and autoclaved (121"C/20 min) cabbage as well as in autoclaved (12l0C/15 min) salted and unsalted cabbage juice during extended storage at 5 and 30°C. According to the authors, both test strains exhibited similar patterns of behavior on sterile cabbage, with popu-

640

Brackett

lations decreasing from approximately 107to 1O4 or 1O5 CFU/g during 42 days of refrigerated storage. This apparent inability of L. monocytogenes to grow on heat-sterilized cabbage at 5°C suggests that heating either decreases the availability of essential nutrients or leads to development of toxic and/or inhibitory constituents in cabbage. In sharp contrast, L. monocytogenes competed well with the normal aerobic flora and lactic acid bacteria of raw cabbage, with Listeria populations increasing approximately 4 orders of magnitude on raw cabbage during the first 25 days of refrigerated storage (Fig. 2). Thereafter, numbers of listeriae failed to decrease appreciably on raw cabbage stored up to 64 days. Similar results also were observed in subsequent studies by Hao et al. [44] and Lovett et al. [58]. Thus, these findings demonstrate that L. rnonocytogenes can grow under conditions normally encountered during shipping and distribution of cabbage. Although both Listeria strains failed to grow in autoclaved cabbage juice containing >5% NaCl during 2 weeks of storage at 3OoC, L. monocytogenes increased to 106CFU/g in the aforementioned homemade salted mushrooms (-7.5% NaCl) during 5 months of cold storage [53]. Hence, behavior of listeriae in raw vegetables appears to be greatly affected by incubation temperature as well as the concentration of salt and various growth constituents [64]. Subsequently, Conner et al. [29] more closely examined the influence of temperature, NaCI, and pH on growth of L. monocytogenes in autoclaved (121"C/ I5 min) clarified and unclarified cabbage juice. As in the previous study, salt-free unclarified cabbage juice again was an excellent growth medium for L. monocytogenes, with initial populations of 104CFU/mL increasing to I09CFU/mL after 8 days of incubation at 30°C. Beyond 8 days, populations in cabbage juice containing low levels of salt decreased rapidly and viable cells were no longer detected after 20 days of incubation at 30°C. Growth rates of

-

-

I

0, 6-1

M

I

el

0

16

32

48

61

Days

FIGURE2 Behavior of L. monocytogenes (O), lactic acid bacteria (m), and total aerobic microorganisms (A)o n raw cabbage incubated at 5°C. (Adapted from Ref. 18.)

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both Listeria strains at 30°C decreased markedly in cabbage juice containing low levels of salt, with inactivation of strains Scott A and LCDC 81-861 occurring in the presence of 1 1 . 5 and 12.5% NaCI, respectively. As expected, behavior of both strains was strongly influenced by acid production, with the pH of samples in which growth had occurred decreasing from 5.6 to 5 4 . 3 after 8 days of incubation at 30°C. Although numbers of both Listeria strains failed to increase in salted and unsalted cabbage juice when the experiment was repeated at 5"C, populations remained relatively stable, generally decreasing only 10- to 100-fold during 70 days of refrigerated storage of cabbage juice containing 3.5-5.0% NaCI. Results from another study [28] suggest that viability of Listeria in similar samples of salted and unsalted cabbage juice can be reduced by adding extracts from several Chinese medicinal plants. Interestingly, growth patterns for L. monocytogenes differed dramatically in clarified cabbage juice, with both strains growing well at 30°C in the presence of up to 2% NaCl. Since similar changes were observed between pH and growth of listeriae in clarified and unclarified cabbage juice, these findings suggest that particulate matter in unclarified cabbage juice may be partly inhibitory to L. monocytogenes in the presence of salt. Using pH-adjusted unclarified cabbage juice, these researchers [25] further demonstrated that L. monocytogenes failed to initiate growth at pH values <5 when inoculated samples were incubated at 30°C. Although more acidic environments (pH 14.8) were lethal, complete inactivation of both Listeria strains did not occur until the pH was reduced to -4.1. When the incubation temperature was lowered to 5"C, L. monocytogenes populations gradually decreased in cabbage juice adjusted to pH (5.2, with the pathogen surviving >63, 49, <21, and <21 days in samples adjusted to pH values of 5.2, 5.0, 4.8, and 4.6, respectively. In contrast, numbers of Listeria remained relatively constant during extended cold storage of cabbage juice adjusted to pH values >5.2. Since inactivation rates were markedly slower at 5 than at 3OoC, it appears that lower temperatures help protect L. monocytogenes from the harmful effects of low pH, as noted in Chapter 6. Concern about behavior of L. rnonocytogenes in fresh produce has extended beyond cabbage and now includes an ever increasing variety of fresh salad vegetables. In 1988, Steinbruegge et al. [72] first reported results of a study which examined the ability of L. monocytogenes to survive and grow on inoculated ( 103- l O5 CFU/g) samples of washed retail head lettuce during storage in sealed and unsealed plastic bags at 5, 12, and 25°C. Although behavior of L. monocytogenes on lettuce was somewhat variable, the pathogen generally grew under conditions simulating proper refrigeration, normal handling, and ambient serving temperatures, with the pathogen increasing 1-4 orders of magnitude following 2 weeks of storage. Similar results were observed when inoculated samples of fresh lettuce juice were held at 5°C for 2 weeks. Salamah [67] later reported similar increases in lettuce juices held at 26°C and up to a 2-log increase at 4°C. Several more recent reports have appeared regarding the fate of L. monocytogenes on various types of salad greens. According to Carlin and Nguyen-the [24], butterhead lettuce (Lnctuca sativa L.) supported growth of L. monocytagenes better than did endive (Cichoriumendivia L.), but the bacterium was unable to grow on lamb's lettuce (Valerianella olitoria L.). In contrast, populations of total aerobic microorganisms were unaffected by salad type. The authors offered no hypothesis as to why lamb's lettuce failed to support growth of L. monocytogenes. Carlin et al. [25] followed up on their previous work by more closely examining several factors that affected growth of L. monocytogenes on endive, with emphasis on storage temperature, age, and quality of the endive leaves, role of epiphytic microflora,

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and the strains and initial concentration of L. monocytogenes present. Overall, the growth rate of L. monocytogenes was essentially the same as that of the natural aerobic microflora at 10 and 20°C but slower than the native microflora at 30 and 6°C. Furthermore, the bacterium grew faster when initially present at lower (10-1000 CFU/g) rather than at higher ( 105CFU/g) populations. In accord with earlier results of Beuchat and Brackett [ 151, Carlin et al. [25] detected no differences in growth among the various strains tested. Carlin and coworkers [26] subsequently investigated in detail the role of indigenous microflora on growth of L. monocytogenes in unsanitized endive leaves and on leaves treated with 10% hydrogen peroxide to reduce or eliminate the indigenous microflora. These investigators also challenged L. monocytogenes with individual strains of pseudomonads and Enterobacteriaceae isolated from endive. The authors observed that reducing the native microflora by disinfection resulted in higher populations of L. monocytogenes on endive leaves. Moreover, they also observed that high populations ( 106- 107 CFU/g) of some strains of indigenous microorganisms reduced growth of L. monocytogenes on endive. A complex mixture of various microorganisms isolated from endive completely inhibited growth of L. monocytogenes in a medium composed of endive leave exudate. In addition to lettuce, tomatoes are among the most popular ingredients in fresh salads. Beuchat and Brackett [16] demonstrated that tomatoes are not a good substrate for growth of L. monocytogenes, probably because of their acidity. Although some growth of Listeria was evident on whole tomatoes after 10-2 1 days of storage at 10 and particularly 21"C, the pathogen was inactivated in chopped tomatoes (-pH 4.1) held at these same temperatures. Additionally, when commercial tomato products were inoculated to contain 1O6 L. monocytogenes CFU/g, populations remained reasonably stable in tomato sauce and tomato juice during 14 days of storage at 21 and particulary 5°C. However, the pathogen survived only 4 and 8 days in samples of ketchup held at 21 and 5"C, respectively, with Listeria inactivation being attributed to higher levels of acetic acid in ketchup as compared with the other tomato products. Information regarding the fate of L. monocytogenes in or on other types of salad vegetables also is limited; however, results from several studies indicate that with the exception of raw carrots [ 14,221, fennel, red cabbage, and Savoy cabbage [22], and beets [2 11, this pathogen will grow and/or survive on most other types of fresh produce including asparagus [9], broccoli [9], cauliflower [9], corn [52], green beans [52], lettuce [15,52], certain types of cabbage [22,29], celery [21], potato juice [67], and radishes [58] during the normal refrigerated shelf life of the product. Gianfranceschi and Aureli [40] similarly noted that survival of L. monocytogenes in spinach was essentially unaffected by freezing at -50°C and extended storage at - 18°C. In addition, Sizmur and Walker [7 11 reported that L. monocytogenes populations in several naturally contaminated vegetable salads purchased from two supermarkets in England increased approximately twofold after 4 days of refrigerated storage. Given the apparent ability of L. monocytogenes to survive and/or grow on most raw salad vegetables and the possible presence of this pathogen on many types of raw produce, health officials need to consider raw vegetables as another possible source of listerial infections. The observation that L. monocytogenes can thrive in various fresh vegetables also led to questions regarding its growth and survival in products prepared from these vegetables. In 1995, Lee et al. [56] published a study on growth and survival of L. monocytogenes in kimchi, a traditional Korean fermented vegetable product. This product can be prepared from various ingredients, but the most common type contains Chinese cabbage and various

-

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flavoring agents such as red pepper, garlic, ginger, NaCl, and pickled seafood. These ingredients are mixed together and subjected to a natural lactic acid fermentation, with the product ultimately reaching a mildly acidic pH [4-51. The authors found that populations of L. monocytogenes Scott A increased during the first 2 days of kimchi fermentation but then decreased. Although the bacterium was eventually inactivated by kimchi ingredients and low pH, the pathogen still persisted after 10 days of fermentation. However, the authors concluded that kimchi could be safely produced by using ingredients of good microbiological quality.

Modified Atmosphere Storage The widespread practice of packaging and/or storing fresh produce in a modified atmosphere has led to dramatic increases in types of produce available to consumers and in their shelf life so that most fresh vegetables (and fruits) are now available throughout the year. However, given the occasional presence of L. monocytogenes on raw produce, the ability of this pathogen to multiply relatively rapidly under microaerobic conditions at refrigeration temperatures and the present popularity of modified-atmosphere packaging, legitimate questions have been raised about the safety of refrigerated produce during longterm storage with modified atmosphere [78]. In response to these concerns, Berrang et al. [9] investigated behavior of L. monocytogenes on inoculated ( 103- l O5 CFU/g) samples of fresh asparagus, broccoli, and cauliflower during extended refrigerated storage in glass jars containing (a) 15% 0 2 : 6 % c o 2 : 7 9 % N2, (b) 11% 0,: 10% C02:79% N2, (c) 18% 02:3% c o 2 : 7 9 % N2, or (d) air. L. monocytogenes behaved similarly on each vegetable when the product was stored in a modified atmosphere or air. All three vegetables supported growth of L. monocytogenes at 15"C, with the pathogen attaining populations of -106 to nearly 109/g when these products were first deemed to be unfit for human consumption 6-10 days after the start of incubation. Although storage in a modified atmosphere at 15°C did not appreciably affect growth of listeriae on any of the three vegetables examined, the ability of such storage conditions to increase the shelf life of these products by 2-4 days beyond that of the products packaged in air led to higher Listeria populations when these vegetables were first declared inedible by subjective evaluations. In contrast, only asparagus supported growth of L. monocytogenes at 4"C, with initial numbers being approximately 10- to 100fold higher at the end of the product's 21-day shelf life. However, numbers of listeriae remained relatively constant on broccoli and cauliflower during 21 days at 4°C regardless of the storage atmosphere. Since these findings and those of the previous study indicate that L. monocytogenes is basically unaffected by controlled-atmosphere storage, the extended shelf life gained with such storage conditions provides additional time for growth of this pathogen which, in turn, increases the public health hazard associated with consumption of' raw vegetables. Similarly, Garcia-Gimeno et al. [39] and Kalander et al. [54] observed that use of modified atmosphere with salads neither enhanced nor repressed growth of L. monocytogenes but that the extended shelflife afforded by modified-atmosphere packaging increased the risk of foodborne illness. Subsequently, Beuchat and Brackett observed that L. monocytogenes behaved similarly when inoculated samples of iceberg lettuce [ 151 and tomatoes [ 161 were stored at 5" or 10°C (lettuce) or 10" and 21°C (tomatoes) in 3% 02:97% N 2 or air. As with cabbage and asparagus, the pathogen grew on lettuce, reaching populations of 1ON- 1O9 CFU/g after 10 days of storage at IOOC, with only slight growth observed in identical samples held

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at 5°C. However, the bacterium only achieved populations of about 105-106 CFU/g in tomatoes regardless of storage atmosphere. The term modijied atmosphere usually refers to systems where atmosphere in which a product is stored is intentionally changed to the desired gas composition. One such example is vacuum packaging of produce. This action has the effect of reducing O2 and thereby slowing respiration and senescence. Aytac and Gorris IS] investigated the effect of a moderate vacuum on growth of L. monocytogenes in chicory endive and mung-bean sprouts; the organism responded differently depending on the product in question. Growth of L. monocytogenes at 5.6"C was enhanced by 400 mB of vacuum in the endive but was repressed in sprouts. The authors pointed out the need for additional barriers when such techniques are used to extend the shelf life of fresh vegetables. The atmosphere also can be changed as a result of metabolic processes of fresh fruits and vegetables. In this instance, gas-permeability characteristics of the packaging material can often drastically affect the atmosphere within the package and, consequently, the microflora in the food. Omary et al. [6 1 ] investigated the influence of packaging material on growth of L. rnonocytogenes in shredded cabbage packaged in films having oxygen transmission ratios (OTR) of 5.6, 1500, 4000, and 6000 cc 02/m2/24h. They found that the type of packaging material used had a significant effect on growth of L. innocua (as a substitute for L. monocytogenes) in cabbage. Populations of L. innocua decreased in all samples after 14 days of storage regardless of packaging film used. However, populations of L. innocua then increased 3.5 logs CFU/g in cabbage packaged in all but the film with the highest ORT. In that instance, populations only increased by about 2 logs CFU/g. In the latter sample, CO2 concentrations had equilibrated at near ambient concentrations, whereas concentrations reached from 30 to 90% when films of lower OTR were used. Although most publications to date indicate that only low populations of L. monocytogenes infrequently contaminate fresh produce, the chance of this pathogen multiplying in products with extended shelf life is significant. Therefore, it appears prudent for handlers of raw produce to store their product in a modified atmosphere and institute sanitation and quality control programs that will decrease the incidence of listeriae in incoming raw vegetables. It also may be necessary to shorten the marketable period for such products even though the food may appear to be acceptable.

Inactivation As afollow-up to the aforementionedstudies dealing with growth and survivalof L. monocytogenes in raw vegetables, scientists also examined different methods by which this pathogen can be eliminatedfrom raw vegetables. Although these methods, which will now be discussed, primarily involve use of heat, chlorine, and lysozyme, information concerning the effect of other methods such as ozone, chlorine dioxide, and irradiation should be forthcoming. In response to the 1981 listeriosis outbreak in Canada involving consumption of contaminated coleslaw, Beuchat et al. [ 181 investigated thermal inactivation of L. monocytogenes in cabbage juice. Flasks of sterile, clarified cabbage juice adjusted to pH 4.0, 4.6, and 5.6 were inoculated to contain approximately 4 X 106 L. monocytogenes CFU/mL; placed in a shaking water bath at 50, 52, 54, 56, or 58°C; and sampled for listeriae at 10-min intervals for up to 60 min. As expected, thermal inactivation rates for Listeria in cabbage juice at 50, 52, 54, and 56°C were faster at lower pH values, with D-values of 25, 14, 6.7, and 3.6 min at pH 4.6 as compared with D-values of 60, 34, 8.4, and 6.8 min at pH 5.6, respectively. No viable cells were detected in cabbage juice held at 58°C for

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10 min. Although inactivation rates were unaffected by addition of 1 or 2% NaCl to cabbage juice, sublethally injured cells were more sensitive to NaCl on a nonselective plating medium (Tryptic Soy Agar) than were uninjured cells. As shown by data in Figure 2, L. monorytogenes can multiply on raw cabbage during extended refrigerated storage; however, results from the study just discussed suggest that normal pasteurization treatments given to cabbage juice and sauerkraut are probably sufficient to eliminate any viable listeriae that may be present. Hence, unlike unfermented raw vegetables, the risk of contracting listeriosis from sauerkraut and other pasteurized fermented vegetable products appears to be minimal. Brackett 1201 investigated the possibility of using hypochlorite solutions to inactivate L. monocytogenes on the surface of Brussels sprouts. In this study, fresh retail Brussels sprouts were inoculated to contain 1O6 L. monocytogenes CFU/g, immersed in a hypochlorite solution containing 200 mg of chlorine/L, removed, air dried for 30 min, and examined for numbers of surviving listeriae. The procedure just described decreased populations of L. monocytogenes on Brussels sprouts approximately I 00-fold. However, since dipping inoculated Brussels sprouts in sterile chlorine demand-free water reduced the number of viable listeriae by approximately 10-fold, the author concluded that many cells were simply washed from the surface rather than inactivated by chlorine. In a followup study with fresh retail lettuce, Beuchat and Brackett [15] also found that L. monocytogenes was still present at levels of 1OS- 1O6 and I 07- 1 Ox CFU/g after extended storage at 5 and 10°C, respectively, regardless of whether or not the lettuce was pretreated with a sodium hypochlorite solution to reduce the population of naturally occurring microflora. Zhang and Farber [801 evaluated several sanitizers and sanitizer combinations, including sodium hypochlorite, chlorine dioxide, trisodium phosphate, and lactic and acetic acids, for inactivation of L. monocytogenes on lettuce and cabbage. Their results generally were similar to those of Brackett [ 181 in that disinfectants were ineffective at reducing populations of L. monocytogenes. None of the treatments tested provided more than a 1.7-log reduction and most were under a 1-log reduction. Trisodium phosphate had no effect on listeriae and surfactants actually reduced efficacy of sanitizers. As indicated in Chapter 6, hypochlorite can be used very effectively to inactivate L. monocytogenes in water supplies and on the surface of previously cleaned equipment. However, current evidence indicates that chlorine dips are relatively ineffective for eliminating L. monocytogenes from contaminated raw vegetables. After demonstrating that egg white lysozyme, a GRAS (generally recognized as safe) food additive, inhibited growth of several foodborne pathogens, including L. monocytogenes, in laboratory media and phosphate buffer [51], Hughey et al. [55] investigated the possibility of using this enzyme during refrigerated storage to inactivate L. monocytogenes on various retail vegetables, including fresh lettuce, cabbage, sweet corn, green beans, and carrots as well as previously frozen corn and green beans. In this study, 1.8-kg portions of coarsely shredded or cut fresh and thawed frozen vegetables were treated to contain 100 mg of lysozyme/kg of vegetables and/or 5 mM of ethylenediamine tetraacetic acid (EDTA), inoculated to contain 103-104L. monocytogenes CFU/g, mixed by hand, and examined for numbers of listeriae during extended storage at 5°C. Overall, lysozyme was fairly effective in decreasing populations of L. monocytogenes on the surface of fresh vegetables, particularly when used in conjunction with EDTA, which presumably facilitated cell lysis by increasing contact between lysozyme and peptidoglycan in the cell wall. Listericidal effects from the combined use of lysozyme and EDTA were most pronounced on lettuce, with the pathogen no longer being detected

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after 12 days of refrigerated storage (Fig. 3). Although lysozyme alone was listeriostatic, use of EDTA alone failed to prevent growth of L. monocytogenes, with the pathogen eventually attaining levels only slightly lower than those observed in untreated lettuce. Listeria behaved similarly on fresh green beans and sweet corn with two exceptions: (a) growth occurred on lysozyme-treated sweet corn and (b) combined use of lysozyme and EDTA never completely eliminated the pathogen from either product. Unlike fresh lettuce, green beans, and sweet corn, numbers of listeriae on EDTA-and lysozyme-treated raw cabbage increased during the first 20 days of refrigerated storage and then decreased 4-5 orders of magnitude during 28 days of incubation at 5°C. Although combined use of lysozyme and EDTA again was most listericidal, 41 days of refrigerated storage were required to rid this lettuce of listeriae. Unlike other fresh vegetables, the pathogen was eliminated within 9 days from untreated raw carrots as well as from those that were treated with lysozyme alone or in combination with EDTA (Fig. 4). Hence, these findings support the notion that carrots probably contain one or more naturally occurring listericidal substances [ 141. In contrast to fresh vegetables, numbers of listeriae remained relatively constant on previously frozen green beans and corn that were treated with lysozyme alone or in

"1

3i \

Control

0 EDTA

2i\ 0

FIGURE3

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15

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Behavior of L. rnonocytogenes on fresh lettuce treated with lysozyme (Lys) and EDTA. (Adapted from Ref. 58.)

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K 0

Control

FIGURE 4 Behavior of L. rnonocytogeneson fresh carrots treated with lysozyme (Lys) and EDTA. (Adapted from Ref. 58.) combination with EDTA. This apparent failure of lysozyme to inactivate listeriae on frozen vegetables may be related to loss of certain lysis-enhancing substances during processing of vegetables. These findings, together with the current use of lysozyme to prevent growth of gas-producing spore-forming bacteria in certain European cheeses, suggest that commercial use of lysozyme in combination with other previously discussed measures should help to inhibit Listeria and other foodborne pathogens on fresh vegetables. Various plant components have long been known to possess antimicrobial properties. Specifically, the essential oils of herbs and spices have been studied most extensively in this regard. Kim et al. [55] demonstrated the antilisterial activity of various essential oil components in vitro and suggested that these components might also be incorporated into foods as barriers to microbial growth. Hao and Brackett [45] and Hao et al. [46] tested this suggestion by determining the efficacy of plant extracts in inhibiting growth of L. monocytogenes in beef and chicken, respectively. They found that eugenol and pimento extracts significantly inhibited growth of the bacterium on cooked chicken breasts during refrigerated storage [46]. However, they also noted that the type of food in which extracts were used was important. Unlike chicken, none of the spice extracts tested effectively inhibited growth of L. monocytogenes in refrigerated, cooked beef [45]. Moreover, some extracts contributed strong odors and flavors which would need consumer approval if actually used for commercial products. As mentioned previously, carrots reportedly possess some inhibitory antilisterial factor(s). Beuchat and Brackett [ 141 were the first to document this when they attempted to artificially inoculate carrots by dipping in suspensions of L. monocytogenes. They noted that populations of the bacterium decreased on both raw whole and shredded carrots but not cooked carrots. Moreover, numbers of L. monocytogenes decreased in the inoculating

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suspensions after dipping of shredded carrots. Based on these results, they suggested that the antilisterial component(s) of carrots is heat sensitive and released when carrot tissue is damaged. The authors suggested that phytoalexin 6-methoxymellein might be one potential compound responsible for the antilisterial action. The results of Beuchat and Brackett were later confirmed by Nguyen-the and Lund [59], who also noted that maceration of carrots in a high-speed blender or in liquid nitrogen likewise destroyed the antilisterial activity. The obvious potential for using carrot juice or its antilisterial component(s) as a natural antimicrobial agent in foods prompted the same two groups of researchers to investigate the mechanism of antilisterial activity. Nguyen-the and Lund [60] found that the antilisterial effect of carrots was suppressed by anaerobiosis, thiol compounds, bovine serum, and the free-radical scavengers histidine and diazabenzocyclooctane. However, the activity was not affected by sodium ascorbate, propyl gallate, catalase, superoxide dismutase, or chelating agents but was enhanced by Tween 20. Despite these results, these authors were unable to determine a specific compound or compounds responsible for the antilisterial activity. Beuchat et al. [ 171 likewise attempted to characterize the antilisterial component(s) of carrots. They found that the lethal and inhibitory effects were greatest in the pH range of 5.0-6.4 and that the optimum concentration of carrot juice needed to inhibit L. monocytogenes growth was about 10%. In addition, they observed that NaCl at concentrations up to 5% protected the listeriae from the antimicrobial action of carrot juice, especially in 10% juice incubated at 5 or 12°C. Despite these observations, they were unable to identify the compound(s) responsible for antilisterial activity or predict the effectiveness of carrot juice as an antilisterial ingredient in food. However, Babic et al. [6] suggested that dodecanoic acid and methyl esters of dodecanoic and pentadecanoic acids identified in purified active extracts of carrots may be responsible for antimicrobial activity. Looking at the potential of using carrot juice as an antilisterial treatment for foods, Beuchat and Doyle [19] determined the influence of dipping shredded lettuce in 20 or 50% carrot juice or adding up to 10% carrot juice to Brie cheese and frankfurter homogenates. Overall, both concentrations of carrot juice significantly repressed growth of L. monocytogenesin shredded lettuce stored at 5 and 12°C but not at 20°C. It is also noteworthy that the carrot juice was rather specific in its activity in that it had no discernible effect on growth of other aerobic microorganisms. In contrast to lettuce, addition of carrot juice to Brie cheese was less effective and was completely ineffective in frankfurter homogenates. Hence, it appears that carrot juice may be of value as an antilisterial agent in some foods.

INCIDENCE OF LISTERIA IN FRUITS Unlike raw vegetables, information concerning the incidence of Listeria spp. on raw fruit is virtually nonexistent. The first documentation was a preliminary report [3] in which CDC officials failed to recover listeriae from seven fruit samples (e.g., cherries, pears, peach, avocado, and tomato) while investigating two clusters of presumed foodborne listeriosis in Los Angeles County, California, and Philadelphia, Pennsylvania. More recently, Schlech [68] mentioned that blueberries, strawberries, and nectarines were implicated in outbreaks of listeriosis. One of the few culture-confirmed cases of listeriosis resulting from consumption of a fruit product occurred in Italy. In this case, DNA fingerprint-

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ing was used to link consumption of pickled olives with a sporadic neonatal case of listeriosis [27]. Although scientific evidence is lacking, two observations, namely, (a) infrequent association between consumption of fruit and listeriosis and (b) most fruits grow well above ground and are therefore not subject to frequent contact with Listeria-contaminated soil or feces, lead one to speculate that the incidence of listeriae on fruit may well be as low or lower than that observed for raw vegetables. Given the probable low incidence of listeriae on raw fruits, it may seem somewhat surprising to learn that FDA officials prompted an Oregon firm to issue a Class I recall for over 500,000 flavored frozen ice and juice bars that were contaminated with L. monocytogenes during the latter stages of manufacture [ 11, which suggests postpasteurization contamination. Since raw milk also was routinely processed into frozen dairy products at this same facility, L. monocytogenes was most likely introduced into the factory environment through the raw milk supply rather than fruit juice. If this is true, then it follows that the incidence of listeriae in highly acidic fruit juice is likely to be extremely low. This view is further supported by results from a recent survey in which Parish and Higgins [62] failed to detect any Listeria spp. in 100 retail samples of reconstituted single-strength orange juice (pH 3.63-3.84) that were pasteurized at 30 geographically distinct dairy and nondairy facilities located across the United States and Canada.

BEHAVIOR OF L. MONOCYTOGENES IN FRUIT JUICES Since we currently lack appreciable information on the incidence of listeriae in raw fruits, it is not surprising that our knowledge of Listeria behavior in these products also is extremely limited. As of January 1997, results from only two studies dealing with viability of listeriae in orange serum and juice have appeared in the scientific literature. In 1989, Parish and Higgins [63] published data from the first of two studies in which orange serum was adjusted to pH values of 3.6-5.0 with hydrochloric acid, inoculated to contain -106 L. monocytogenes CFU/mL, and examined for numbers of listeriae during prolonged incubation at 4 and 30°C. As was true for cabbage juice [29], behavior of listeriae in orange serum also was markedly influenced by incubation temperature and pH. Overall, L. monocytogenes failed to grow in refrigerated orange serum adjusted to pH 5 4 . 6 and was completely eliminated from these samples after 18 to 70 days of storage, with lowest pH values proving most detrimental to survival of Listeria (Fig. 5). However, modest growth of listeriae was observed in orange serum samples at pH 5 , with the pathogen still being present at levels of 102-103CFU/mL in the two least acidic samples after 90 days of refrigerated storage. Incubation at 30°C led to Listeria increases of approximately 10- and 100-fold in orange serum samples adjusted to pH values of 4.8 and 5.0, respectively. As was true for unclarified cabbage juice [29], overall viability of listeriae again was greatly reduced by raising the incubation temperature, with the pathogen generally being eliminated from orange serum samples at pH 3.6-4.0 and 4.2-5.0 after 5 and 8 days of incubation at 3OoC, respectively. These same authors [62] subsequently used several enrichment procedures to determine viability of listeriae in single-strength reconstituted samples of commercial frozen concentrated orange juice that were inoculated to contain 1-- 10 L. monocytogenes CFU/ mL. Although numbers of inherent microorganisms (primarily lactic acid bacteria and yeast) increased from - 102to 10' CFU/mL after 4 weeks of incubation at 4OC, L. monocytogenes was eliminated from reconstituted frozen orange juice (average pH of 4.06) after

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20

30

40

50

60

70

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FIGURE5 Behavior of L. monocytogenes in pH-adjusted orange serum during extended incubation at 4°C.(Adapted from Ref. 63.) 42 days of refrigerated storage. These findings appear to be consistent with those from the previous study involving orange serum.

BEHAVIOR OF L. MONOCYTOG€N€S IN OTHER PRODUCTS OF PLANT ORIGIN Increased consumer demand for precooked, long shelf life, ready-to-eat foods containing a minimum of preservatives is making development of microbiologically safe products increasingly difficult. Not too many years ago, it was thought that no foodborne pathogen could multiply in properly refrigerated food. However, this belief has been proven invalid by emergence of L. monocytogenes and Yersinia enterocolitica, in particular, as causes of foodborne illness. Thus public health officials have become concerned about the safety of many cook-chilled and ready-to-eat foods of animal origin, including fermented and unfermented dairy products, luncheon meats, sausage, and precooked chicken as well as peel-and-eat shrimp. These concerns have since spread to products of plant origin, including soy milk, precooked delicatessen products such as ravioli (prepared in part from flour), and food colorants derived from red beets. Since there is an increased use of soy milk by individuals who cannot tolerate cow's milk, the ability of Listeria to proliferate in this product should be known. Hence, Ferguson and Shelef [38] inoculated commercially available pasteurized and sterile soy milk to contain 1 O2 or l O4 L. monocytogenes CFU/mL and incubated the products at 5 and 22°C. The pathogen attained maximum populations similar to those previously observed in cow's milk, reaching levels of 7 X 10'- 3 X 109and 8 X 10' CFU/mL of soy milks following 3 and 30 days of incubation at 5 and 22"C, respectively. Not surprising, generation times for L. monocytogenes in soy milk, 1.55 h at 22°C and 37.68 h at 4OC, are similar to those previously reported for the same strain of L. monocytogenes in cow's milk (see Chap. 10). No one has yet investigated the incidence of Listeria spp. on soybeans; however, since listeriae are relatively common in soil, the potential exists for these organisms to find their way into soy milk-processing factories and also into the finished product as a postpasteurization contaminant. If this is true, then rapid growth of L. monocytogenes in soy milk at

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refrigeration temperatures suggests that this product should not be overlooked as a possible vehicle of human listerial infections. Concern about behavior of Listeria in delicatessen products marketed in England and the United States prompted Beuchat and Brackett [13] to investigate viability and thermal inactivation of L. monocytogenes in commercially prepared meat, cheese, and egg ravioli purchased from Atlanta-area delicatessens. The growth portion of this study involved quantitation of L. monocytogenes in inoculated (- 104and 1 O6 CFU/g) samples of ravioli during 14 days of incubation at 5°C. For thermal inactivation tests, the three types of ravioli were inoculated to contain 3 X 105L. monocytogenes CFU/g, stored 0 or 9 days at 5OC, and then boiled up to 7 min using cooking procedures that might be practical in the home. Overall, numbers of viable listeriae decreased < 10-fold during the 9-day refrigerated shelf life of the three types of ravioli. Results of thermal inactivation studies indicated that normal cooking procedures (7 min of boiling) were adequate to destroy L. monocytogenes populations of 105CFU/g in all three types of ravioli regardless of whether or not ravioli was refrigerated 0 or 9 days before cooking. Although this study provides valuable information concerning behavior of Listeria in ravioli, there appears to be an urgent need for more work of this type to address the microbiological safety of precooked and/or ready-to-eat delicatessen products such as sandwiches, filled rolls, pizza, garlic bread, desserts, confectionery products, and chocolate, since work in England [41] and elsewhere has shown that all of these products can harbor L. monocytogenes. Increased use of plant-based food colorants prompted El-Gazzar and Marth [35] to investigate behavior of Listeria in a commercial aqueous extract from the red beet root (Beta vulgaris) to which vitamin C, citric acid, and sodium propionate ( 51.5%) were added as preservatives. As in their previous work with milk coagulants [33,34] and annatto colorants [32], samples of beet extract were inoculated to contain 103-107L. monocytogenes strain CA, V7, or Scott A CFU/mL and examined for numbers of survivors during prolonged storage at 7°C. Not surprisingly, the combined effect of a relatively low pH of 4.3-4.8 and sodium propionate prevented growth of listeriae in all samples of beet colorant. However, although 42-56 days of incubation at 7°C was sufficient to rid these extracts of 10'-104 strain CA CFU/mL, this strain was still detected in 56-day-old samples that contained larger initial populations. In contrast, strains V7 and Scott A were far more resistant to the listericidal action of beet extract, with both strains being recovered at levels of 10'-104 CFU/mL, depending on initial inoculum, following 56 days of storage. Hence, unlike highly alkaline annatto extracts in which L. monocytogenes was inactivated almost instantaneously (see Chap. 12), prolonged survival of listeriae in beet colorants makes it imperative that these extracts be processed and handled carefully to prevent their contamination with this pathogen. The sporadic nature of listeriosis suggests that L. monocytogenes can be an infrequent problem in unusual foods of plant origin as well as fruits and vegetables. Whereas this viewpoint is supported by one sporadic case of listeriosis in Canada that was linked to alfalfa tablets [30,36], the fact that L. monocytogenes can be present in virtually any ecological niche suggests that occasional cases of listeriosis are likely to be associated with unusual as well as common plant-based foods.

-

REFERENCES 1.

Anonymous. 1987. Frozen ice, juice and fudge bars recalled. FDA Enforcement Report, June 3,

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2. Anonymous. 1990. Chicken, potato salads recalled by Campbell unit due to Listeria. Food Chem. News 32(9):61. 2a. Anonymous. 1997. Hummus with roasted peppers recalled. FDA Enforcement Report, Aug. 13. 2b. Anonymous, 1997. Potato salad recalled. FDA Enforcement Report, Sept. 2. 2c. Anonymous. 1998. Fresh frozen coconut recalled. FDA Enforcement Report, Jan. 14. 2d. Anonymous. 1998. Hummus dips and salads recalled. FDA Enforcement Report, June 12. 2e. Anonymous. 1998. Vegetable hummus recalled. FDA Enforcement Report, April 29. 2f. Anonymous. 1998. Sprouts recalled. FDA Enforcement Report, Sept. 5. 3. Archer, D.L. 1988. Review of the latest FDA information on the presence of Listeria in foods. WHO Working Group on Foodborne Listeriosis. Geneva, Feb. 15-19. 4. Arumugaswamy, R.K., G.R.R. Ali, and S.N.B.A. Hamid. 1994. Prevalence of Listeria monocytogenes in foods in Malaysia. Int. J. Food Microbiol. 23:117-121. 5. Aytac, S.A., and L.G.M. Gorris. 1994. Survival of Aeromonas hydrophilu and Listeria monocytogenes on fresh vegetables stored under moderate vacuum. World J. Microbiol. Biotechnol. 10:670-672. 6. Babic, I.C., C. Nguyen-the, M.J. Amiot, and S. Aubert. 1994. Antimicrobial activity of shredded carrot extracts on food-borne bacteria and yeast. J. Appl. Bacteriol. 76:135-141. 7. Bendig, J.W.A., and J.E.M. Strangeways. 1989. Listeria in hospital lettuce. Lancet 1:616617. 8. Bennik, M.H.J., E.J. Smid, F.M. Rombouts, and L.G.M. Gorris. 1995. Growth of psychrotrophic foodborne pathogens in a solid surface model system under the influence of carbon dioxide and oxygen. Food Microbiol. 12509-5 19. 9. Berrang, M.E., R.E. Brackett, and L.R. Beuchat. 1989. Growth of Listeria monocytogenes on fresh vegetables stored under controlled atmosphere. J. Food Prot. 52:702-705. 10. Beuchat, L.R. 1996. Listeria monocytogenes: incidence on vegetables. Food Control 7(4/5): 223-228. 11. Beuchat, L.R. 1996. Pathogenic microorganisms associated with fresh produce. J. Food Prot. 59:204-216. 12. Beuchat, L.R., M.E. Berrang, and R.E. Brackett. 1990. Presence and public health implications of Listeria monocytogenes on vegetables. In A.J. Miller, J.L. Smith, and G.A. Somkuti. Foodborne Listeriosis. Elsevier, New York, pp. 175- 181. 13. Beuchat, L.R., and R.E. Brackett. 1989. Observations on survival and thermal inactivation of Listeria monocytogenes in ravioli. Lett. Appl. Microbiol. 8: 173- 175. 14. Beuchat, L.R., and R.E. Brackett. 1990. Inhibitory effects of carrots on Listeria monocytogenes. Appl. Environ. Microbiol. 56: 1734-1742. 15. Beuchat, L.R., and R.E. Brackett. 1990. Survival and growth of Listeria monocytogenes on lettuce as influenced by shredding, chlorine treatment, modified atmosphere packaging and temperature. J. Food Sci. 55:755-758, 870. 16. Beuchat, L.R., and R.E. Brackett. 1991. Behavior of Listeriu monocytogenes inoculated into raw tomatoes and processed tomato products. Appl. Environ. Microbiol. 57: 1367-1 37 1. 17. Beuchat, L.R., R.E. Brackett, and M.P. Doyle. 1994. Lethality of carrot juice to Listeria monocytogenes as affected by pH, sodium chloride and temperature. J. Food Prot. 57:470-474. 18. 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. 19. Beuchat, L.R., and M.P. Doyle. 1995. Survival and growth of Listeria monocytogenes in foods treated or supplemented with carrot juice. Food Microbiol, 12:73-80. 20. Brackett, R.E. 1987. Antimicrobial effect of chlorine on Listeria monocytogenes. J. Food Prot. 50~999-1003. 21. Breer, C. 1988. Occurrence of Listeria spp. in different foods. WHO Working Group on Foodborne Listeriosis, Geneva, Feb. 15- 19. 22. Breer, C., and A.A. Baumgartner. 1992. Vorkommen und Verhalten von Listeria monocyto-

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genes auf Salaten und Gemusen sowie in frischgepresten Gemusesaften. Archiv. Lebensmittelhyg. 43:97- 120. Buchanan, R.L., H.G. Stahl, M.M. Bencivengo, and F. del Corral. 1989. Comparison of lithium chloride-phenylethanol-moxalactamand modified Vogel Johnson agars for detection of Listeria spp. in retail-level meats, poultry and seafood. Appl. Environ. Microbiol. 55599-603. Carlin, F., and C. Nguyen-the. 1994. Fate of Listeria monocytogenes on four types of minimally processed green salads. Lett. Appl. Microbiol. 18:222-226. Carlin, F., C. Nguyen-the, and A. Abreu da Silva. 1995. Factors affecting the growth of Listeria monocytogenes on minimally processed fresh endive. J. Appl. Bacteriol. 778:636-646. Carlin, F., C. Nguyen-the, and C.E. Morris. 1996. Influence of background microflora on Listeria monocytogenes on minimally processed fresh broad-leaved endive (Cichorium endivia var. Zatifolia). J. Food Prot. 59:698-703. Casolari, C., R. Neglia, M. Malagoli, and U. Fabio. 1994. Foodborne sporadic neonatal listeriosis confirmed by DNA fingerprinting. Annual Meeting of the American Society of Microbiologists, Las Vegas, NV, May 23-27, p. 382, Abstr. P-77. Chung, K.-T., W.R. Thomasson, and C.D. Wu-Yuan. 1990. Growth inhibition of selected food-borne bacteria by plant extracts. J. Appl. Bacteriol, 69:498-503. 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. Czajka, J., and C.A. Bott. 1994. Verification of causal relationships between Listeria monocytogenes isolates implicated in food-borne outbreaks of listeriosis by randomly amplified DNA patterns. J. Clin. Microbiol. 32:1280- 1287. De S i m h , M., C. Tarrag6, and M.D. Ferrer. 1992. Incidence of Listeria monocytogenes in fresh foods in Barcelona (Spain). Int. J. Food Microbiol. 16:153-156. El-Gazzar, F.E., and E.H. Marth. 1989. Fate of Listeria monocytogenes in some food colorants and starter distillate. Lebensm. Wiss. Technol. 22:406-4 10. El-Gazzar, F.E., and E.H. Marth. 1989. Loss of viability by Listeria monocytogenes in commercial bovine-pepsin rennet extract. J. Dairy Sci. 72: 1098-1 102. El-Gazzar, F.E., and E.H. Marth. 1989. Loss of viability by Listeria monocytogenes in commercial microbial rennet. Milchwissenschaft 44:83-86. El-Gazzar, F.E., and E.H. Marth. 1991. Survival of Listeria monocytogenes in food colorant derived from red beets. J. Dairy Sci. 74:81-85. Farber, J.M., A.O. Carter, P.V. Varughese, F.E. Ashton, and E.P. Ewan. 1990. Listeriosis traced to the consumption of alfalfa tablets and soft cheese. N. Engl. J. Med. 322:338. 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. Ferguson, R.D., and L.A. Shelef. 1990. Growth of Listeria monocytogenes in soy milk. Food Microbiol. 7:49-52. Garcia-Gimeno, R.A., G. Zurera-Cosano, and M. Amaro-L6pez. 1996. Incidence, survival and growth of Listeria monocytogenes in ready-to-use-mixed vegetable salads in Spain. J. Food Safety 16:75-86. Gianfranceschi, M., and P. Aureli. 1996. Freezing and frozen storage on the survival of Listeria monocytogenes in different foods. Ital. J. Food Sci. 4:303-309. Gilbert, R.J. 1990. Personal communication. Gohil, V.S., M.A. Ahmed, R. Davies, and R.K. Robinson. 1985. Incidence of Listeria spp. in retail foods in the United Arab Emirates. J. Food Prot. 48:102-104. Gray, M.L. 1960. A possible link in the relationship between silage feeding and listeriosis. J. Am. Vet. Med. Assoc. 136:205-208. 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.

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45. Hao, Y .-Y., and R.E. Brackett. 1995. Efficacy of plant extracts and cultured whey of antagonistic organisms to inhibit Listeria monocytogenes in refrigerated, cooked poultry. Annual Meeting of American Society of Microbiology, Washington, DC, May 21-25, p 388., Abstr. P-37. 46. Hao, Y.-Y., R.E. Brackett, and M.P. Doyle. 1994. Efficacy of plant extracts to inhibit psychrotrophic pathogens in refrigerated, cooked beef. Annual Meeting of American Society of Microbiology, Las Vegas, NV, May 23-27, p 379., Abstr. P-58. 47. Harvey, J., and A. Gilmore. 1993. Occurrence and characteristics of Listeria in foods produced in Northern Ireland. Int. J. Food Microbiol. 19:193-205. 48. 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. 49. 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. 50. Hofer, E. 1975. Study of Listeria spp. on vegetables suitable for human consumption. VI Congress0 Brazil de Microbiologia, Salvador, July 27-3 I , Abstr. K- 1 1. Cited in Ralovich, B. 1984. Listeriosis Research, Present Situation and Perspective, Akadimiai Kiado, Budapest, p. 73. 51. Hughey, V.L., and E.A. Johnson. 1987. Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease. Appl. Environ. Microbiol. 53:2 1652170. 52. 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: 63 1-638. 53. Junttila, J., and M. Brander. 1989. Listeria monocytogenes septicemia associated with consumption of salted mushrooms. Scand. J. Infect. Dis. 21 :339-342. 54. Kallander, K.D., A.D. Hitchens, G.A. Lancette, J.A. Schmieg, G.R. Garcia, H.M. Solomon, and J.N. Sofos. 1991. Fate of Listeria monocytogenes in shredded cabbage stored at 5 and 25°C under a modified atmosphere. J. Food Prot. 54:302-304. 55. Kim, J.M., M.R. Marshall and C.-I. Wei. 1995. Antibacterial activity of some essential oil components against five foodborne pathogens. J. Agric. Food Chem. 43:2839-2845. 56. Lee, S.-H., M.K. Kim, and J.F. Frank. 1995. Growth of Listeria monocytogenes Scott A during kimchi fermentation and in the presence of kimchi ingredients. J. Food Prot. 58:12151218. 57. Lin, C.-M., S.Y. Fernando, and C.-i. Wei. 1996. Occurrence of Listeria monocytogenes, Salmonella spp., E. coli and E. coli 0157:H7 in vegetable salads. Food Control 7(3):135-140. 58. Lovett, J., D.W. Francis, and J.G. Bradshaw. 1988. Outgrowth of Listeria monocytogenes in foods. In A.J. Miller, J.L. Smith, and G.A. Somkuti. Foodborne Listeriosis. Elsevier, New York, pp. 183-187. 59. Nguyen-the, C., and B.M. Lund. 1991. The lethal effect of carrot on Listeria species. J. Appl. Bacteriol. 70:479-488. 60. Nguyen-the, C., and B.M. Lund. 1992. An investigation of the antibacterial effect of carrot on Listeria monocytogenes. J. Appl. Bacteriol. 73:23-30. 61. Omary, M.B., R.F. Testin, S.F. Barefoot, and J.W. Rushing. 1993. Packaging effects on growth of Listeria innocua in shredded cabbage. J. Food Sci. 58:623-626. 62. Parish, M.E., and D.P. Higgins. 1989. Extinction of Listeria monocytogenes in single-strength orange juice: Comparison of methods for detection in mixed populations. J. Food Safety 9: 267-277. 63. Parish, M.E., and D.P. Higgins. 1989. Survival of Listeria monocytogenes in low pH model broth systems. J. Food Prot. 52: 144-147. 64. Petran, R., and E. Zottola. 1989. A study of factors affecting growth and recovery of Listeria monocytogenes Scott A. J. Food Sci. 54:458-460.

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65. Petran, R.L., E.A. Zottola, and R.B. Gravani. 1988. Incidence of Listeria monocytogenes in market samples of fresh and frozen vegetables. J. Food Sci. 53: 1238-1240. 66. Ryu, C.-H., S. Igimi, S. Inoue, and S. Kumagai. The incidence of Listeria species in retail foods in Japan. Int. J. Food Microbiol. 16:157-160. 67. Salamah, A.A. 1993. Isolation of Yersinia enterocolitica and Listeria monocytogenes from fresh vegetables in Saudi Arabia and their growth behavior in some vegetable juices. J. Univ. Kuwait (Sci.) 20:283-290. 68. Schlech, W.F. 1996. Overview of listeriosis. Food Control 7: 183- 186. 69. Schlech, W.F., P.M. Lavigne, R.A. Bortolussi, A.C. Allen, E.V. Haldane, A.J. Wort, A.W. Hightower, S.E. Johnson, S.H. King, E.S. Nichols, and C.V. Broome. 1983. Epidemic listeriosis: evidence for transmission by food. N. Engl. J. Med. 308:203-206. 70. Simpson, D.M. 1996. Microbiology and epidemiology in foodborne disease outbreaks: the whys and why nots. J. Food Prot. 59:93-95. 71. Sizmur, K.I., and C.W. Walker. 1988. Listeria in prepackaged salads. Lancet i: 1167. 72. Steinbruegge, E.G., R.B. Maxcy, and M.B. Liewen. 1988. Fate of Listeria monocytogenes on ready to serve lettuce. J. Food Prot. 5 1596-599. 73. Van Netten, P., I. Perales, A. van de Moosdijk, G.D.W. Curtis, and D.A.A. Mossel. 1989. Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp. Int. J. Food Microbiol. 8:299-3 16. 74. 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 :211217. 75. Weis, J. 1975. The incidence of Listeria monocytogenes on plants and in soil. In M. Woodbine, ed. Problems of Listeriosis. Surrey, UK: Leicester University Press, pp. 61-65. 76. Weis, J., and H.P.R. Seeliger. 1975. Incidence of Listeria monocytogenes in nature. Appl. Microbiol. 30:29-32. 77. Welshimer, W.J. 1968. Isolation of Listeria monocytogenes from vegetation. J. Appl. Bacteri01. 95:300-303. 78. Willcox, F., P. Tobback, and M. Hendrickx. 1994. Microbial safety assurance of minimally processed vegetables by implementation of the hazard analysis critical control point (HACCP) system. Acta Aliment. 23:221-238. 79. Wong, H.-C., W.-L. Chao, and S.-J. Lee. 1990. Incidence and characterization of Listeria monocytogenes in foods available from Taiwan. Appl. Environ. Microbiol. 56:3 101-3 104. 80. Zhang, S., and J.M. Farber. 1996. The effects of various disinfectants against Listeria monocytogenes on fresh-cut vegetables. Food Microbiol. 13:31 1-321.

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17 Incidence and Control of Listeria in Food-Processing Facilities ROBERTGRAVANI Cornell University, Ithaca, New York

INTRODUCTION Overwhelming evidence indicates that when L. monocytogenes and other Listeria spp. are present in commercially processed foods, this happens primarily because the product was contaminated after processing rather than because these organisms survived heat treatments that normally render the product safe. This view is strongly supported by the lack of scientific evidence indicating that minimum required heat treatments given to dairy, meat, poultry, seafood, and other products are inadequate to inactivate levels of listeriae that might be reasonably expected to occur in such products before heat processing. Although L. monocytogenes is clearly more heat tolerant than most other non-spore-forming foodborne pathogens, to date, no recalls of commercially prepared, Listeria-contaminated products have been unequivocally linked to the inadequacy of minimum required heat treatments. However, the clearest indication that L. monocytogenes and other Listeria spp. enter commercially processed foods as postprocessing contaminants comes from the fact that apparently healthy, non-thermally injured cells have been routinely recovered from many thermally processed dairy, meat, poultry, and seafood products and that these organisms have been found in the working environments of virtually all processing facilities that have produced foods involved in Listeria-related recalls. L. monocytogenes is a particularly difficult organism to control in food-processing facilities [61d]. Refrigerated food plants, in particular, provide conditions which allow for 657

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L. rnonocytogenes ’ survival and growth. The organism can adhere to food-contact surfaces and form a biofilm or coating which impedes the effectiveness of sanitation procedures [4 lc]. The refrigerated, moist environment, coupled with organic soil deposition, allows L. rnonocytogenes to survive and grow. L. rnonocytogenes is also a frequent contaminant of raw materials used in processing plants, so there is constant reintroduction of the organism into the plant environment [41b]. To control this pathogen, every potential avenue of entry and cross contamination must be controlled. This final chapter has been specifically designed for plant managers, sanitation workers, and quality control/quality assurance personnel employed in the food industry. In keeping with the format of Chapters 11 through 16, results from recent American and European surveys concerning the incidence of listeriae in environments of dairy-, meat-, poultry-, seafood-, vegetable-, and fruit-processing facilities as well as household kitchens will be described first. This will be followed by some general guidelines for reducing levels of listeriae and other microbial contaminants in working areas that are common to virtually all food-processing facilities. The wide variations in microbial load and types of microorganisms present in similar raw and finished products manufactured at different facilities along with the fact that no two factories are exactly alike in terms of design, equipment, maintenance, product flow, sanitation practices and procedures, distribution patterns, and managerial policies suggest that a discussion of current cleaning and sanitation programs used at particular food-processing facilities would be of little benefit. Instead specific problem areas within processing plants such as pasteurizers, fillers, sausage peelers etc., associated with the manufacture of particular products will be identified. Then a brief discussion of how good manufacturing practices (GMPs), prerequisite programs, and the Hazard Analysis Critical Control Point (HACCP) programs can all be used to decrease sharply the microbial load in any food, thereby reducing the possibility of producing a product contaminated with L. rnonocytogenes or any other foodborne pathogen.

INCIDENCE OF LISTERIA SPP. IN VARIOUS TYPES OF FOOD-PROCESSING FACILITIES IN THE UNITED STATES Interest in the extent of Listeria contamination in various food-processing facilities is of recent origin, since L. rnonocytogenes was not identified as a serious foodborne pathogen until 41 cases of listeriosis in Canada, including 17 deaths, were linked to consumption of contaminated coleslaw in 1981. Despite further evidence 2 years later suggesting possible involvement of pasteurized milk in an outbreak of listeriosis in Massachusetts, public health officials in the United States and elsewhere did not yet regard the presence of L. rnonocytogenes in food as a major threat to public health. However, this situation changed dramatically in June of 1985 when up to 300 cases of listeriosis, including 40 deaths, were eventually linked to consumption of contaminated Mexican-style cheese in southern California. This listeriosis outbreak, along with America’s major outbreak of salmonellosis in which over 16,000 individuals in the Chicago area became ill during March and April of 1985 after consuming a particular brand of pasteurized milk contaminated with Salmonella typhirnuriurn [59], prompted U.S. Food and Drug Administration (FDA) officials to begin testing various types of domestic and imported cheese for Listeria. FDA officials also developed the Dairy Safety Initiative Program, which included microbiological surveillance of finished dairy products and the factory environment in which they were produced

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along with in-depth inspection of fluid milk factories and eventually all types of dairyprocessing facilities located throughout the United States. With the subsequent discovery of L. monocytogenes in cooked, ready-to-eat meat, poultry, and seafood products by FDA and U.S. Department of Agriculture (USDA) officials, manufacturers of foods other than dairy products also became concerned about the incidence of listeriae in their products and processing facilities. Once FDA and USDA officials announced their plans to review GMPs that were presumably being used by most American firms, the food industry launched a Herculean effort to identify Listeria spp. and eliminate such problems within the food-processing environment before governmental inspectors arrived. Considering the adverse publicity and potential monetary losses that could result from discovery of L. monocytogenes in the finished product and the factory environment, it is not surprising that very little information concerning the incidence of listeriae and other microbial contaminants in food (except Class I recalls) and food-processing facilities has been released to the scientific community. Hence, although vast amounts of data have been generated since 1985 by local, state, and federal government inspectors as well as private microbiological testing and consulting laboratories and the food manufacturers themselves, much of the information which now follows is either of a general nature describing particular niches within food-processing facilities from which listeriae have been isolated or consists of limited results from academic surveys which describe the actual incidence of Listeria contamination in a relatively small number of food-processing facilities.

Dairy-Processing Facilities Following several dairy-related outbreaks of salmonellosis and listeriosis, FDA officials in cooperation with state governments and the dairy industry intensified surveillance of various types of dairy-processing facilities under the Dairy Safety Initiative Program which began April 1, 1986 [57]. Under this program, state officials were requested to sponsor a series of statewide meetings to discuss foodborne illness associated with Grade A and non-Grade A dairy products and to intensify their surveillance and inspection efforts in dairy-processing facilities. Nationally, FDA officials vowed to (a) conduct intensified check ratings in every interstate milk shipment (IMS) milk pasteurization plant, (b) conduct similar inspections at non-Grade A (non-IMS) milk pasteurization plants, (c) initiate a microbiological surveillance program designed to detect pathogenic microorganisms in finished product (see Chaps. 11 and 12), (d) intensify and upgrade training and standardization practices for federal and state milk specialists, rating officers, and sanitarians, and (e) regularly prepare national reports which summarize the status of the United States dairy industry. In the first of these reports [4] covering the 6-month period from April to September 1986, 9 of 357 (2.5%) milk pasteurization factories examined produced various dairy products contaminated with L. monocytogenes. A subsequent report in February 1987 indicated generally similar contamination rates with 16 of 620 (2.6%) and 3 of 620 (0.48%) dairy-processing facilities manufacturing finished products containing L. monocytogenes and L. innocua, respectively [ 5 ] .Eight months later, FDA officials reported that 11 of 604 (1 3%)IMS and 18 of 412 (4.4%) non-IMS milk pasteurization factories had produced products contaminated with Listeria spp., principally L. rnonocytogenes [6]. Extensive follow-up efforts in milk-processing plants producing Listeria-contaminated products uncovered various defects in factory design and pasteurization equipment.

Gravani

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Nevertheless, FDA officials have maintained that listeriae entered these products as postpasteurization contaminants. This view is strongly supported by FDA’s success in isolating Listeria from numerous floor drains in processing and other areas, wooden (porous) walls, floors and ceilings, wooden pallets, external surfaces of milk cartons, and sweetwater (refrigerated water) from leaking pasteurizer plates. Although not clearly identified in FDA’s “list’ ’ of environmental samples that harbored Listeria, FDA officials [75] noted the following problem areas related to environmental, postpasteurization contamination of dairy products with listeriae: (a) improperly operating high-temperature short-time (HTST) and/or vat pasteurizers, (b) leaking and/or cracked storage tanks, jacketed vessels, and valves, (c) inadequate sanitizing regimens, (d) cross-connecting pipes which allow commingling of raw and pasteurized product, (e) use of contaminated rags and sponges, (f) exposure to contaminants in unfiltered air and condensate, (g) filling and packaging operations, (h) conveyor belts, (i) use of returned product and reclaiming operations, (i) walls, floors, and ceilings particularly in walk-in refrigerators, (k) formation of aerosols, (1) traffic patterns within the factory, (m) entrances and floor mats, and (n) personal cleanliness of employees and others in the factory. In reality, L. monocytogenes and other foodborne pathogens have been detected in environmental samples from many of these problem areas as indicated in the following surveys of dairy factories in California and Vermont. In response to these federal programs, officials of the Milk and Dairy Foods Control Branch of the California Department of Food and Agriculture published [38] results from a statewide survey in which 597 environmental samples were collected from 156 milkprocessing facilities during the first half of 1987 and analyzed for listeriae. Overall, Listeria spp. were identified in the working environment of 46 (29.5%) milk-processing facilities with 31 of these 46 (67.4%) Listeria-positive factories being contaminated with L. monocytogenes (Table 1). Furthermore, L. monocytogenes and other Listeria spp. were most frequently observed in factories producing fluid milk products followed by those that manufactured frozen dairy products (i.e., ice cream and novelty desserts) and cultured milk products (i.e., yogurt and cottage cheese), with lowest contamination rates being associated with production of miscellaneous products and cheese. In all likelihood, this unusually low incidence of listeriae in California cheese factories was a direct result of

TABLE 1 Incidence of Listeria in Various Types of Milk-Processing Facilities in California, January to July 1987

Type of facility Fluid milk Frozen milk products Cheese Cultured milk products Miscellaneous productsa Total

No. of facilities examined

L. rnonocytogenes

All Listeria spp.

63 30 41 9 13

19 (30.2) 7 (23.3) 2 (4.9) 2 (22.2) l b (7.7)

27 (42.9) 11 (36.7) 4 (9.8) 3 (33.3) lb (7.7)

156

31 (19.9)

46 (29.5)

No. (%) of positive facilities

Includes butter, nonfat dry milk, whey products, and condensed milk. Positive sample from a butter factory. Source: Adapted from Ref. 38. a

Listeria in Food-Processing Facilities

66 I

massive clean-up efforts that were instituted following the 1985 listeriosis outbreak in the Los Angeles area involving Mexican-style cheese. A comparison of the incidence of listeriae in different milk-processing areas and sample sites (Table 2) supports the widespread belief that listeriae most frequently enter products after rather than before pasteurization, with the prevalence of these organisms in the factory environment increasing as the product passes through processing, filling, packaging, and storage areas. This apparent movement of listeriae through milk-processing facilities is most readily seen in results obtained from sampling conveyor belts and floor drains. However, sporadic isolation of listeriae from condensate as well as wooden blocks, pallets, case dollies, and utility tables points to additional routes by which these organisms can be disseminated in dairy processing plants. Although the low incidence of listeriae in raw milk receiving rooms as compared to other areas of the factory may at first seem surprising, these findings most likely reflect difficulties encountered in adequately cleaning and sanitizing equipment in processing, filling, and packaging areas of factories rather than what could be interpreted as a near absence of listeriae in California raw milk. In an environmental survey of 39 frozen milk product plants in California, Walker et al. [77a] collected 922 samples and found 111 (12%) positive for Listeria spp. Listeria monocytogenes was the only species recovered from 5 (12.8%) plants and L. innocua was the only species recovered from 13 (33.3%) plants. Both species were isolated from 9 (23.1%) plants. The highest recovery rates of Listeria were found in the batch flavoring, freezing, ingredient blending, and packaginglfilling areas of plants surveyed. Working at the University of Vermont, Klausner and Donnelly [56] made a large-

TABLE 2 Incidence of Listeriae in Different Milk-Processing Areas and Sample Sites

Facility working area and sample site Raw milk receiving room Drain Condensate Othei Processing room Drain Condensate Other" Filling/Packaging room Drain Condensate Conveyor Othei Cold storage room Drain Condensate Conveyor

No. of samples examined

No. (%) of positive samples

L. monocytogenes

,411 Listeria spp. I (3.3) 1 (4.5) 0

30 32 1

1 (3.3) 0 0

150 76 21

4 (2.7) 1 (1.3) 3 (14.3)

14 (9.3) 3 (3.9) 6 (28.6)

60 36 15 10

7 (11.7) 1 (2.8) 5 (33.3) 0

12 (20.0) 1 (2.8) 7 (20.0) 2 (20.0)

105 44 14

12 (11.4) 0 4 (28.6)

17 (16.2) 1 (2.3) 9 (64.3)

a Includes wooden blocks, pallets, case dollies, and utility tables. Source: Adapted from Ref. 38.

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scale survey to identify sources of Listeria (and Yersinia) contamination in fluid milk-, cheese-, and non-cheese-processing facilities. Overall 66.7, 9.5, and 23.8% of samples collected from floors and other non-food-contact surfaces at 34 fluid milk, cheese, and non-cheese factories were positive for Listeria spp., with L. rnonocytogenes and L. innocua being identified in 1.4 and 16.1%, respectively, of 361 samples examined. As expected, the percentage of Listeria-positive samples was higher among those from floors (12.0-27.9%) than from other non-food-contact surfaces (8.1%) (Table 3) and wet (85.7%) rather than dry (14.3%) areas. According to these investigators, paper filler beds, whey drainage pans on cheese presses, and case-washing areas were particularly prone to contamination with Listeria and Yersinia. The fact that 20.9% of all positive samples contained both Listeria and Yersinia suggests that yersiniae might be somewhat useful as a potential indicator of Listeria contamination within the dairy-processing environment. As noted, L. rnonocytogenes, Yersinia spp., and most other foodborne pathogens are more commonly found in wet than dry processing areas. However, the fact that listeriae (a) were recovered from whey drainage pans, (b) were routinely shed in whey during cheese-making experiments, (c) grew in samples of refrigerated milk and whey, and (d) survived the typical spray-drying process used to manufacture nonfat dry milk suggests that these organisms should be of concern to manufacturers of dry dairy products. In the light of these concerns, Gabis et al. [44] determined the incidence of Listeria in the working environment of 18 dry milk- and whey-processing facilities throughout the United States. The authors supplied environmental sampling kits containing sterile cellulose sponges, fabric-tipped swabs, and other necessities to all firms participating in the survey along with instructions as to how and where to collect samples. All samples were then sent to a central laboratory and within 48 h of collection were analyzed for listeriae according to the FDA procedure. Overall, only 2 of 410 (0.24%) samples examined were positive for Listeria spp., with L. monocytogenes and a species other than L. rnonocytogenes being isolated from floor drains in a raw milk receiving area and from a composite sample from several floor drains and trenches in a powder production area, respectively (Table 4). Allowing factory employees to choose specific sampling sites as well as the number of samples to be analyzed may have somewhat biased these results;

TABLE 3 Incidence of Listeria and Yersinia on Floors and Non-

Food-Contact Surfaces of 34 Fluid Milk, Cheese, and Non-Cheese Factories i n Vermont

Type of sample

Floor areas Coolers Processing Entrances MatdFootbaths Other areas Non-food-contact surfaces Total

No. of samples analyzed

No. (%) of positive samples

Listeria

Yersinia

43 117 64 25 38 74

12 (27.9) 21 (17.9) 1 1 (17.2) 3 (12.0) 10 (26.3) 6 (8.1)

9 (20.9) 14 (12.0) 18 (27.7) 1 (4.0) 6 (15.8) 4 (5.4)

36 1

63 (17.5)

52 (14.4) ~~

Source: Adapted from Ref. 56.

~

Listeria in Food-Processing Facilities

663

TABLE4 Incidence of Listeria in the Working Environment of 18 Nonfat Dry Milk- and Whey-Processing Facilities Located Throughout the United States

Work area Raw milk receiving Wet processing Cheese factory Whey factory Dryer room Bagging room Heating, ventilating and air conditioning system Miscellaneous Total

No. of samples analyzed

L. monocytogenes

Other Listeria spp.

62 151 22 23 38 27 53

1 (1.6) 0 0 0 0 0 0

0 0 0 1 (4.3) 0 0 0

0

0

1 (0.24)

I (0.24)

34 410

No. (%) of positive samples

Source: Adapted from Ref. 44.

however, the incidence of listeriae and hence the risk of postprocessing contamination appears to be many times lower in dry rather than wet dairy-processing facilities. Nevertheless, since manufacturers of nonfat dry milk and dry whey are not immune to the Listeria problem, they should take appropriate action to eliminate this organism from the processing environment, thereby greatly reducing the chance of producing a contaminated product. In a follow-up study, Pritchard et al. [67a] sampled 30 dairy processing plants in Vermont. Of the 346 sites tested, 122 (35.3%) contained one or more species of Listeria. Coolers and freezers had the highest rate, with 14 of 30 sites (46.7%) being positive for Listeria (Table 5). Other sites that resulted in high positive rates included dry storage areas (39.6%) and raw milk receiving and storage areas (39.4%). Pritchard et al. [67a]

TABLE5 Evaluation of Listeria Species Isolates Based on Area of Processing Plant Area designation

No. of sites

No. of positive

% Positive

Processing Entrances to processing Entrances not to processing Raw milk receiving/storage Coolers and freezers Dry storage Othera

145 53 37 33 30 16 31

52 21 11 13 14 7 4

35.9 39.6 29.7 39.4 46.7 43.8 12.9

Total

346

122

--

Includes areas such as common hallways, testing laboratories, and wheels of forklifts. Source: Adapted from Ref. 67a.

a

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also noted that plants producing dairy ingredients, frozen milk products or fluid milk, had significantly higher incidence rates of Listeria than expected. Facilities producing cultured dairy foods or a combination of cultured dairy foods and fluid milk were found to have significantly lower incidence rates of Listeria than expected. These researchers also observed that when dairy farms were contiguous to the processing facilities, these plants were more likely to be contaminated than plants without on-site dairy farms.

Meat-Processing Facilities Unlike dairy-processing facilities in which raw milk is pumped into the factory, pasteurized, and then either packaged immediately or pumped to closed vats for processing into cream, butter, ice cream, cheese, or other dairy products, meat-processing factories are in actuality open-air disassembly line operations in which animals are slaughtered, eviscerated, and broken down to obtain various cuts of meat, hides for leather, and other items of commercial value. Considering that domestic cattle, sheep, and pigs frequently shed L. monocytogenes asymptomatically in fecal material, it is not surprising that surveys have shown this pathogen to be not only ubiquitous but also endemic to slaughterhouses and meat-packing facilities. Initiation of the USDA-FSIS testing program for listeriae in cooked and ready-toeat meat products in September of 1987 (see Chap. 13) prompted immediate action by the meat industry. However, even before government testing began, meat processors became concerned about the incidence of listeriae in the working environment. In June 1987, results from a large-scale survey were reported in which nearly 2300 environmental samples were collected from over 40 meat-processing facilities nationwide and analyzed for listeriae [9]. Fourteen processing areas within these factories yielded evidence of being contaminated with L. monocytogenes or other Listeria spp. Overall, listeriae were recovered from -21% of all environmental samples examined. (These results also compare favorably with those of a much smaller survey [15] in which Listeria spp. were detected in 9 of 27 (33%) meat-processing environmental samples.) Problem areas in which 220% of the samples were positive included drains, trenches, floors, exhaust hoods, cleaning aids (sponges, brooms, hoses, and mops), product-contact surfaces (peelers, conveyors, and slicers), and wash areas. Sampling of surfaces in contact with sliced luncheon meat revealed Listeria contamination rates of 9.3, 32.3, and 23.6% before, during, and after production, respectively. Similarly, listeriae were recovered from 2.8, 14.5, and 25.5%, respectively, of food-contact surfaces examined before, during, and after production of frankfurters. From September 1987 to October 1991, USDA-FSIS inspectors sampled over 15,000 processed meat products, including cooked beef, sliced hams from cans, cooked sausage, jerky, cooked poultry, salads and spreads, and imported meats [74a]. The overall incidence of L. monocytogenes during this sampling period was 1.6%, with 235 products testing positive for L. monocytogenes This led to 25 recalls of product from the market during 1989-1991 [74a]. Data from the USDA’s microbiological monitoring of over 13,000 lots of ready-toeat meat products, from January 1, 1993 to September 30, 1997, indicated an incidence of 2.9% of the lots testing positive for L. monocytogenes (Levine, personal communication 1998). Results from a large-scale 1987 survey sponsored by the American Meat Institute [2,15] support the notion that Listeria spp. are widely distributed within the environment of many meat-processing facilities, and as in the earlier study by Flowers [9], also point

Lister ia in Food-Processing Facilities

665

to floors, drains, cleaning aids, wash areas, sausage peelers, and food-contact surfaces as significant problem areas, with between 20 and 37% of such samples harboring listeriae (Table 6). With the identification of listeriae in condensate and compressed air and on walls and ceilings, there can be no doubt that these organisms are ubiquitous in at least some meat-processing facilities. Recognizing the potential opportunity for Listeria to contaminate meat during packaging, one major manufacturer of processed meat products attempted to obtain “nearoperating room conditions” in its packaging room by cleaning the area for 3 days and then fogging the entire packaging room with 200 ppm quaternary ammonium compound [8,9]. In spite of these efforts, listeriae were still detected in 1 of 19 environmental samples obtained from the packaging room. After this exercise, the firm packaged processed meat products in this room over a 2-week period. Despite adherence to normal cleaning and sanitizing procedures at the end of each working day, the overall incidence of listeriae in the packaging room increased, with 3 of 20 (15%), 6 of 20 (30%), and 8 of 20 (40%) samples being positive for Listeria spp. 3,6, and 8 days after the room was initially cleaned and fogged, respectively. Owing to the increased concern for L. monocytogenes in meat products, there has been a concerted effort to minimize the risk of postprocess contamination during the production of processed meats. In one study, cited by Tompkin et al. [74a], swab samples were collected from packaging lines and floors where exposed ready-to-eat product was transported, chilled, stored, or packaged. The incidence of Listeria at these locations, from August 1989 to January 1992, is summarized in Figure 1. The overall trend is toward improved control of Listeria with fewer positive samples being evident following the inception of a Listeria control program. The results show a strong seasonal effect for the presence of Listeria in the finished product environments, with fewer positive samples being detected during the winter months. In addition, Tompkin et al. [74a] also reported results of a three-year study in which about 100 packaging lines were tested for Listeria (Table 7). The percentage of Listeria-negative samples increased from 44 in 1989 to 64 in 1991. The percentage of lines that exceeded the company’s established criterion of 5% of samples positive for Listeria decreased from 29 in 1989 to 13 in 1991. As improvements were made, attention was given to chronically positive lines. Examples of contaminated

TABLE 6 Incidence of Listeria spp. in

Post-Heat-Processing Areas of 41 Meat Factories Examined in the United States During 1987

Area Floors Drains Cleaning aids Wash areas Sausage peelers Food contact surfaces Condensate Walls and ceilings Compressed air Source: Adapted from Ref. 2.

Positive samples (%)

37 37 24 24 22 20 7 5 4

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666

30 25 %

20

P 0

I

T I

v

E

15

10

5

n

D

J

E C

U N

D

J

D

J

D

E C

U N

E C

U N

E C

1989

1990

1991

FIGURE1 Incidence of Listeria on packaging lines and in the environment (floors) from August 1989 to January 1992 inclusive. (From Ref. 74a.) sites on packaging lines included hollow rollers for conveyors, on/off valves and switches, rubber seals around doors, fibrous conveyor belts, and areas of equipment which were inaccessible to cleaning. The authors also noted that occasional lapses in cleaning and sanitizing procedures resulted in a fairly rapid loss of control. They observed that a certain sequence of events can lead to periodic contamination of packaging lines. The floor is particularly difficult to render Listeria-negative, and this situation provides a ready source of organisms to contaminate the packaging line during production or while cleaning, allowing the establishment of sites for microbial multiplication. This sequence of events can be prevented by striving for Listeria-negative floors, effectively cleaning and sanitizing the packaging lines at the end of each day’s production, eliminating inaccessible sites in the equipment, and by providing adequate preventive maintenance of the equipment.

TABLE 7 Listeria Contamination on Packing Lines from 1989 to 1991 % of Lines positive for Listeria at

Year

No. of lines

0%

15%

>5%a

1989 I990 1991

96 I06 97

44 59 64

27 27 23

29 14 13

Exceeds company guideline. Source: Adapted from Ref. 74a.

a

Listeria in Food-Processing Facilities

667

The authors summarized their report with the comment, ". . . for the present, it must be concluded that existing technology cannot eliminate Listeria from the cooked product environment of processing plants." Since Listeria spp., including L. monocytogenes, have been found in up to 50% of raw beef, pork, and lamb marketed in the United States, complete elimination of listeriae from meat-processing environments appears highly improbable. However, the American Meat Institute has developed a series of interim guidelines [2], which, if followed, will reduce the incidence of listeriae and decrease the overall microbial load in the working environment. A detailed description of these guidelines appears later in this chapter.

Poultry-Processing Facilities Reports have shown that up to 50% of all raw poultry sold in the United States contains various Listeria spp., including L. monocytogenes, with fecal material from infected flocks cited most frequently as the source of contamination. Unfortunately, information concerning the incidence of listeriae in American poultry-processing facilities is presently limited to results from two California surveys. In these studies, researchers at the University of California-Davis investigated the prevalence of listeriae in processing samples from one chicken [46] and one turkey slaughterhouse [47] during three or four separate visits. According to these investigators, no Listeria spp. were isolated from feathers, incoming chiller water, or scalding water, the latter of which aids in feather removal (Table 8). Nonetheless, L. monocytogenes and L. innocua were identified in samples of overflow chiller water and feather picker drip water obtained from the chicken slaughterhouse, with both organisms being detected in recycled water used to clean gutting equipment. Incidence rates for L. monocytogenes in chicken- and turkey-processing facilities were generally similar, with the percentage of Listeria-positive samples increasing approximately 2- to 2.5-fold during the latter stages of processing. However, L. welshimeri and L. innocua were absent from most chicken- and turkey-processing samples, respectively. Although only two poultry slaughterhouses were examined in this survey, inability of these researchers routinely to detect L. welshimeri in fresh chicken meat and L. innocua in fresh turkey meat processed at these facilities suggests that L. welshimeri and L. innocua might be able preferentially to colonize the gastrointestinal tract of turkeys and chickens, respectively. These findings, along with the ability of these investigators to further demonstrate an increasing incidence of Listeria spp. on the gloves and hands of poultry workers from the beginning to the end of processing (Table 9) confirms that these contaminants move along the processing line with the raw product. Unfortunately, neither the USDA nor the poultry industry have released any data regarding the incidence of listeriae within the general working environment of poultryprocessing facilities. However, considering the fecal carriage rate for listeriae in domestic birds, the current assembly line methods for processing poultry, and the fact that Listeria spp. (including L. monocytogenes) and salmonellae have be.en isolated from up to about half of all raw chickens marketed in the United States, one can speculate that the poultry and meat industries face similar problems in controlling the spread of listeriae and other organisms in the work environment. If one draws a parallel between methods used to process meat and poultry, then floors, drains, cleaning aids, wash areas, and food-contact surfaces emerge as likely niches for Listeria spp., including I,. monocytogenes, in poultryprocessing facilities. These predictions may be supported by published scientific data in the future.

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668

TABLE 8 Incidence of Listeria spp. in One Chicken and One Turkey Slaughterhouse in California No. of chickenhurkey slaughterhouse samples analyzed

Sample ~~~~~~~

Scalding water overflow Feather picker drip water Incoming chiller water Overflow chiller water Recycled water for cleaning gutters Source: Adapted from Refs. 46 and 47.

~

16/15 16/15 16/0 16/15 16/15

~

No. (%) of positive samples

L. monocytogenes ~

~

o/o

0/1 (6.7)

o/o

2 (12.5)/0 1 (6.3)/2 (13.3)

L. innocua

o/o

3 (18.8)/0

o/o

010 5 (31.3)/0

L. welshimeri

o/o

0/1 (6.7)

o/o

0/1 (6.7) 0/3 (20.0)

Total

010 3 (18.8)/2 (13.3) 010 2 (12.5)/1 (6.7) 6 (37.5)/5 (33.3)

Listeria in Food-Processing Facilities

669

TABLE 9 Incidence of L. rnonocytogenes and L. innocua on the Hands and Gloves of Poultry Meat Processors Assigned to Three Different Stations in a Slaughterhouse

Sample

No. of chickedturkey slaughterhouse samples analyzed

L. monocytogenes

L. innocua

L. welshimeri

Total

20/30 11/30 44/30

2 (10.0)/3 (10.0) 4 (36.4)/3 (10.0) 20 (45.5)/5 (16.7)

2 (10.0)/0 1 (9.1)/0 1 1 (25.0)/0

012 (6.7) 0/7 (23.3) 0/7 (23.3)

4 (20.0)/5 (16.7) 5 (45.5)/10 (33.3) 31 (70.5)/12 (40.0)

Postchilling handlers Leg/wing cutters Leg/wing packers Source: Adapted from Refs. 46 and 47.

No. (%) of positive samples

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Egg-Processing Facilities The discovery of L. innocua and, to a lesser extent, L. monocytogenes in 15 of 42 (36%) samples of frozen, raw, commercial liquid whole egg obtained from 6 of 11 manufacturers located throughout the United States suggests that listeriae-as well as salmonellae-contaminated poultry feces may contaminate the surface of eggs before breaking, and that these organisms in turn may be spread to various areas within the egg-processing environment. Fortunately, the Egg Products Inspection Act of 1970 led to regulations which now require that all egg products be pasteurized to eliminate salmonellae (and L. monocytogenes). However, as is true for fluid milk, there is ample opportunity for recontamination of liquid egg products with listeriae, salmonellae, and nonpathogenic organisms after pasteurization which can greatly decrease the shelf life and/or microbial quality of the finished product. Although Listeria spp. have not yet been recovered from commercially prepared pasteurized egg products or the associated manufacturing environment, prudent producers of such products should be certain that floors, drains, cleaning aids, wash areas, and food-contact surfaces as well as egg-breaking and egg-separating, pasteurization, and packaging equipment are thoroughly cleaned and sanitized on a regular basis to eliminate potential problems involving listeriae, salmonellae, and high levels of spoilage organisms.

Seaf ood-Processing FaciIities After L. monocytogenes was recovered from fresh frozen crabmeat in May of 1987, FDA officials began testing a wide range of domestic and imported fish and seafood products for listeriae and other organisms of public health significance. The results from these analyses led to numerous Class I recalls of Listeria-contaminated products, and government officials also released additional findings that were obtained during visits to various seafood-processing facilities. Between January and April of 1988, inspectors from the Oregon Department of Agriculture analyzed 480 environmental swab samples from 17 seafood-processing facilities located throughout Oregon [ 10,43,77]. Although only 4% of all samples were positive for Listeria spp., 10 of 17 (60%) factories yielded evidence of Listeria contamination in the work environment. Specific locations from which listeriae were isolated included (a) a fiberglass tote in a walk-in cooler, (b) a drain in a walk-in cooler, (c) a phosphate recirculation system on a shrimp-processing line, (d) an ice tote in a cold room, (e) a floor gutter near a shrimp peeler, (f) a wooden door frame in a crab-freezing room, (g) tires on heavy machinery, (h) a cold saturated (-23%) brine solution, (i) the framework of a fish dumpster, (j) floor and wall junctions in a cooler, and (k) seagull droppings on an office manager’s window. Additional environmental niches within processing plants that are strongly suspected of harboring listeriae include walls, floors, ceilings, condensate, pooled water, and processing wastes. Hence, this information along with other observations that virtually all Listeria cells recovered from processed seafoods have been healthy rather than thermally or otherwise injured suggest that the presence of listeriae in processed seafood is almost exclusively the result of recontamination after processing. Although L. monocytogenes and other Listeria species have been isolated from different types of raw and processed seafood, the main source of contamination is unknown. Several studies [41a,41d] have been conducted to detect the potential sources of this pathogen in seafood-processing plants so product contamination could be minimized. Eklund et al. [41d] surveyed coldsmoked salmon-processing plants to determine the occurrence and sources of L. monocytogenes. These authors observed that cleaning and sanitizing procedures adequately elimi-

Listeria in food-Processing facilities

671

nated L. monocytogenes from the processing line and equipment, but recontamination occurred soon after processing was resumed. They also identified the external surfaces of fresh and frozen fish as the primary source of L. monocytogenes in cold-smoked fishprocessing plants (Table 10). During the filleting, rinsing, and brining operations, the bacterium is transferred to the exposed flesh, and as the product moves through the processing steps, the equipment, personnel, and other surfaces which the product contacts become contaminated and these then serve as secondary sources of contamination. Destro et al. traced the transmission of L. monocytogenes in a shrimp-processing plant [41a 1, using two molecular typing methods: random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Of the 115 L. monocytogenes isolates examined, 25 were recovered from the plant environment (floors, walls, and pipes); 15 were from equipment and utensils, including,tables, plastic boxes, knives, and trays; 9 were found in water used in shrimp processing; 7 were isolated from the hands of employees; and 59 were from the shrimp. The results from this interesting study indicated that environmental strains all fell into composite groupings unique to the environment, whereas strains from both water and utensils shared another composite profile group. The L. monocytogenes isolates from fresh shrimp belonging to one profile group were found in different areas of the processing line. This same profile group was also present on the hands of employees from the processing and packaging areas of the plant. This study showed that there were many different sources of L. rnonocytogenes in the shrimp-processing plants. Information on preventing postprocessing contamination of fish, seafood, and other fishery products is presented in the second half of this chapter.

Vegetable- and Fruit-Processing Facilities Although consumption of coleslaw prepared from contaminated cabbage was directly linked to the first documented outbreak of foodborne listeriosis in 1981, the incidence of

TABLE 10 incidence of Listeria in a Cold-Smoked Sal mon-Processi n g Pia nt

Area in Plant ~~~~

~

L. monocytogenes

L. innocua

11/59 212 1 /9 616 317

15/59 012 019 316 417 3 /4 3I9 14/26

~~

Raw Product and Processing Area Thawing water for fish (from tank) Rack from bottom of thawing tank Filleting table Rinse water Skins from raw salmon Slime from raw salmon Drip from raw salmon Trimming from raw salmon Finished Product and Processing Area Salmon sides from smokehouse Trim table Trim machine Skins from skinning machine Fillet midline trimmings Product trimmings from slicers Source: Adapted from Ref. 41d.

414

819 15/26 919 1/8 6/15 29/30 8/20 17/35

719 018

2/15 8/30 0120 20135

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listeriae in raw vegetables and fruits and particularly the prevalence of these organisms in work environments of vegetable- and fruit-processing facilities have received relatively little attention. Nevertheless, the long-recognized association of listeriae with soil and the discovery of Listeria spp., including L. monocytogenes on raw vegetables suggest that these organisms are almost certainly in vegetable- and fruit-processing facilities. Unfortunately, the extent of Listeria contamination in such facilities in the United States is currently unknown. However, soil and production-area samples from one potato-processing factory in the Netherlands have yielded L. monocytogenes, L. innocua, and L. seeligeri (see Tables 14 and 15).

INCIDENCE OF LISTERIA SPP. IN WESTERN EUROPEAN AND AUSTRALIAN FOOD-PROCESSING FACILITIES As is true for the United States, information concerning the extent of Listeria contamination in European food-processing facilities also is limited. However, existing information indicates that European and American food companies are experiencing similar problems regarding listeriae in the manufacturing environment. Furthermore, since similar food production, processing and packaging methods as well as cleaning and sanitation practices are employed in both Western Europe and North America, much of the following information regarding the incidence of Listeria contamination within Western European food-processing facilities is probably applicable to manufacturers of similar products in the United States and Canada.

Western Europe Interest in the incidence of listeriae within European food-processing facilities has developed in parallel with the discovery of these organisms in foods destined for human consumption. As noted in Chapter 12, large quantities of French Brie cheese were contaminated with L. monocytogenes in 1986. Therefore, emphasis was first placed on determining the prevalence of listeriae in cheese factories. The results of one small-scale environmental survey of French cheese factories [32] identified L. monocytogenes in one floor sample and L. innocua was recovered from boards, wheels, and equipment (7 of 22 samples), brushes (1 of 6 samples), and filtered air (1 of 19 samples). From 1988 to 1990, a French cheese factory was sampled for Listeria contamination [53a]. Of the 344 samples collected and analyzed for Listeria, 61 strains (44 L. monocytogenes and 17 L. innocua) were isolated from four varieties of cheese, cheese brines, processing equipment, and the plant environment. The L. monocytogenes strains were recovered from the ripening and rind washing stages and not before, so Jacquet et al. [53a] theorized that the cheese contamination occurred at these points in the manufacturing process. During a survey of German factories producing soft smear-ripened cheese, Terplan 1741 also isolated nonpathogenic Listeria spp. from smear liquid, various pieces of machinery (especially smearing machines), and floor drains, with L. monocytogenes being detected far less frequently than other listeriae (Table 11). Hence, opportunity exists for contamination of both mold and bacterial surface-ripened cheese during the latter stages of manufacture and storage. Although such published information is limited, some unpublished data are available on the prevalence of listeriae in other Western European cheese factories. As mentioned in Chapter 12, Swiss officials who were tracing the source of contamination in the 1987 listeriosis outbreak involving consumption of Vacherin Mont d’Or soft-ripened cheese

Listeria in Food-Processing Facilities

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TABLE 11 Prevalence of L. rnonocytogenes and Nonpathogenic Listeria spp. Within the Working Environment of German Factories Producing Soft SmearRipened Cheese ~

~~

No. (%) of positive samples

Environmental sample Smear liquid and smearing machines Other machinery Ripening boards Condensate and cooling water Floor drains

No. of samples Nonpathogenic analyzed L. rnonocytogenes Listeria spp. 2 10 25 1 69 36 74

2 (0.9) 12 (4.8) 0 1 (2.8) 3 (4.1)

33 (15.7) 31 (12.3) 2 (2.9) 2 (5.6) 29 (39.2)

Total 35 43 2 3 32

(16.7) (17.1) (2.9) (8.3) (43.2)

Source: Adapted from Ref. 74.

recovered the epidemic strain of L. monocytogenes from smear brine, curing brine, wastewater sinks, wooden cheese hoops, and wooden boards used in 10 different cheese factories that manufactured Listeria-contaminated cheese [37]. Additionally, nearly half of the 12 cellars used to ripen cheese contained listeriae, with the pathogen being detected on 6.8% of the wooden shelves and 19.8% of the brushes used in the ripening cellars. Although not noted in the report, one would suspect that L. mc,nocytogenes also was present in commonly recognized environmental niches such as drains, floors, stagnant water, and various food-contact surfaces within cheese factories and ripening cellars. Thus brushing cheese with saltwater and ripening hooped cheese on wooden shelves appear to be two important means for dissemination of listeriae within cheese factories. In 1988, Cox [39,40] presented some preliminary data concerning the prevalence of Listeria spp. within one blue and six soft cheese factories in Western Europe as well as in one ice cream factory and eight chocolate factories. As expected, listeriae generally occupied similar environmental niches in both soft and blue cheese factories; however, Listeria contamination was far more common in ripening than production areas of the one blue cheese factory examined (Table 12). Ripening practices for blue cheese, including maintenance of a relatively moist environment, appear to be the likely reason for higher rates of Listeria contamination in ripening than production areas. Although some environmental niches in this blue cheese factory were not sampled, results for soft cheese factories point to walls, air coolers, stagnant water, and condensate as possible problem areas in blue cheese factories as well. During a similar investigation, samples from at least half of the drains, conveyors, stagnant water, floors, and residue and waste products from one Western European ice cream factory contained populations of Listeria spp. ranging from 10 to >106 CFU/g or mL (Table 13). This factory manufactured all of its ice cream from commercially produced reconstituted powdered milk (a product from which Listeria has not yet been isolated) rather than fresh milk. Hence, these findings strongly suggest that Listeria contamination in dairy-processing facilities is not always linked to incoming raw milk or milk haulers. Listeria spp., including L. monocytogenes, also have been detected in commercially produced chocolate that was marketed in England [48]. Furthermore, a 1988 report by Cox [39] indicated that 8 of 32 (25%) and 10 of 59 (17%) samples obtained from damp, wet, and dry areas of eight Western European chocolate factories were positive for Listeria spp. Although growth of listeriae in chocolate is very unlikely, contamination of the fin-

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TABLE 12 Incidence of Listeria spp. in Several Western European Blue and Soft Cheese Factories ~~~

~

Percentage of samples yielding Listeria spp. Environmental sample Drains Floors Residues Equipment Walls Air coolers Stagnant water Condensate Brine Miscellaneous

Soft cheese factory

Blue cheese factory

22 20 NA' 0 33 22 14 5 NA 19

71 5/83 23/46 O/NA NA/NA NAINA NAINA NAINA O/NA NAINA

NA, not applicable. a Production areas. Ripening areas. Not analyzed. Source: Adapted from Refs. 39 and 40.

ished product during packaging is clearly possible. The relatively low risk of producing Listeria-contaminated chocolate can be further reduced by development of adequate cleaning and sanitation programs and by maintaining production and packaging areas as dry as possible. In one of the largest European surveys reported thus far, Cox et al. [41], during the latter half of 1986, investigated the incidence of Listeria spp. in the processing environment of 17 establishments in the Netherlands that produced fluid dairy products, ice cream, Italian-style cheese, frozen food, potato products, and dry culinary foods. A total of 608 samples were collected from drains, floors, condensed and stagnant water, residues, processing equipment, and/or other areas and were analyzed for listeriae using the original

TABLE 13 Incidence of Listeria spp. i n the Production Environment of One Western European Ice Cream Factory

Environmental sample

Percentage of samples yielding Listeria spp.

Drains Conveyors Stagnant water Floors Residuedwaste products NR, Not reported. Source: Adapted from Refs. 39 and 40.

100 75 66 63 50

Listeria populations (CFU/g or ml) ?lob 1o2 NR

10-1O6 10-104

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USDA or FDA method with or without modification. All presumptive Listeria isolates were then speciated according to results from conventional biochemical tests. Despite use of GMPs in these factories, Listeria spp. were recovered from all types of food-processing facilities examined with the exception of two that produced dry culinary products. Overall, 181 of 608 (29.8%) samples yielded Listeria spp. with L. innocua, L. monocytogenes, and L. seeligeri being identified in 87.3, 14.9, and 0.5% of all positive samples, respectively. Although only five samples contained both L. monocytogenes and L. innncua, the actual number of such samples is probably somewhat greater, since a limited number of presumptive Listeria isolates from each sample were chosen for confirmation. As shown in Table 14, L. innocua was most prevalent in establishments that produced processed potato products followed by those that produced ice cream, frozen food, Italian-style cheese, and fluid dairy products, with the organism generally being isolated most frequently from drains, floors, and condensed and stagnant water. In contrast, L. monocytogenes was detected in 11.8% of all environmental samples obtained from one ice cream factory but was found in 2.9, 3.0, 3.3, and 3.7% of similar samples from establishments that manufactured fluid dairy products, potato products, frozen food, and Italian-style cheese, respectively (Table 15). Although only one ice cream factory was examined in this survey, the results are as expected when one recalls that Cox [39,40] previously found that listeriae were widespread in another Western European ice cream factory and also were present in very large numbers, particularly in floor drains (Table 13). Given such populations of listeriae in ice cream factories and the current extruding, niolding, and freezing methods used to produce ice cream, and particularly ice cream novelties, one can easily postulate many routes whereby listeriae may recontaminate the finished product, as has been reported in the United States. Results concerning the incidence of Listeria spp. as well as L. innocua and L. munocytogenes in various work environments of all 15 food-processing facilities are summarized in Table 16. Overall, these findings are comparable to what has been previously noted for similar food-processing facilities in the United States; for example, Listeria spp. and L. innocw were most frequently recovered from drains followed by condensed and stagnant water, floors, residues, and processing equipment. With a few minor exceptions, which probably resulted from the number of samples analyzed, this same trend is readily apparent for all five types of food-processing facilities listed in Table 14. Thus a logical pattern emerges in which L. innocua moves from floor drains to pools of condensed and stagnant water, which then come into direct contact with floors and residues. Once present in open areas of the work environment, L. innocua is spread by employees to processing equipment that comes into direct contact with the product. Unlike L. innocua, L. monocytogenes was far less prevalent in all types of food-processing facilities and was distributed fairly evenly within the factory environment with incidence rates ranging between 2.3 and 7.7%. Although L. innocua is by definition nonpathogenic, the fact that L. innocua and L. monocyto,qenes (and possibly other Listeria spp.) occupy similar environmental niches indicates that detection of listeriae anywhere within the manufacturing environment should prompt immediate corrective action, the details of which will be discussed shortly. In one of the remaining few Western European surveys reported, Hudson and Mead [511 determined the incidence of Listeria spp. at 10 different sites within one large English poultry-processing facility. According to these authors, scald water, feathers, and chill water as well as swab samples from defeathering machines and conveyors leading to the chiller were free of listeriae; however, L. monocytogenes was routinely isolated from automatic carcass openers and also was present in samples from evisceration-line drains,

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TABLE 14 Incidence of L. innocua in Working Environments of 15 Food-Processing Facilities in the Netherlands No. of positive samples/No. of samples analyzed (%)

Environmental sample

Fluid dairy factory na = 5

Ice cream factory n = l

Drains Condensed/stagnant water Floors Residues Processing equipment Miscellaneous

2/4 (50.0) 2/5 (40.0) 0/2

0/13

4/4 (100.0) 4/8 (50.0) 8/16 (50.0) 4/12 (33.3) 7/20 (35.0) 2 B d (25.0)

Total

4/34 (11.8)

29/68 (42.6)

NAb o/ 10

NA, not applicable. a Number of factories analyzed. Not analyzed. Includes one sample positive for L. seeligeri. Conveyor belt (two of two positive). Raw milk (two of two positive), untreated effluent. Potato delivery soil (two of three positive), sand from effluent treatment (two of two positive). Source: Adapted from Ref. 4 1.

Italian-style cheese factory n= 5

Frozen food factory

19/42 (45.2) 7/20 (35.0) 14/44 (31.8) 16/71 (22.5) 6/68 (8.8) 12/103' (11.7)

2/3 (66.7)

1/6 (16.7) 15/78 (19.2)

4/ 17' (23.5)

74/348 (2 1.3)

20/91 (22.0)

32/68 (47.1)

n = 3 NA

2/4 (50.0)

NA

Potato-processing factory n = l 7/13 7/10 9/13 5/15'

(53.8) (70.0) (69.2) (33.3)

NA

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TABLE15 Incidence of L. monocytogenes in Working Environments of 15 Food-Processing Facilities in the Netherlands No. of positive samples/No. of samples analyzed (%)

Fluid dairy factory na = 5

Ice cream factory n = l

Italian-style cheese factory n= 5

Frozen food factory n = 3

Drains Condensed/stagnantwater Floors Residues Processing equipment Miscellaneous

0/4 0/5 0/2 NAb 0/10 1/13 (7.7)

0/4 0/8 1/16 (6.3) 0/12 6/20 (30.0) 1/8' (12.5)

2/42 (4.8) 0/20 2/44 (4.5) 7/71 (9.9) 2/68 (2.9) O/ 103

1/3 (33.3) NA 0/4 NA 0/6 2/78 (2.6)

1/13 (7.7) 0/15 NA 1/17d(5.9)

Total

1/34 (2.9)

8/68 (1 1.8)

13/348 (3.7)

3/91 (3.3)

2/67 (3.0)

Environmental sample

NA, not applicable. Number of factories analyzed. Not analyzed. Sponge (one of one positive). Potato delivery soil (one of three positive). Source: Adapted from Ref. 4 1 .

Potato-processing factory n = l 0/13

o/ 10

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TABLE 16 Overall Incidence of Listeria spp. in Working Environments of 15 Food-Processing Facilities in the Netherlands

No. of samples analyzed

Listeria spp.

L. innocua

Drains Condensed/stagnant water Floors Residues Processing equipment Miscellaneous

66 43 79 97 104 219

36 (54.5) 20 (46.6) 36 (45.6) 32a (33.0) 20 (19.2) 37 (16.9)

34 (51.5) 20 (46.5) 33 (41.8) 24 (24.7) 14 (13.5) 33 (15.1)

3 (4.5) 0 4 (5.1) 7 (7.2) 8 (7.7) 5 (2.3)

Total

608

181h(29.8)

158 (26.0)

27 (4.4)

Environmental sample

No. (%) of positive samplesa

L. rnonocytogenes

One sample yielded L. seeligeri. Five samples yielded both L. monocytogenes and L. innocua. Source: Adapted from Ref. 4 1.

neck-skin trimmers, and conveyors on which carcasses travel to the packing area (Table 17). Although only one to three samples from each site were analyzed in three successive visits, the areas from which L. monocytogenes was recovered in this poultry-processing facility are generally similar to those observed by Genigeorgis et al. [46,47] for chicken and turkey slaughterhouses in California (see Table 8 ) .

Australia Information concerning the prevalence of listeriae in food-processing facilities located in other parts of the world is currently limited to a few Australian studies. Following the isolation of L. monocytogenes from ricotta cheese in 1987, the Victorian Dairy Industry Authority and the Department of Agriculture and Rural Affairs conducted a joint survey to determine the extent of Listeria contamination in the working environments of 5 2 Melbourne-area factories producing pasteurized milk and different types of cheese [76]. Overall, various Listeria spp. were detected in 141 of 763 ( 1 8.5%) environmental samples from 21 of 5 2 (40.4%) factory environments, with L. monocytogenes, L. seeligeri, and L. iva-

TABLE17 Incidence of Listeria spp. in the Working Environment of One Poultry-ProcessingFacility in England

Type of sample Transport crates Automatic carcass opener Evisceration-line drain Neck-skin trimmer Conveyor to packing area Source: Adapted from Ref. 5 I .

No. of samples analyzed

L. monocytogenes

L. innocua

9 3 3 3 3

0 3 (100) 2 (66.7) 2 (66.7) 1 (33.3)

I (11.1) 0 0 0 0

No. (%) of positive samples

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novii being identified in 132 (93.6%), 8 (5,7%), and 1 (0.7%) of these Listeria-positive samples, respectively. More important, L. rnonocytogenes was present in all but one of the ListeriLz-positive factories. As expected from other surveys conducted in the United States and Western Europe, factory sites most frequently contaminated with listeriae once again included drains and floors in coolers, surfaces of manufacturing and packaging equipment, and conveyors. Even though strict cleaning and sanitizing programs were implemented at many of these facilities, Listeria spp. were very difficult to eliminate from the working environment, with these organisms being continuously isolated from one factory over a period of 5 months. Sutherland and Porritt [73a] conducted a 3-year study in 12 Australian dairy-processing facilities to assess the environmental diversity and identify the major environmental niches for L. rnonocytogenes. A total of 565 environmental samples were collected and tested. The overall incidence of Listeria-positive samples was 21% (Table 18). Approximately half of these samples (12%) were positive for L. monocytogenes. Cheese, ice cream, and mixed-product plants all had similar incidences of L. rnonocytogenes and Listeria spp. The incidence of L. rnonocytogenes in mixed-product factories (18%) was comparable to the higher levels found in milk factories. Sutherland and Porritt [73a] also highlighted four major ways that L. rnonocytogenes enters a dairy-processing facility, including: 1. Ingredients-especially raw milk 2. Inward goods-including milk crates and crate washers, vehicles (trucks, road, and rail tankers), and wooden pallets 3. Environment-including air and internal air quality 4. Personnel-especially outside contractors and visitors Once L. rnonocytogenes is inside the processing plant, these authors [73a] found numerous areas in which this pattern can survive, grow, and potentially contaminate product. Conveyor systems, drains, and floors were the most common isolation sites. Other areas of concern related to were traffic flow, cooking units, and internal air quality. Complete elimination of listeriae from dairy-processing facilities may, in some instances, be nearly impossible; however, the likelihood of producing Listeria-contaminated products can be greatly reduced by following GMPs, which include implementation of rigorous cleaning and sanitizing programs for equipment used at critical points during manufacture and packaging of the foods in question.

INCIDENCE OF LISTERIA IN HOUSEHOLD KITCHENS Thus far, this chapter has dealt exclusively with Listeria contamination in commercial food-processing facilities; however, because of the relatively high incidence of Listeria spp. (including L. monocytogenes), salmonellae, and other foodborne pathogens in fresh beef, pork, lamb, and poultry available to the general public at butcher shops and supermarkets, safe home preparation of these foods must be reemphasized. In 1989, Cox et al. [41] isolated nine strains of listeriae from 7 of 35 (20%) household lutchens surveyed in the Netherlands. Overall, L. rnonocytogenes was recovered from four dishcloths and one refrigerator, with two dishcloths and two dustbins from two other households yielding L. innocua and L. welshirneri, respectively. Considering results from commercial food-processing facilities, one might expect to recover Listeria spp. from such household kitchen

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TABLE 18 Three-Year Study in 12 Australian Dairy-Processing Plants to Determine Environmental Diversity and Identify Major Environmental Niches of L. rnonocytogenes

Factory type Cheese Milk Ice cream Mixed product Total

No. of factories

No. of samples

7 2 1 2

319 87 53 106

12

565

Source: Adapted from Ref. 30a.

Listeria spp. (%)

34 51 9 22

L. monocytogenes (%o)

(11) (59) (17) (21)

26 (8) 20 (23) 3 (6) 19 (18)

116 (21)

68 (12)

L. innocua

L. grayii

(%)

(%)

Mixed

(%I

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areas as drains, U-tubes, and drain boards. If this is true, then garbage disposal systems could conceivably lead to problems from production of aerosols. Although further work is needed to clarify the public health significance of listeriae in the kitchen environment, you may recall from Chapter 13 that L. rnonocytogenes was found in many refrigerated foods belonging to an Oklahoma woman who contracted listeriosis after consuming contaminated turkey frankfurters that were eventually recalled nationwide. Centers for Disease Control and Prevention (CDC) officials also isolated L. rnonocytogenes from 15 of 25 (60%)refrigerators that were used by apparent victims of foodborne listeriosis [25]. Hence, consumers should regularly clean and sanitize kitchen areas, sinks, and refrigerators. Such efforts should help prevent potential problems involving listeriosis and other forms of foodborne illness in the home.

CONTROL OF LISTERIA IN FOOD-PROCESSING FACILITIES The discovery of Listeria spp., including L. rnonocytogenes, in various fermented and unfermented dairy products, raw and ready-to-eat meats, poultry products, seafoods, and vegetables has prompted food manufacturers to renew their concern about factory hygiene and product safety. Although failsafe procedures for the production of Listeria-free foods largely do not yet exist, specific guidelines have been developed for controlling listeriae and other microbial contaminants within American dairy- [4,18,36,49,57,68,73,75],meat[ I ,2], poultry- [ 191, and seafood- [43,45,77] processing facilities with Denmark [21], England [53,58,67], France [ 171, and Australia [30a] also addressing the elimination of listeriae from fluid milk and cheese operations during all facets of production, distribution, and retail sale. In response to the discovery of L. rnonocytogenes in ready-to-eat foods and delicatessen products, European public health officials have expressed particular concern about contamination of these products during retail slicing and storage. They also have warned grocery store managers to give particular attention to storage temperatures for refrigerated foods in display cases and the potential sale of products beyond their normal code dates. Most of these guidelines stress the need to (a) decrease the possibility that raw products will contain listeriae, (b) minimize environmental contamination in food-processing facilities, and (c) use processing methods that will eliminate listeriae from food. Following these proposed guidelines, which will be discussed in detail shortly, will decrease the possibility of producing foods contaminated with L. monocytogenes and other foodborne pathogens. I11 addition, diligent attention to cleaning and sanitation and overall GMPs will lead to lower microbial populations in processed foods which will in turn increase the shelf life of the finished product. Any approach to controlling the spread of listeriae and other microorganisms in food-processing facilities is complicated by the enormous variety of foods being processed today along with variability in quality of incoming raw products, design of the factory, sanitary design of the processing and packaging equipment, and processing methods. However, this subject can be simplified by first focusing on problem areas such as factory design, general factory environment, heating and air-conditioning systems, traffic patterns, and personnel cleanliness that are common to all food-processing facilities. Once Listeriacontrol measures for these problem areas are understood, attention can be given to specific processing steps which are unique to the dairy, meat, poultry, seafood, and vegetable industries.

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General Guidelines Factory Design Every food processor should be firmly committed to the long-term production of safe, wholesome food. The first step toward such a goal is an adequately designed factory to produce the particular product. Although newly constructed buildings offer countless advantages in that they can be designed for production of specific products, existing buildings also can be used for safe production of food provided that such facilities have been properly modified to meet certain basic requirements. Design features that are widely considered to be essential for all types of foodprocessing facilities include (a) a raw product receiving area that is completely isolated from processing and packaging areas of the factory; (b) tight-fitting exterior windows and doors that will prevent animals and insects from entering processing and packaging areas; (c) easily cleaned and sanitized walls, floors, and ceilings that are constructed of tile, metal, or concrete and not porous materials such as wood; (d) floors designed to drain rapidly and prevent pooling of water; (e) floor drains located away from packaging equipment, especially if processed foods are exposed to factory air; (f) proper screens, debris baskets, and traps on floor drains; (g) a quality control and/or quality assurance laboratory that is well isolated from other areas of the factory; and (h) proper means of waste disposal outside the factory to discourage congregation of insects, rodents, birds, and other animals that may harbor Listeria and other pathogenic microorganisms. In addition to these concerns, the heating, ventilating and air-conditioning (HVAC) system also must be properly designed to minimize airborne contamination [68]. Features considered to be essential for such a system include (a) intake air vents on the roof of the building that are located upwind from prevailing air currents but away from dumpsters, raw product receiving areas, and vents that are discharging factory air; (b) installation of screens and filters inside incoming air vents to remove particulate matter and condensate; (c) easily cleanable HVAC systems; and (d) proper location of dehumidifiers and airconditioning systems so that these units drain away from processing and packaging areas. Most important, all HVAC systems must be designed to produce a higher positive air pressure in processing and packaging rather than in receiving areas. This design readily prevents movement of airborne contaminants from raw product areas to the cleanest areas of the factory where foods are processed and packaged.

Factory Environment Various bacteria, yeasts, and molds can be found in most food-processing areas other than those associated with aseptic packaging, with populations normally being many times higher in receiving than in processing and packaging areas. Furthermore, most of these microorganisms will grow in the factory environment if given a suitable temperature and enough time along with an adequate supply of nutrients and water. Although microbial contamination will always occur in food-processing facilities, eliminating microbial growth by altering (a) temperature, (b) time that the organism is present in the environment, (c) availability of nutrients, and/or (d) availability of water will sharply decrease the incidence of L. rnonocytogenes and other foodborne pathogens as well as spoilage organisms in the factory environment. Hence, production of a safe food product with a long shelf life depends largely on control of timehemperature constraints and elimination of available nutrients and/or water through the concerted effort of everyone involved. Since air, water, waste products, and anything else that comes in contact with the

Listeria in Food-Processing Facilities

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finished product must be considered as a potential source of contamination, food processors must strive to prevent the spread of microbial contaminants from heavily contaminated raw product receiving areas to processing and packaging areas. In addition to construction of physical barriers between such areas in food-processing plants, all incoming cases, pallets, containers, forklifts, and cleaning materials such as brushes and other equipment must be assumed to harbor listeriae along with other microbial contaminants and therefore should never be allowed to enter processing and packaging areas. Ideally, separate equipment, including tools employed by maintenance persons, should be available for use in raw and finished product areas. If this is not possible, then all equipment should be cleaned and sanitized before entering processing and packaging areas. As previously stressed, all areas within food-processing facilities should be kept dry and as free as possible from processing waste to minimize microbial growth. Also, floor drainage problems that lead to pooling of water must be eliminated as well as cracks and holes in floor tiles and grouting in which water and food particles can accumulate. Since L. monocytogenes has been recovered from condensate in dairy factories, it is imperative to keep all processing and packaging equipment and walls, floors, and ceilings as condensate-free as possible. In the event that dripping condensate cannot be prevented by manipulation of temperature and humidity in processing and packaging areas, deflector shields should be installed to prevent direct contact between exposed product and dripping condensate. Aerosols provide another ready means for disseminating listeriae and other microbial contaminants throughout critical areas of food-processing facilities [55],with L. rnonocytogenvs surviving 3.42 h in experimentally produced aerosols of reconstituted skim milk [7 11. ‘Therefore, high-pressure sprays should never be used in processing and packaging areas for cleaning floors or drains, since both are major sources of listeriae and other microbial contaminants and resulting aerosols can contaminate food-contact surfaces of equipment. Operation of unshielded centrifugal pumps in such areas also is discouraged. In addition to the building itself, all equipment within the factory should be designed to minimize cross contamination between the factory environment and product and also should be constructed of stainless steel or other easily cleaned and sanitized nonabsorbent, nontoxic materials such as certain types of bonded rubber and plastic. All piping in foodprocessing facilities should be free-draining and designed to eliminate trapping of food and cleaning and sanitizing solutions used in clean-in-place (CIP) systems. It also is important that equipment such as product conveyors is positioned high enough above the floor to minimize cross contamination from floors and drains. The air supply within the factory must be considered as a potential source of Listeria and other microbial contaminants. Hence, all HVAC ducts and accompanying air filters should be kept in good repair and cleaned regularly to eliminate excessive dust and dirt. Compressed air lines and filters should also be inspected regularly and be free of moisture, oil, and debris.

Cleaning a n d Sanitizing Cleaning can be defined as the physical removal of visible dirt, impurities, and other extraneous matter (i.e., nutrients for growth of microorganisms, including Listeria) through proper use of solutions of soaps, detergents, surfactants, and abrasive agents. In contrast, sanitizing causes inactivation of most microorganisms left on cleaned surfaces by exposing them to heat or chemical agents such as chlorine, iodine (iodophor), acid anionic, or quaternary ammonium compounds. Hence, the routine use of good cleaning and sanitizing practices is of utmost importance in controlling microbiological safety and

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quality of finished products. In establishments such as those that produce fluid milk and ice cream, adherence to good cleaning and sanitation practices that involve both equipment and the factory environment is the only means of preserving product quality beyond initial pasteurization of ingredients. Each food-processing facility needs to institute and enforce an effective cleaning and sanitizing program that will ensure production of safe products. As part of this program, management personnel need to develop standard operating procedures for every job in the factory along with master schedules with the frequency of cleaning and sanitizing procedures so that the workers will recognize their individual responsibilities and will maintain accurate records regarding routine sanitation practices. Management personnel also need to instill in their employees the great importance of good cleaning and sanitizing practices through the use of continuing education programs that deal with current issues such as Listeria. Such cleaning and sanitizing responsibilities should never be assigned to new untrained employees. Floors, drains, walls, ceilings, and each piece of equipment in the factory should be cleaned and/or sanitized on a regular basis with the frequency of cleaning and sanitizing being dependent on the extent to which the particular item becomes contaminated during normal operation and whether or not a product is likely to come in contact with the item during processing and/or packaging. All food-contact surfaces such as tables, peelers, slicers, collators, overhead shielding, conveyors, conveyor belts, chain rollers, supports, and other intricate equipment directly associated with processing, filling, and packaging operations need to be cleaned and sanitized daily and in some instances more often, particularly around filling and packaging operations. A regular cleaning and sanitizing schedule also must be adopted for non-food-contact surfaces such as floors, walls, ceilings, floor drains, pipes, blowers, HVAC ducts, coils and pans from dehumidifying and air-conditioning units, light fixtures, material handling equipment, and wet and dry vacuum canisters. As indicated in the first half of this chapter, Listeria spp., including L. monocytogenes, have been most frequently isolated from floor drains and floors, thus suggesting that these areas may function as reservoirs for listeriae in food-processing facilities. Although all floors and drains, including drain covers and baskets, in production and refrigerated storage areas should be thoroughly cleaned and sanitized daily, high-pressure hoses should never be used in these areas, since such practices readily promote the spread of listeriae to nearby equipment and other areas of the factory through splashing and the production of aerosols. Managers of food-processing facilities must be sure that proper equipment is available for daily cleaning and sanitizing operations. Absorbent articles such as sponges and rags should never be used in the factory environment, since these items function as virtual “microbial zoos.” Various types of metal scrappers can be used for removing hard mineral deposits, with disposable paper towels being best suited for eliminating excess moisture and accidental spills. Unlike sponges and rags, brushes are readily cleaned and sanitized and are therefore suitable for widespread use in the factory. However, to avoid cross contamination, separate color-coded brushes with nonporous plastic or metal handles should be used for scrubbing (a) exterior and interior surfaces of equipment, (b) raw and finished product areas, (c) food-contact and non-food-contact equipment surfaces, and (d) floor drains. Brushes, particularly those used to scrub floor drains, are best cleaned and stored in sanitizing solution after use. Sanitizing is the final step in eliminating L. monocytogenes, other foodborne pathogens, and the myriad of spoilage organisms present in the production environment. Since the presence of organic debris, particularly if proteinaceous, readily decreases the effec-

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tiveness of sanitizing agents against most microorganisms, including listeriae [33],it is important to remember that every item must first be thoroughly cleaned before it is sanitized. Research has demonstrated that L. monocytogenes is sensitive to sanitizing agents commonly employed in the food industry. According to several authors [6 1,651, chlorinebased, iodine-based, acid anionic, and/or quaternary ammonium-type sanitizers were effective against L. monocytogenes when used at concentrations of 100 ppm, 25-45 ppm, 200 ppm, and 100-200 ppm, respectively. Although these concentrations may have to be adjusted to compensate for in-plant use as well as oxidation and reduction factors relating to water quality and hardness, recommended concentrations should not be markedly exceeded, since the use of extremely concentrated sanitizing solutions heightens the danger to employees, increases the risk of chemical contamination of food, and in some instances causes corrosion of equipment. Since foaming chlorine-based sanitizers are corrosive, their use should be primarily confined to floors, floor drains, walls, and ceilings. Alternatively, these areas can be flooded or foamed with quaternary ammonium-type sanitizers (-300 ppm); however, fogging exterior surfaces with quaternary ammonium-type sanitizers is frequently regarded as being ineffective and dangerous for employees. Quaternary ammonium-based sanitizers also are not recommended for use on food-contact surfaces and should never be used in cheese or sausage factories, since lactic acid starter culture bacteria are rapidly inactivated by small residues of these sanitizers. In contrast, acid anionic and iodine-type sanitizers are best suited for equipment surfaces, with the former readily neutralizing excess alkalinity from cleaning compounds and preventing formation of alkaline mineral deposits. Although also effective, the use of steam should be confined to closed systems because of potential hazards associated with aerosol formation. Sanitizing with hot water is not advised, since sufficiently high water temperatures cannot be easily maintained. Custom-designed CIP systems have been installed in many food-processing facilities, particularly dairies, for automated cleaning and sanitizing of pipelines, tanks, vats, heat exchangers, homogenizers, and other equipment in processing lines. Although presumably adequate by design, CIP systems also should be reviewed for proper timing, flow rate, temperature, pressure, and sanitizer strength as recommended by chemical suppliers. Furthermore, proper operation of the entire system should be verified from data collected on recording charts, which can be stored for future reference. Regardless of how well these recommendations for cleaning and sanitizing are followed, every food-processing facility should verify the effectiveness of its cleaning and sanitation program through daily microbiological analysis of both product and environmental samples gathered from all areas of the facility. During environmental sampling, the efficacy of cleaning and sanitizing procedures can be easily determined through the use of ATP bioluminescence monitoring systems that are available from a number of manufacturers [42d]. Particular attention should be given to floor drains, floors, filling and packaging areas, and any processing equipment that is difficult to clean. Although environmental samples are most easily collected using swabs or sponges, only polyurethane or expanding cellulose sponges should be used for such a purpose, since other types, including retail cellulose sponges, contain inhibitory agents that not only prevent recovery of L. monocytogenes and Staphylococcus aureus but also interfere with recovery of Brochothrix thermosphacta, Aerornonas hydrophila, Pseudornonus putrefuciens, and P. jhorescens as well as Escherichia coli, Serratia marcescens, and Enterobacter cloacae [60]. It is important to stress that laboratory personnel should never attempt to isolate

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pathogenic microorganisms from such samples unless the laboratory is in a separate building and completely removed from the factory. Although analysis of environmental and, if necessary, food samples for microbial pathogens is best left to outside commercial testing laboratories that are FDA approved or otherwise certified, coliform and standard aerobic plate counts should be obtained for samples from the factory environment and the food during all stages of production to monitor the extent of postprocessing contamination and thus to quickly identify any problems associated with inadequate cleaning and sanitizing. Coliform organisms are commonly regarded as being indicators of postprocessing contamination and the possible presence of pathogens; however, the presence or absence of coliforms in food or environmental samples does not guarantee the presence or absence of foodborne pathogens. In fact, often little if any correlation has been observed between the presence of coliforms and Listeria in finished product. Therefore, routine testing of environmental samples for Listeria spp. and other foodborne pathogens by outside laboratories remains a critical component of any sanitation verification program.

Traffic Patterns Employee movement within food-processing facilities also can have a major impact on the microbiological quality of finished products. Therefore, traffic patterns need to be developed that restrict or preferably eliminate movement of workers between raw, processing, filling, packaging, and shipping areas. Managers need to educate employees about the spread of Listeria and other microbial contaminants from clothing, boots, and tools to all areas of the factory, and they need to situate locker rooms, changing areas, and lunch and break rooms to minimize traffic through production areas. Issuing differentcolored outer garments to workers in various areas of the factory has proven helpful in monitoring employee movement. Since L. monocytogenes and other microbial pathogens are commonly associated with raw products of both plant and animal origin, employees working in raw product receiving areas (including maintenance personnel) and individuals who deliver raw products, particularly milk haulers, should be denied access to all processing areas. When necessary, employee movement between raw product and processing areas of the factory should only be allowed after completely changing outer garments as well as scrubbing and disinfecting boots. All workers should be encouraged to use disinfectant-containing footbaths that should be placed in all doorways leading into the factory as well as between raw product and production areas. These footbaths need to be monitored daily for sanitizer strength and cleanliness. Since a great variety of microorganisms are carried on street clothing, it also may be prudent for managers to consider limiting the number of visitors and tour groups going through the factory. Large glass observation windows provide ample opportunity for visitors to view processing areas while at the same time prevent introduction of additional microbial contaminants.

Personnel Clean Iiness Factory managers and supervisors must stress good employee hygiene and also set a good example for other workers. All individuals with obvious illnesses, infected cuts, or abrasions need to be excluded from working in processing areas or from doing other tasks that may lead to contamination of food, food-contact surfaces, or packaging materials or equipment. Furthermore, the use of tobacco and chewing gum as well as the consumption of food should be banned in processing areas along with the wearing of hairpins, rings, earrings, watches, and other jewelry. Above all, employees should always wash their hands thoroughly before starting work, on returning to work, and after touching floors, walls,

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light switches, any other unclean surface, and garbage. To further promote their use, handwashing facilities should be properly designed and conveniently located near work stations. All factory workers need to be provided with hair and/or beard nets as well as clean clothes, suitable footwear, and disposable gloves. Special attention also is needed to assure that street clothes do not enter processing areas and that factory clothing, including footwear, remain inside the factory. All factory clothing should be changed daily or more often if soiled, with the responsibility of laundering being left to the employer. Although these recommendations may, in some instances, be difficult for food processors to follow and enforce, this task will be made much easier if management can instill in workers the conviction that each employee is personally responsible for both the quality and safety of the foods that are produced and ultimately consumed by the public.

INDUSTRY-SPECIFIC EQUIPMENT, PROCESSING METHODS, AND PRODUCTS It now is appropriate to briefly examine some of the industry-specific equipment and processing methods, many of which have been cited as critical control points for the production of Listeria-free dairy, meat, poultry, seafood, and vegetable products. Although this information will be useful to enhance the effectiveness of preexisting cleaning and sanitation programs, the reader is reminded that food-processing facilities, even though they manufacture similar products, are all unique in terms of factory design, raw product quality, and product flow, handling, and processing methods. Therefore, no universally acceptable cleaning and sanitation program can be developed for the safe production of a given product.

Dairy Industry Farm Environment Since listeriae are widespread in the environment, any quality control program should first contain a plan to minimize contamination of raw milk with Listeria and other microorganisms on the dairy farm. Along with good animal husbandry practices, including the use of only high-quality feed and silage, farm workers also should give attention to cleanliness of the milkhouse and milking equipment. Most important, teats and udders of all cows should be properly sanitized and dried before milking equipment is attached. Bulk tanks in which raw milk is stored also need to be properly maintained and inspected regularly.

CIa rif iers and Separat0 rs All raw milk should be filtered and subsequently clarified and separated by centrifugation to remove extraneous matter and somatic cells (i.e., leukocytes) before pasteurization. Since L. monocytogenes is sometimes found in leukocytes, clarifiers and separators should be well isolated from the pasteurizer and all finished product areas of the factory. Sealed containers should be used to dispose of all clarifier and separator waste, both of which may contain high levels of listeriae. Special care also should be used in cleaning and sanitizing separators, clarifiers, and surrounding areas.

Pasteurization Proper pasteurization using a vat or high-temperature short-time (HTST) pasteurizer is the only commercially practical means by which all non-spore-forming pathogens, includ-

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ing L. monocytogenes, can be inactivated in raw milk. Thus it is imperative that all pasteurization equipment be designed, installed, maintained, and operated properly. Although continuous-flow HTST pasteurization is used to process virtually all fluid milk and ice cream mix, vat (or batch) pasteurization is employed by many smaller firms, particularly those involved in cheesemaking, when the volume of incoming raw milk is too small to justify the use of a continuous-flow HTST system. If vat pasteurization is used, raw milk must be heated to a minimum of 623°C (145°F) and then held at that temperature for at least 30 min. In theory, vat pasteurization is a relatively simple process with raw milk being pumped into a steam- or hot water-jacketed vat and held for the prescribed time. However, FDA inspections conducted as part of the Dairy Initiative Program mentioned earlier have uncovered numerous problems with vat pasteurizers, including improper equipment design, the absence of proper outlet valves and air space thermometers, and improperly operated air space heaters. The latter problem is particularly critical, since the air space temperature above the product in the vat must be at least 23°C (5°F) higher than that of the product at all times to assure proper Pasteurization. Operators of such pasteurizers should be made accountable for proper performance as well as proper cleaning and sanitizing of the equipment. In addition, recording charts showing time and temperature relationships along with other data for each vat of product pasteurized should be kept for at least 3 months. As mentioned earlier, continuous-flow HTST pasteurization at 71.7"C (161OF) for a minimum of 15 s is the principal method for processing raw milk. Although an in-depth discussion of the many intricate problems associated with HTST pasteurization equipment is beyond the scope of this book, a basic knowledge of HTST pasteurization is essential to appreciate the seriousness of some of the recently identified problems that have been linked to faulty maintenance and/or operation of the equipment. Interested readers may consult the HTST Pasteurizer Operation Manual [50] for more detailed information on HTST pasteurization. All HTST pasteurizers consist of five basic components, as shown in Figure 2: (a) plate heat exchanger-a series of thin, gasketed stainless steel plates divided into three sections (heater, regenerator, and cooler) for heating incoming raw milk and cooling outgoing pasteurized milk; (b) constant level tank-provides a constant level of raw milk to the HTST system; (c) timing pump-a positive displacement pump that establishes the holding time of the time and temperature relationship for pasteurization; (d) holding tube-a length of pipe in which fully heated milk is held for the required holding time; and (e) flow diversion valve-a three-way valve that will allow properly pasteurized milk to enter the regenerator section of the plate heat exchanger or divert improperly pasteurized milk to the constant level tank for repasteurization. In addition to these five components, a source of steam and/or hot water is required to heat incoming raw milk, a safety thermal limit recorder is needed to activate the flow diversion value in the event of improper pasteurization, and a cold milk recorder is required to record the temperature of outgoing pasteurized milk. Finally, auxiliary components that may be added to HTST units for additional processing of milk or milk products include a booster pump, homogenizer as a timing pump, stuffing pump, and flavor treatment or vacuum units. Inspections of HTST pasteurizers conducted in conjunction with the FDA Dairy Initiative Program uncovered numerous problems relating to proper installation and maintenance of these units. Problems most commonly associated with HTST pasteurization equipment have included stress cracks and/or pinholes in the heat exchanger plates, leaking gaskets, improper flow diversion valves, and inadequate cleaning and sanitizing of

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Diversion Line

Cold Past,

Flow-Diversion valve

-I Holding Tube

Cooler

Regenerator

Heater Timing Pump

Raw Milk Constant Level Tank

FIGURE2 Schematic diagram o f milk flow through an HTST pasteurizer. (Adapted f r o m Ref. 50.) the pasteurization unit. Although not a strict regulatory requirement, positive pressure should be maintained between the product and heating medium as well as the product and cooling medium (sweetwater) to prevent Listeria-contaminated raw milk or sweetwater from mixing with pasteurized product in the event that some of the heat exchanger plates contain stress cracks or pinholes. Operators should examine all pasteurization plates for defects every 6 months using the standard dye test. Sweetwater and glycol solutions also should be routinely examined for microbial contaminants, since these coolants may harbor L. monocytogenes, Yersinia enterocolitica, and Salmonella typhimurium for extended periods along with large populations of psychrotrophs [ 18,661; the latter are particularly detrimental to product shelf life. As was true for vat pasteurization, operators of HTST pasteurizers must be responsible for proper operation of these units and retain accurate records and chart recordings for each lot of pasteurized product for at least 3 months. Although the inability of L. monocytogenes to survive the minimum allowable HTST heat treatment given to commercially available raw milk ( 7 1 . 7 W 1 5 s) is now generally accepted, most fluid milk processors in the United States are pasteurizing milk at -76.7"C for 20 s, which is well above the minimum requirements established in the Pasteurized Milk Ordinance. This more severe heat treatment markedly extends the shelf life of the finished product by inactivating larger numbers of spoilage organisms than does minimal HTST pasteurization. However, the psychrotrophic nature of L. monocytogenes increases the need to prevent introduction of listeriae into the product after pasteurization.

Pipeline and Cross Connections Many large dairy processors have installed up to several miles of pipeline in the factory to handle movement of raw milk from storage tanks to the pasteurizer and pasteurized milk from the pasteurizer to various holding tanks, mixing tanks, and product areas located

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throughout the factory. Considering the enormous quantities of product that can be manufactured at such facilities during one production period, careful attention must be given to each stage of manufacture, since an error made during these operations could adversely affect thousands of people, as was true for the 1985 outbreak of milkborne salmonellosis in Chicago. FDA inspections have uncovered numerous violations related to pipelines, including cross connections between raw and pasteurized milk lines and/or storage and holding tanks as well as cross connections between CIP and product lines and other potentially hazardous circuits. Since many of these lines allow easy bypass of raw product around the pasteurizer thus permitting postpasteurization contamination in the event of equipment failure or operator error, factory managers, engineers, or other qualified people need to walk through the factory and construct an up-to-date detailed blueprint of raw and pasteurized product flow throughout the entire factory. Once the blueprint is constructed, any unwanted piping, dead ends, illegal cross connections, or unauthorized changes made to initial installations should be promptly identified and eliminated. Most important, all pasteurized product lines need to be separated from raw and CIP lines by a physical break. In many plants, pipes are physically labeled with the type of product (raw or pasteurized) that flows through them. To be of continued use, blueprints must be routinely updated and reviewed for accuracy by “walking” the blueprints through the factory. Finally, no piping changes should ever be made without prior review by qualified authorities.

Filling a n d Packaging Postpasteurization contamination frequently occurs during filling and packaging operations when products are exposed to difficult-to-clean surfaces on equipment, the manufacturing environment, and airborne contaminants [54]. Areas associated with product contamination have included mandrels, drip shields, bottom and top breakers, prefilling coding equipment, deflecter bars, and cutting blades as well as overhead shielding, conveyors, conveyor belts, chain rollers, supports, and lubricants. Product extruder heads are particularly prone to contamination and therefore should be sanitized frequently during filling operations. Such practices will lead to the production of safe products with markedly increased shelf lives.

Reclaimed a n d Reworked Product Salvage programs, by their nature, are high-risk operations that can put an entire company in jeopardy if not done in a sanitary manner. Potential hazards associated with such salvage operations include (a) failure to repasteurize returned product before reuse; (b) inadvertently pumping returned but not repasteurized product through pasteurized product lines without proper cleaning and sanitizing between use; (c) accidental reuse of outdated product; (d) reuse of product returned from retail stores that may have been temperature abused, tampered with, or exposed to chemical or biological contamination; and (e) the use of product from contaminated, leaking, or otherwise damaged containers. Therefore, any product that left the possession and control of the processor or has been mishandled, inadequately protected from Contamination, or exposed to temperatures of 27.2”C (45°F) should be discarded. Dairy processors also should seriously consider confining the use of reclaimed and repasteurized milk to dairy products prepared from non-Grade A milk. According to the Pasteurized Milk Ordinance, American dairies involved in reclaiming programs now must have separate areas or rooms isolated from Grade A milk operations for receiving, handling, and storing all returned products. Outdated products

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and those which have left the control of the processor and later are returned to the dairy for disposal should never reenter the factory. Given the recent isolation of L. monocytogenes and other microbial contaminants from the external surface of cartons containing returned product, along with the proven ability of L. monocytogenes and Salmonella spp. to survive up to 14 days on the external surface of both waxed cardboard and plastic-type milk containers [72], the process of opening containers and emptying reclaimed product into vats for reprocessing will likely introduce many new unwanted microbial contaminants into the factory environment. Therefore, it is imperative that all returned products be handled similar to raw milk and be repasteurized, preferably using times and temperatures well above the required minima. After reprocessing, all equipment including tanks, pumps, and pipelines used in the reclaiming operation should be thoroughly cleaned and sanitized. In view of the problems associated with salvage operations, each dairy processor needs to reevaluate the advantages and disadvantages involved in reclaiming products and then decide whether or not the monetary benefits gained by such practices will outweigh the potential public health and other risks.

Frozen Dairy Products Although few bacterial species can grow at temperatures below OOC, most microorganisms, including listeriae, can survive for long times in frozen dairy products such as ice cream, ice cream novelties, and sherbet. Unlike fluid milk, frozen dairy products are particularly susceptible to microbial contamination during freezing and filling operations. All barrel freezers used to make frozen dairy products should be thoroughly sanitized before use, since hand assembly of the many intricate freezer parts is likely to introduce numerous contaminants. The source of air for the barrel freezer is another likely source of contamination. Hence, in addition to maintaining positive air pressure in this area and keeping the surrounding area as clean and sanitary as possible, all air lines connected to the barrel freezer should be equipped with dryers and bacterial filters to prevent airborne contaminants from entering the product. Ingredient feeders are perhaps the greatest source of Contaminants in frozen dairy products. Therefore, fruits, nuts, candy, and other ingredients that are added directly to frozen ice cream mix need to be closely monitored for coliforms, pathogens, and other microbial contaminants. Exposure of ingredients to the factory environment also should be minimized. Strict adherence to GMPs is necessary during the production of molded, extruded, and/or dipped ice cream novelties, since many such products have been recalled because of contamination with L. monocytogenes (see Table 5 in Chapter 11). Condensate in and around hardening rooms as well as conveyor belts appears to be a likely source for such contaminants. Finally, handling of product rerun exiting the freezer needs to be assessed at each factory. Although rerun product should never be added directly back to tanks containing unfrozen mix, frozen rerun product can be reclaimed by blending it with fresh mix, which is then repasteurized. Any rerun that is not reclaimed should be clearly separated from reclaimable material and properly disposed.

Fermented Dairy Products Fortunately, the incidence of Listeria contamination in yogurt, cultured cream, cultured buttermilk, and other fermented fluid milk products appears to be quite low with very few recalls being issued for these products. The species of lactic acid bacteria used in manufacturing these products as well as the bacteriostatic and bactericidal effects of vari-

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ous organic acids produced during fermentation and the resultant lowering of pH are undoubtedly responsible for the near-absence of such recalls. However, since bacterial pathogens (including L. monocytogenes) and various spoilage organisms may inadvertently contaminate fermented milk products during any stage of manufacture, producers of such products need to follow GMPs and be readily aware of potential problems regarding improper cleaning and sanitizing of equipment and the processing plant environment as well as potential sources of postpasteurization contamination (e.g., filling and packaging areas) discussed earlier in this chapter. The production of Listeria-free cheese, particularly soft and semisoft varieties surface-ripened with mold (e.g., Brie, Camembert) or bacteria (e.g., brick, Limburger), is difficult, since environmental conditions required for proper cheese ripening also promote the growth of L. monocytogenes and other unwanted organisms. Swiss officials who investigated the 1987 listeriosis outbreak involving consumption of Vacherin Mont d' Or softripened cheese (see Chapter 12) eventually isolated the epidemic strain of L. monocytogenes from wooden shelves and cheese hoops found in over half of the caves used to ripen the tainted cheese. Thus the basic problem associated with soft cheese manufacture is to prevent postprocessing contamination by eliminating L. monocytogenes from the ripening room and particularly the shelves on which such cheese must be ripened. Considering the ability of L. monocytogenes to grow very rapidly both inside and on the surface of Brie, Camembert, brick, Limburger, and other similar cheeses during ripening, manufacturers of such products should test a portion of each lot for listeriae before releasing the product for sale. In addition to these concerns, several studies have demonstrated that L. monocytogenes can survive well beyond 60 days in brick, Cheddar, and other varieties of cheese that were prepared from pasteurized milk inoculated with the pathogen. Certain cheeses, primarily hard and semihard varieties, can be manufactured from raw milk in the United States and elsewhere if the finished product is aged a minimum of 60 days at or above 1.7"C (35°F) to eliminate pathogenic microorganisms. However, since experimental evidence has indicated that this process is inadequate to free contaminated cheese from viable cells of L. monocytogenes, cheesemakers should consider preparing cheese from pasteurized milk whenever possible.

Meat Industry Since Listeria spp., including L. rnonocytogenes, are virtually endemic to slaughterhouse environments, meat processors are faced with an almost impossible challenge of producing Listeria-free raw meats. Direct application of lactic and/or acetic acid to animal carcasses is one of the few economically feasible means by which meat processors can effectively reduce populations of listeriae and other surface contaminants, including common spoilage organisms [ 16,28,62]. Nevertheless, although adoption of this procedure and following the general guidelines for controlling listeriae in food-processing establishments will benefit slaughterhouse operators, it appears unlikely that rigid enforcement of even the most stringent slaughter, dressing, cleaning, and sanitizing procedures will completely eliminate L. monocytogenes from wholesale and retail cuts of raw beef, pork, and lamb. Therefore, consumers of such products need to understand the potential health hazards associated with consumption of less than thoroughly cooked meats and also must follow appropriate hygienic practices in the kitchen to prevent the spread of listeriae from raw meats to readyto-eat foods. Firms producing processed meat products must assume that all incoming raw meat

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is potentially contaminated with listeriae, including L. monocytogenes. Since most Listeria contamination of finished product appears to result from postprocessing contamination rather than from the organism surviving various processing treatments, it is essential to segregate raw and finished products as well as employees working in raw and finished product areas of the factory. Although there is no “magic bullet’’ for Listeria control, the incidence of listeriae in all areas of the factory can be greatly reduced through conscientious enforcement of a stringent cleaning and sanitation program. One six-step program that has been recommended for cleaning food contact surfaces [7] includes (a) an initial dry clean-up step to remove as much product residue as possible, followed by (b) a warm water rinse (with minimum splashing) to mobilize fat and remove product; (c) cleaning with an appropriate foaming detergent; (d) warm or hot water rinse with minimum splashing; (e) disinfecting with an appropriate sanitizing agent (i.e., chlorine or quaternary ammonium compound); and finally (f) thorough drying of the cleaned and sanitized area. According to Boyle et al. [34,35], L. monocytogenes populations in inoculated samples of carcass rinse fluid, Hobart meat grinder rinse fluid, and floor drain waste water obtained from a beef- and lamb-processing facility increased one to four orders of magnitude during 24 h of incubation at 8 and 35°C with the pathogen exhibiting shorter generation times in waste fluids containing 3.1 rather than 5 I .4% protein. Hence, although the procedure just described may seem adequate, routine random testing for Listeria and coliforms as well as an estimation of the general microbial load on cleaned and sanitized food-contact surfaces should be done as an integral part of any sanitation program. In 1987, the American Meat Institute published some interim guidelines for controlling the incidence of listeriae and other pathogenic and nonpathogenic microbial contaminants during production of ready-to-eat meat products [2]. Although the recommendations in this report regarding facility requirements, factory environment, food-contact and nonfood-contact surfaces, cross contamination, airborne contamination, condensation control, cleaning and sanitizing, traffic patterns, and personnel cleanliness are generally similar to those already presented in this chapter as General Guidelines, this report also outlined some of the critical operations associated with the production of specific categories of ready-to-eat meat products

Roast Beef, Corned Beef, a n d Other Rebagged Products Products such as roast beef and corned beef that are repackaged after cooking are particularly prone to contamination with listeriae and other microorganisms. Therefore, attention must be given to proper sanitation and prevention of cross Contamination when these products are removed from bags in which they were cooked. The outside surface of all bags should be thoroughly washed and sanitized before the bags are opened. In addition to a sanitary working environment, repackaging of cooked product requires use of clean clothing as well as frequently sanitized utensils and gloves. Trimming and cutting of cooked product just before rebagging are two more critical steps where listeriae and other contaminants can enter and compromise the integrity of the final product. Therefore, contact between cooked product and unsanitized surfaces must be avoided during rebagging operations. Since repackaging is by nature a wet process, this operation also needs to be well isolated from other processing areas to reduce cross contamination.

Frankfurters and Other Link Products Sausages such as frankfurters and other link varieties are typically prepared from a finely ground mixture (or emulsion) of beef and/or pork, which is stuffed into artificial or natural casings. After twisting the casing at approximately 6-inch intervals, the links are

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cooked using steam or hot water and then hung for smoking. To obtain skinless frankfurters, the artificial casing must be mechanically peeled from the congealed meat mixture. Although prompt attention to cleaning, sanitizing, and cross-contamination problems is required during all stages of frankfurter production, the product is particularly vulnerable to contamination with listeriae and other microorganisms during the peeling process. It is imperative to keep the area around peeling machines as dry and as free from meat scraps and juices as possible. Peeling machine operators also need to change protective garments and gloves frequently. Hoods on peeler machines have been cited as a source of listeriae and should therefore be eliminated if at all possible. Manufacturing practices also should be reviewed to ensure that losses from floor contamination and reworked product are minimized. Although unpeeled frankfurters that touch the floor or other unclean surfaces can be reworked (i.e., washed and peeled after all other frankfurters have been peeled), any peeled frankfurters that come in contact with the floor or other unclean surfaces should be destroyed. This latter recommendation is supported by data indicating that L. monocytogenes is difficult to destroy on the surface of frankfurters during cooking without making the product organoleptically unacceptable [ 131. In addition to these concerns, brine chillers also have been cited as a potential source of listeriae, thus leading to contamination of casings and product surfaces. Finally, all packaging and heat-shrinkmg equipment should be cleaned and sanitized daily to avoid spreading contaminants from steam and water to packaging lines.

Luncheon Meats Concerns regarding control of listeriae and other contaminants in luncheon meats are generally similar to those just discussed for frankfurters and other link products. However, in addition, slicing equipment should be kept dry and free of scraps and juices that may serve as potential nutrients for microbial contaminants, including listeriae.

Poultry Industry Potential sources of listeriae contamination during processing of raw poultry are in many ways similar to those just discussed for the meat industry. Since a substantial percentage of birds harbor Listeria spp. (including L. monocytogenes) and Salmonella in their intestinal tract, enforcement of proper clean-up (i.e., elimination of water, condensate, and waste) and cleaning and sanitizing programs will likely decrease the incidence of contamination but will never completely eliminate these pathogens from raw poultry-processing facilities or the raw product. Most modern poultry-processing facilities are continuous line operations in which incoming birds are shackled, electrically stunned, bled, scalded to facilitate feather removal, plucked of feathers, eviscerated, inspected, washed, chilled, dried, and packaged for sale. Processing steps during which L. rnonocytogenes, Salmonella spp., and other pathogens are most likely to contaminate the product include scalding, defeathering, evisceration, and chilling [63,64]. In 1988, USDA officials proposed processing changes that may be helpful in decreasing the incidence of Salmonella (and presumably Listeria) in raw poultry [ 191. These changes included (a) segregating and processing pathogen-infected flocks at different times from noninfected flocks; (b) examining the potential benefits of adding bactericidal concentrations of organic acids to chill water tanks; (c) experimentation with different scalding methods (e.g., hot water sprays, steam scalders, or scald additives); (d) routine sanitizing of all equipment and utensils with hot water or bactericidal

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agents; (e) reemphasis of employee hygiene programs with routine handwashing and sanitizing required by all evisceration line workers; (f) elimination of off-line processing; and (g) installation of equipment designed automatically to transfer carcasses from the picking line to the evisceration line. Additional work is needed to streamline further processing of poultry carcasses and minimize cross contamination during their processing. With increasing consumption of poultry both in and outside the home, persons preparing these products must take special precautions to prevent the spread of L. monocytogenes, Salmonella spp., and other foodborne pathogens from raw poultry to other products (e.g., fruits and vegetables) that are frequently consumed without heating. The common practice of washing and rinsing raw poultry before coohng has been questioned, since this step fails to reduce microbial populations markedly on poultry skin and also leads to increased contamination of kitchen sinks, faucets, and other food preparation areas [79]. Since all foodborne pathogens commonly associated with raw poultry (including L. monocytogenes) are readily susceptible to heat, thorough cooking appears to be the best means of assuring that such products are free of hazardous microorganisms. An Oklahoma breast cancer patient contracted listeriosis in December of 1988 after consuming Listeria-contaminated turkey frankfurters. Thus producers of processed poultry products (e.g., turkey and chicken frankfurters and rolls) need to take precautions similar to those previously described for the manufacture of roast beef, corned beef, frankfurters, link sausage, and luncheon meats with special attention being given to the cleanliness of rebagging operations and sausage peelers.

Egg Industry As stated earlier, the contents of intact whole eggs are normally sterile unless the laying hen infects the yolk with Salmonella enteritidis. Foodborne pathogens, including L. monocytogenes and S. enteritidis, have frequently been isolated from commercially broken, raw liquid whole egg, with contamination most likely resulting from the presence of the organisms in the manufacturing environment or on eggshells. Although pasteurization as required for commercially broken, raw liquid whole egg is likely sufficient to eliminate normally encountered populations of L. monocytogenes and salmonellae in raw liquid egg, all egg-breaking operations need to be well isolated from pasteurization and filling and packaging areas to minimize recontamination of finished product. Since L. monocytogenes and other foodborne pathogens probably enter egg-processing facilities as eggshell contaminants, egg receiving and washing sections of the factory also should be segregated from other processing areas. Considering the potential for postpasteurization contamination, many of the previously described guidelines for cleaning and sanitizing dairy factories also appear to be applicable to manufacturers of pasteurized liquid egg products.

Fish and Seafood Industry L. monocytogenes and other foodborne pathogens such as Vibrio, Salmonella, Shigella, Staphylococcus aureus, Clostridium botulinum, Aeromonas hydrophila, and certain strains of Escherichia coli have been isolated from raw and/or cooked finfish, shrimp, crab, lobster, oysters, and scallops. An integrated approach to product safety is needed to minimize contamination of seafood from harvest to the time of consumption. On December 18, 1997, the FDA adopted the final regulations to ensure the safe and sanitary processing of fish and fishery products [75a]. The regulations mandate the application of HACCP principles to the processing of seafood. Seafood processors and

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importers need to evaluate the kinds of hazards that could affect their products, institute controls to keep these hazards from occurring, or significantly to minimize their occurrence, monitor the performance of those controls, and maintain records of this monitoring as a matter of routine practice. Limiting postharvest contamination of freshly caught fish and seafood is the first step toward producing a safe, high-quality endproduct. Adherence to good sanitation and hygienic practices aboard fishing vessels is imperative. Contact between freshly caught seafood and waterfowl such as pelicans and seagulls should be minimized, because these birds are intestinal carriers of L. monocytogenes and other foodborne pathogens. All seafood should be either frozen or refrigerated immediately after harvest to stop or retard growth of microbial contaminants, including spoilage organisms. Two observations, namely, (a) the routine recovery of healthy rather than thermally or otherwise injured listeriae from processed seafood and (b) the discovery of L. monocytogenes in the manufacturing environment of all American factories that have been involved in Listeria-related recalls, indicate that this pathogen enters the product primarily after processing through improper handling. Inadequate separation between raw and finished product resulting from faulty factory design and indifferent attitudes of employees toward proper sanitation have been most frequently cited as factors that promote postprocessing contamination. The general guidelines that were discussed previously regarding factory design, processing environment, proper cleaning and sanitizing, employee traffic patterns, and personnel cleanliness also are valid for the seafood industry. In addition to these recommendations, seafood processors also are urged to (a) eliminate processing waste, pooled water, and condensate from walls, floors, and ceilings as well as from processing and refrigerated areas; (b) eliminate the use of high-pressure sprays; (c) reduce airborne contamination; (d) cover outside dumpsters to decrease problems involving seagulls and other wildlife; (e) assign specific equipment (i.e., product totes) for use in either raw or cooked product areas of the factory; and (f) if possible, replace wooden totes with fiber totes, which can be easily cleaned and sanitized. Listeria spp., including L. monocytogenes, have been isolated most frequently from crabmeat and cooked and peeled shrimp (see Table 4 in Chapter 15). This observation is not surprising if one considers how these products are processed and packaged for the consumer. Processing of Dungeness crab generally begins by immersing and cooking either sections of or the entire crab in boiling water for approximately 7-9 or 17-20 mins, respectively. Although current information indicates that such a heat treatment is sufficient to destroy listeriae [43], underprocessing may lead to survivors. After cooking, the crab is cooled in a water bath and either “picked” immediately or iced and refrigerated in a walk-in cooler until the meat can be hand picked from the shell. Extensive handling of the product by workers during picking, subsequent inspection, and packaging affords many opportunities for postprocessing contamination. Although lactic acid dips appear to be somewhat useful in reducing populations of L. monocytogenes and other microorganisms on the surface of crabmeat as well as fresh and frozen shrimp, such treatments will not completely eliminate listeriae from the finished product [43]. Therefore, strict adherence to GMPs, which include proper employee hygiene and cleaning and sanitizing of picking equipment, must be observed in and among picking areas to avoid negating the benefits of cooking. Unfortunately, crab processing varies widely with the species of crab-Dungeness, blue, stone, king, and golden crab. Hence, some of the critical control points discussed for Dungeness crab are not applicable to other species. For example, blue crabmeat is

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typically removed from the animal in the raw state, placed in sealed containers, and then pasteurized (85"C/ 1 min) to eliminate L. monocytogenes and other microbial pathogens. Since pasteurization of blue crabmeat becomes a critical control point in processing, it may be prudent to certify and/or license crabmeat pasteurization operators or their supervisors, as has been required for operators of retorts in the canning industry for many years. Problems regarding postprocessing contamination also are encountered during the production of cooked and peeled shrimp. After shrimp are cooked, those destined for breading are mechanically peeled and sometimes deveined by splitting and removing the vein-like intestine. Unfortunately, many mechanical shrimp peelers have design flaws which necessitate almost continuous movement of the operator between both raw and cooked sides of the equipment, thus affording ample opportunity for postprocessing contamination. Proper cleaning and sanitizing of the equipment (particularly protective covers over flumes and gutters) and the surrounding area are essential for producing high-quality microbiologically safe products. Even when handled under the best possible conditions, raw seafood such as crab, shrimp, lobster, clams, oysters, and the myriad of finfish currently available to consumers probably will never be completely free of L. monocytogenes or other foodborne pathogens. Considering that many individuals are not ' 'seafood-smart,' ' processors and marketers of seafood have an obligation to educate the general public and provide consumers with proper haridling and cooking instructions. Individuals who insist on consuming unprocessed fish (e.g., sushi) and seafood (e.g., oysters) also should be made aware of potential health problems associated with consumption of such products.

Fruit and Vegetable Industry Despite a limited amount of information concerning incidence of L. monocytogenes in raw fruits, the pathogen has been recovered from raw vegetables including cabbage, cucumbers, inushrooms, potatoes, and radishes. Other than the 198 1 Canadian listeriosis outbreak involving coleslaw and one isolated case in Finland linked to consumption of raw salted mushrooms, no additional confirmed cases of vegetableborne listeriosis have been documented in the literature. Thus the scientific community and the public at large have been, until recently, somewhat less concerned about Listeria contamination in vegetables than in dairy, meat, poultry, and seafood products. Routine examination of raw vegetables for L. monocytogenes and other foodborne pathogens is unlikely to reduce the risk of foodborne illness to any great extent. However, since raw sheep manure was the probable source of L. monocytogenes in the Canadian coleslaw outbreak, vegetable processors should have some assurance that incoming raw vegetables have been grown, irrigated, fertilized, harvested, packaged, and transported to the firm using hygienically sound agricultural practices. Vegetable processors should consider rejecting raw vegetables that probably will be consumed without cooking if the grower fails clearly to demonstrate the use of good agricultural practices. Consumption of vegetables that will be adequately cooked before eating is of little concern, since L. monocytogenes and other non-spore-forming pathogens are destroyed during cooking. Although routine washing of raw vegetables in potable water is recommended for commercial establishments and homes, this practice generally fails to reduce the microbial load on raw vegetables by more than 10-fold. Therefore, persons handling and preparing raw produce and salad vegetables should follow good hygienic practices during slicing, dicing, chopping, and grating operations to prevent the spread of potentially

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hazardous microorganisms to other foods. Finally, all knives, cutting boards, and other food-contact surfaces should be thoroughly cleaned and sanitized after use to inactivate organisms inadvertently introduced into the kitchen environment during preparation of raw produce.

PRACTICAL APPROACHES TO FOOD SAFETY Traditional Approaches The traditional approaches to controlling microbiological hazards associated with food products involve the simultaneous use of employee education and training programs, frequent inspection of facilities and operations, extensive microbiological testing of raw ingredients, and unfinished and finished products. Employee education and training programs should be directed toward a thorough understanding of food hygiene, factory cleaning and sanitation requirements, and various causes of microbial contamination, including growth and survival patterns of potential contaminants such as listeriae. Trained employees also should be able to select and apply control methods that will provide consumers with safe, high-quality products. The second means of controlling microbiological hazards, frequent inspection of facilities and operations, is necessary to ensure that GMPs (i.e., procedures that consistently yield safe products of acceptable quality) are being followed. GMPs to produce specific foods have been outlined in both advisory and regulatory documents such as GMP guidelines and the various codes of hygienic practice developed by the Codex Alimentarius Committee on Food Hygiene. The final means of controlling microbial hazards in finished products is through rigorous microbiological testing of ingredients as well as unfinished and finished product. Analysis of samples for pathogens or, more commonly, indicator (coliforms, fecal streptococci) or spoilage organisms is crucial to ascertaining that good manufacturing, handling, and distribution practices are being followed.

Hazard Analysis Critical Control Point Concept Although the traditional approaches for controlling microbial contaminants are being used by many food companies around the world, cases of foodborne illnesses still occur. The need for a modified approach to food safety assurance led to the development of the Hazard Analysis Critical Control Point Concept (HACCP) which can be used to identify and control biological, chemical, and physical hazards in foods from raw material production, procurement, and handling to manufacturing, distribution, and consumption of the finished product. Although a detailed discussion of HACCP is beyond the scope of this book, a brief explanation will assist the reader in understanding how the concept, along with GMPs and prerequisite programs, can be used to reduce the level of L. monocytogenes in food processing facilities and subsequently in cooked, ready-to-eat foods. The HACCP concept was developed by the Pillsbury Company with the cooperation and participation of the National Aeronautics and Space Administration (NASA), the Natick Laboratories of the U.S. Army, and the U.S. Air Force Space Laboratory Project Group [32a]. The development of the HACCP system began in 1959 when Pillsbury was asked to produce a food that could be used in the space program. There needed to be assurance (as close to 100% as possible) that the food produced for the space program would not be contaminated with bacterial or viral pathogens, toxins and chemical or physi-

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cal hazards that could cause illness or injury. After much research and evaluation, the HACCP concept was developed and first presented to the scientific community at the 1971 Conference for Food Protection [74b]. The HACCP concept was first used in the acidified and low-acid canned food industry and was then adopted by a number of companies during the 1970s and early 1980s. After an important 1985 National Academy of Sciences publication strongly recommended the HACCP concept, the food industry expressed considerable interest in the application of HACCP. The National Advisory Committee on Microbiological Criteria for Foods (NACMCF) was established and embraced the HACCP concept. In 1989, the committee developed a HACCP document as a guide for maintaining uniformity of the principles and definitions of terminology [61a]. Since then, the NACMCF has made several refinements and improvements in the HACCP concept and published revisions in 1992 [61b] and 1997. The 1997 document, entitled HACCP Principles and Guidelines [61c], contains many additions and includes a section on prerequisite programs. Prerequisite programs are essential to the successful development and implementation of a HACCP plan [70b] and form the foundation upon which a HACCP plan is built. Many of the prerequisite programs are based on the current GMPs in the Code of Federal Regulations [43a] and in the Codex Alimentarius General Principles of Food Hygiene [53b] for foods intended for international trade. In addition to specific items in the GMPs, prerequisite programs can include other activities such as ingredient specifications, supplier approval programs, ingredient-to-product traceability, and consumer complaint management programs. A summary of prerequisite program activities is presented in Table 19 [70b]. Prerequisite programs are not part of the formal HACCP system and are established and maintained separately. There are some circumstances where the existence of a prerequisite program does not preclude the use of specific activities with a HACCP system [70b]. For example, although sanitation procedures are normally part of a prerequisite program, some manufacturers manage selected sanitation procedures as critical control points (CCPs) in their HACCP systems. This has been done frequently in the meat and dairy industries where sanitation procedures for meat slicers, ice cream fillers, and other pieces of equipment were established as CCPs to help prevent recontamination of processed products by L. rnonocytogenes [70b]. The existence and effectiveness of prerequisite programs should be assessed during the design and implementation of each HACCP plan. Well-developed and consistently performed prerequisite programs can simplify the HACCP plan, so it is imperative that all food processors establish, document, and maintain effective prerequisite programs to support their HACCP plans [70b]. HACCP is a management system that is designed for use in all segments of the food industry from production agriculture to consumption of the finished product. The HACCP approach for controlling biological hazards in food is based on seven principles [61c]:

1. 2. 3. 4. 5. 6. 7.

Conduct a hazard analysis Determine the critical control points Establish critical limits Establish monitoring procedures Establish corrective actions Establish verification procedures Establish record-keeping and documentation procedures.

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TABLE 19 Summary of Prerequisite Program Activities -~

Facilities Adjacent properties Building exterior Building interior Traffic flow patterns Ventilation Waste disposal facilities Sanitary handwashing facilities Water, ice, culinary steam Lighting Raw Materials Controls Specifications Supplier approval Receipt and storage Temperature control Testing procedures Sanitation Master schedules Pest control program Environmental surveillance activities Chemical control programs Training Personal safety GMPs HACCP Production Equipment Sanitary design and installation Cleaning and sanitation Preventive maintenance Calibration of equipment Production Controls Product zone controls Foreign material control Metal protection program Allergen control Glass control Storage and Distribution Temperature control Transport vehicle cleaning and inspection Product Controls Labeling Product traceability Customer and consumer complaint investigations Source: Adapted from Ref. 70b.

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Principle 1: Conduct a Hazard Analysis The hazard analysis is the key element in developing an effective HACCP plan. The purpose of the hazard analysis is to determine which of the potential hazards associated with a food or a manufacturing process presents a significant risk to consumers. The hazard analysis involves two stages. The first stage, hazard identification, involves the review of ingredients used in the product, the activities conducted at each step in the process and the equipment used, the final product and its method of storage and distribution, and the intended use and consumers of the product. Hazard identification focuses on developing a list of potential food hazards associated with each process step. In stage two, the hazard evaluation, each potential hazard is evaluated based on severity and its likelihood of occurrence. It is important that food processors conduct a hazard analysis on all existing and any new products to be manufactured, since ultimate microbiological safety of nonthermally processed foods is directly related to the quality of raw materials. Any hazard analysis must begin with identification of hazards associated with raw materials, with particular attention being given to raw products of animal origin (i.e., milk, meat, poultry, and seafood), all of which may harbor L. monocytogenes and other foodborne pathogens. Although heat. treatments, acidulation, fermentation, salting, and drying are designed to destroy or inhibit growth of pathogenic and spoilage microorganisms, other operations such as slicing and dicing, cooling of cooked products, and filling and packaging may allow pathogenic organisms to contaminate the final product. Therefore, all hazards associated with manufacturing procedures and postprocessing contamination, as previously discussed, must be fully understood along with the consequences of processing failures and/ or errors. Food processors should also be familiar with the effect of various physicochemical factors (i.e., pH, water activity, preservatives, and type of packaging with or without modified atmosphere) on the behavior of pathogenic organisms, including L. monocytogenes, in the product during processing, distribution, storage, and use by the consumer. The National Advisory Committee HACCP document [61c] contains a series of questions regarding ingredients, intrinsic factors of the food during and after processing, processing procedures, microbial content of the food, faulty design, equipment design and use, and packaging sanitation that can be used when conducting a hazard analysis. It should be noted that any change in raw materials, product formulation, processing, packaging, distribution, or intended use of the product should prompt an immediate reassessment of hazards, since these changes have the potential to affect product safety adversely.

Principle 2: Determine CCPs A CCP is a step at which control can be applied and is essential to prevent or eliminate a food safety hazard or reduce it to an acceptable level. Complete and accurate identification of CCPs is fundamental to controlling food safety hazards, and CCPs must be carefully developed and documented. Examples of CCPs may include thermal processing, chilling, and producl formulation control.

Principle 3: Establish Critical Limits A critical limit is a boundary of safety and is used to distinguish between safe and unsafe operating conditions at a CCP. Each CCP will have one or more control measures to assure that the identified hazards are prevented, eliminated, or reduced to acceptable levels. Critical limits may be based on factors such as temperature, time, physical dimensions,

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humidity, moisture level, water activity (aw), pH, titratable acidity, salt concentration, available chlorine, viscosity, or preservatives. Critical limits must be scientifically based and may be obtained from regulatory standards and guidelines, the scientific literature, experimental results, and experts.

Principle 4: Establish Monitoring Procedures Monitoring is a planned sequence of observations or measurements to assess whether a CCP is under control and to produce an accurate record for future use in verification. Monitoring facilitates the tracking of an operation and is used to keep the process in control. Monitoring is also used to determine when there is loss of control and a deviation occurs at a CCP (i.e., exceeding or not meeting a critical limit). Monitoring procedures must be effective to determine deviations and then corrective actions must be taken. Most monitoring procedures need to be rapid and often include visual observations and measurement of temperature, time, pH, and moisture level. Microbial tests are seldom effective for monitoring owing to their time-consuming nature and problems with assuring detection of contaminants.

Principle 5: Establish Corrective Actions When there is a deviation from an established critical limit, corrective actions are necessary. Through the establishment of corrective actions, foods that may be hazardous are prevented from reaching consumers. When there is a deviation from critical limits, corrective actions are needed to: Determine and correct the cause of noncompliance. Determine the disposition of noncompliant product. Record the corrective actions that are taken. Specific corrective actions should be developed for each CCP and included in the HACCP plan.

Principle 6: Establish Verification Procedures Verification determines the validity of the HACCP plan and is used in evaluating whether the facility’s HACCP system is functioning according to the HACCP plan. An effective HACCP system requires little endproduct testing, since sufficient validated safeguards are built in early in the process. Firms should rely on frequent reviews of their HACCP plan, verification that the plan is being correctly followed, and review of CCP monitoring and corrective action records. Another important aspect of verification is the initial validation of the HACCP plan to determine that the plan is scientifically and technically sound, that all hazards have been properly identified, and that if the HACCP plan is properly implemented, these hazards will be effectively controlled. Subsequent validations are performed and documented by the HACCP team or independent expert as needed. Validations are conducted when there is an unexplained system failure, when a significant product, process, or packaging change occurs, or when new hazards are recognized.

Principle 7: Establish Record-Keeping and Documentation Procedures The establishment of an effective record-keeping system is an integral part of a HACCP system. Records are the only reference available to trace the production history of a fin-

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ished product. If questions arise concerning the safety of a product, a review of records may be the only way to prove that the product was prepared and handled in a safe manner in accordance with the company's HACCP plan [51a]. A well-developed and implemented HACCP plan built on a strong foundation of GMPs and prerequisite programs can reduce the level of L. monocytogenes in foodprocessing facilities and in cooked, ready-to-eat foods.

SAMPLING PLANS FOR L. MONOCYTOG€/V€SIN FOODS Prevention of microbiological hazards is clearly of considerable importance when one considers the serious health problems that may develop in certain individuals who consume Listeria-contaminated foods. Consequently, the International Commission on Microbiological Specifications for Foods (ICMSF) proposed that L. monocytogenes be placed in the same category with Brucella, Clostridiurn botulinurn, C. pe$ringens type C, Salrnonella typhi, Shigella dysenteriae, Vibrio cholera, and hepatitis A virus [ 121, all of which pose severe health hazards. In February 1988, the ICMSF considered application of its sampling plans to assess acceptability of foods with respect to L. monocytogenes. (The reader must be cautioned from the start that no microbiological sampling plan other than one which involves total destructive sampling of all products manufactured can ever provide complete consumer protection. j According to terminology developed previously by the ICMSF, sampling plans for L. monocytogenes would follow the recommendations made for cases 13, 14, and I5 [52]. Case 13 applies when conditions under which the product is normally handled and consurned after sampling reduce the degree of hazard associated with the product, whereas cases 14 and 15 refer to situations in which hazard levels remain constant or increase, respectively. Using a statistically based two-class attribute sampling plan, n (i.e., number of sample units to be examined from a particular lot) would equal 15, 30, or 60 for cases 13, 14, and 15, respectively, and c (i.e., the maximum allowable number of sample units containing L. rnonocytogenes) would equal 0 for all three cases. A three-class attribute sampling plan also was proposed in the United States by a working group of the National Advisory Committee on Microbiological Criteria for Foods [22]. According to this plan, which was developed for ready-to-eat shrimp and crabmeat, n (i.e., number of samples for foods produced in facilities employing HACCP and GMP systems) would equal 10, whereas c (i.e., mandatory standard for L. monocytogenes that should not be exceeded) would equal 0. Thus, with the exception of n, this plan is similar to the two-class attribute plan proposed by the ICMSF. Before recommending any Listeria-sampling plan, there must be good epidemiological evidence indicating that the product or product group to be sampled has been implicated in foodborne listeriosis. In addition, there must be good reason to believe that introduction of a sampling program will substantially reduce the risk of contracting listeriosis from consumption of such products. Based on information collected in 1988, the ICMSF made a series of recommendations concerning sampling plans for listeriae in milk, soft cheeses, and vegetables [ 121. Although I,. monocytogenes is commonly found in raw milk, minimum required pasteurization (7 1.7"C/ 15 s) should eliminate this hazard. Therefore, the ICMSF recommended that manufacturers institute monitoring programs to prevent postpasteurization contamination rather than routine sampling plans of pasteurized endproducts as the most appropriate means of protecting the consumer.

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Many varieties of Listeria-contaminated cheese also have been identified since 1985, with contamination most frequently being reported in surface-ripened cheeses. Since L. monocytogenes can grow rapidly in Brie and Camembert cheese during the late stages of ripening, a two-class attribute sampling program should be considered if such cheeses are destined for consumption by pregnant women, immunocompromised adults, or the elderly. However, since no sampling program can ensure that such products are completely free of L. monocytogenes, public health interests are far better served by application of HACCP principles during cheese manufacture and ripening. Although the ICMSF recognized that raw vegetables also may become contaminated with L. monocytogenes, routine testing of raw vegetables is unlikely to markedly reduce the risk of contracting listeriosis. Hence, consumers of raw vegetables are urged to wash all such products vigorously before consumption. Endproduct-sampling programs are not the answer to protecting consumers from listeriosis or other types of foodborne illness. However, microbiological sampling is recommended by many regulatory agencies and the World Health Organization as part of the HACCP approach to prevent opportunities for contamination by, survival, and growth of L. monocytogenes as well as other microbial pathogens in raw materials, factory environments, and food products during manufacture, storage, distribution, sale, and use.

Status of L. monocytogenes in Foods The status of L. monocytogenes in cooked, ready-to-eat foods is still being discussed and debated in scientific communities and regulatory agencies around the world. In the United States, public health and regulatory agencies have a zero tolerance for this organism, that is based on its ability to produce a life-threatening illness at a presumably low, but as yet unknown infectious dose and can grow at refrigeration temperatures. France, Germany, and the Netherlands accept up to 100 cfu/g [69a, 69bl. In Germany, if the level of L. monocytogenes is in the range of 100- 1000 cfu/g, the product is reprocessed. In the Nordic countries, a working group has recommended action be taken for food containing > 100 L. monocytogenes cfu/g [69a]. Denmark has established a zero tolerance for foods that have received a listericidal treatment after packaging. If > 10 organismdg are found, corrective HACCP actions are required. If the level of L. monocytogenes is > 100 organisms/ g, the product is recalled. The Danish position also states that if the organism can grow in a product and the shelf life exceeds 1 week, then there is also a zero tolerance with specific sampling plans. If the organism cannot grow, levels between 10 and 100 may be acceptable [69b]. (This is a simplification of the Danish position, as there are six food categories with associated sampling plans and acceptance criteria.) Italy has a zero tolerance policy in effect. In the United Kingdom, < 100 cfu/g of L. monocytogenes is considered fairly satisfactory, whereas 100- 1000 cfu/g is unsatisfactory and > 1000 cfu/g is unacceptable [69b]. In Australia, the Australia, New Zealand Food Authority (ANZFA) is developing a food standards code that specifies a zero tolerance for L. monocytogenes in ready-to-eat foods such as meat pastes, piit&,smoked fish, marinated smoked mussels, and cheese made from thermized milk that has a moisture content >40% and a pH >5 (V.N. Scott, personal communication, 1998). In Canada, the compliance criteria for L. monocytogenes in ready-to-eat foods are composed of three categories [42a]:

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Category 1. Ready-to-eat foods causally linked to listeriosis, including soft cheeses, liver piite, coleslaw mix with a shelf life > 10 days, and jellied pork tongue. Action Level >O cfu/50 g Category 2. All other ready-to-eat foods supporting growth of L. rnonocytogenes with refrigerated shelf life of >10 days. Action Level >O cfu/25 g Category 3. Ready-to-eat foods supporting growth of L. rnonocytogenes with refrigerated shelf life < 10 days and all ready-to-eat foods not supporting growth. Action Level 5 1 0 0 cfu/g depending on the plant’s GMPs In Brazil, for cheeses with a moisture content of >36%, no L. rnonocytogenes is allowed in a 25-g sample [69a]. In Asian countries, there are no policies for the level of L. rnonocytogenes in foods as yet [69a]. Owing to the ubiquitous nature of L. rnonocytogenes, most developed countries around the world are strongly emphasizing the use of GMPs and HACCP systems for reducing the levels of this organism in food-processing facilities. There is still considerable debate among scientists in industry, academia, and regulatory agencies on developing “acceptable” levels of L. rnonocytogenes in foods [70a], since the infectious dose is not yet known. At the present time, all intervention strategies should be used to reduce the level of this organism in foods. In conclusion, to reduce the incidence of L. rnonocytogenes in the food supply, food processors must develop and implement HACCP plans that are built on a strong foundation of GMPs and prerequisite programs which in turn will decrease the incidence of listeriosis.

REFERENCES 1. Adams, C.E. 1990. Use of HACCP in meat and poultry inspection. Food Technol. 44(5): 169- 170. 2. American Meat Institute. 1987. Interim guideline: microbial control during production of ready -to-eat meat products. Controlling the incidence of Listeria monocytogenes. American Meat Institute, Washington, DC. 3. Anonymous. 197 I . Workshop 2, Prevention of contamination of commercially processed foods. In Proceedings of the 1971 National Conference on Food Protection, U.S. Government Printing Office, Washington, DC, p. 56. 4. Anonymous. 1986. Food and Drug Administration dairy product safety initiatives-Preliminary status report. FDA Center for Food Safety and Applied Nutrition-Milk Safety Branch. Washington, DC, September 22. 5. Anonymous. 1987. FDA continues to find Listeria during dairy plant inspections. Food Chem. News 29( 1):47-48. 6. Anonymous. 1987. FDA convinced dairy industry can avoid Listeria contamination. Food Chem. News 29(39):3-4. 7. Anonymous. 1987. FSIS to give firms 5 days for clean-up before resampling for Listeria. Food Chem. News 29(32):7-9. 8. Anonymous. 1987. Ice cream industry seeks parity with meat industry on Listeria policy. Food Chem. News 29(23): 15. 9. Anonymous. 1987. Meat industry research shows Listeria widespread, control difficult. Food Chem. News 29( 17):27-29. 10. Anonymous. 1988. FDA regional workshops to discuss microbial concerns in seafoods. Food Chem. News 30(43):27-3 1.

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35. Boyle, D.L., G.R. Schmidt, and J.N. Sofos. 1990. Growth of Listeria monocytogenes inoculated in waste-fluids from clean-up of a meat grinder. J. Food Sci. 55:277-278. 36. Bradley, R.L., Jr. 1986. The hysteria of Listeria. Dairy Field 169(11):37, 57. 37. Breer, C. 1988. Occurrence of Listeria spp. in different foods. WHO Working Group on Foodborne Listeriosis, Geneva, Switzerland, Feb. 15- 19. 38. Charlton, B.R., H. Kinde, and L.H. Jensen. 1990. Environmental survey for Listeria species in California milk processing plants. J. Food Prot. 43: 198-201. 39. Cox, L.J. 1988. Listeria monocytogenes-a European viewpoint. General Assembly of IOCCC, Hershey, PA, April 28-30. 40. Cox, L.J. 1988. Prevention of foodborne listeriosis-the role of the food processing industry. WHO Informal Working Group on Foodborne Listeriosis, Geneva, Switzerland, February 15-19. 41. Cox, L.J., T. Kieiss, J.L. Cordier, C. Cordellana, P. Konkel, C. Pedrazzini, R. Beumer, and A. Siebenga. 1989. Listeria spp. in food processing, non-food and domestic environments. Food Microbiol. 6:49-6 1. 41a. 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. 62:705-7 1 1. 41b. Doyle M.P. 1988. Effect of environmental and processing conditions on Listeria monocytogenes. Food Technol. 42: 169- 171. 41c. Eckner, K.F. 1990. Biofilms and food sanitation. Silliker Tech. Bull., SCOPE 5 : 1-4. 41d. Eklund, M.E., F.T. Poysky, R.N. Paranjpye, L.C. Lashbrook, M.E. Peterson, and G.A. Pelroy . 1995. Incidence and sources of Listeria monocytogenes in cold-smoked fishery products and processing plants. J. Food Prot. 58502-508. 42. Facinelli, B., P.E. Varaldo, M. Toni, C. Casolari, and V. Fabio. 1989. Ignorance about Listeria. Hr. Med. J. 299:738. 42a. Farber, J.M., and J. Harwig. 1996. The Canadian position on Listeria monocytogenes in ready-to-eat Foods. Food Control. 7(4/5):253-258. 42b. Flickinger, B. 1996. Plant sanitation comes to light: evaluation of ATP-bioluminescence systems for hygiene monitoring. Food Quality March:22-36. 42c. Flickinger, B. 1997. Light up your plant, part 11: into the laboratory. Food Quality June/ July:20-22. 42d. Flowers, R., L. Milo, E. Myers, and M.S. Curiale. 1997. An evaluation of five ATP bioluminescence systems. Food Quality June/July:23-33 43. Food and Drug Administration. 1988. Proceedings of the National Meeting on Cooked/ Processed Seafood, Food and Drug Administration, Center for Food Safety and Applied Nutrition, Washington, DC, December 16. 43a. Food and Drug Administration. 1997. Current Good Manufacturing Practices in Manufacturing, Packing or Holding Human Food. Code of Federal Regulations. No. 21, Part 110. U.S. Government Printing Office, Washington, DC. 44. Gabis, D.A., R.S. Flowers, D. Evanson, and R.E. Faust. 1989. A survey of 18 dry dairy product processing plant environments for Salmonella, Listeria and Yersinia. J. Food Prot. 52: 122- 124. 45. Garrett, S.E., and M. Hudak-Roos. 1990. Use of HACCP for seafood surveillance and certification. Food Technol. 44(5): 159-165. 46. Genigeorgis, C.A., D. Dutulescu, and J.F. Garayzabal. 1989. Prevalence of Listeria spp. in poultry meat at the supermarket and slaughterhouse level. J. Food Prot. 52:618-624, 630. 47. Genigeorgis, C.A., P. Oanca, and D. Dutulescu. 1990. Prevalence of Listeria spp. in turkey meat at the supermarket and slaughterhouse level. J. Food Prot. 53:282-288. 48. Gilbert, R.J. 1990. Personal communication. 49. Goff, H.D. 1988. Hazard analysis and critical control point identification in ice cream plants. Dairy Food Sanit. 8: 131- 135.

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Listeria in Food-Processing Facilities 67. 67a. 68. 69. 69a. 69b. 70. 70a. 70b. 71. 72. 73. 73a. 74. 74a. 74b. 75. 75a. 76. 77. 77a. 78. 79.

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Prentice, G.A. 1989. Living with Listeria. J. Soc. Dairy Technol. 4255-58. Pritchard, T.J., C.M. Beliveau, K.J. Flanders, and C.W. Donnelly. 1994. Increased incidence of Listeria species in dairy processing plants having adjacent farm facilities. J. Food Prot. 57:770-775. Radmore, K., W.H. Holzapfel, and H. Luck. 1988. Proposed guidelines for maximum acceptable air-borne microorganism levels in dairy processing and packaging plants. Intern. J. Food Microbiol. 6:91-95. Rank, R.J. 1989. Liquid eggs deviating from the standard of identity; temporary permit for rnarket testing. Fed. Reg. 54: 1794- 1795. Ryser, E.T. 1998. Personal communication. Scott, V.N. 1998. Personal communication. Shapton, N. 1988. Hazard analysis applied to control of pathogens in the dairy industry. J. Soc. Dairy Technol. 41 :62-63. Skinner, R. 1996. Listeria: UK Government’s approach. Food Control. 7(4/5):245-247. Sperber, W.H., K.E. Stevenson, D.T. Bernard, K.E. Deibel, L.J. Moberg, L.R. Hontz, and V.N. Scott. 1998. The role of prerequisite programs in managing a HACCP system. J. Dairy Food Environ. Sanit. 18:4 18-423. Spurlock, A.T., E.A. Zottola, and R.K.L. Petran. 1989. The survival of Listeria monocytogenes in aerosols. J. Food Prot. 52:751 (Abstr.). 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. Surak. J.G., and S.F. Barefoot. 1987. Control of Listeria in the dairy plant. Vet. Hum. Toxicol. 29:241-249, Sutherland, P. and R. Porritt. 1995. Dissemination and ecology of Listeria monocytogenes in Australian dairy factory environments. Proceedings of the XI1 International Symposium on Problems of Listeriosis Perth, Australia. pp. 29 1-297. Terplan, G. 1988. Listeria in the dairy industry-situation and problems in the Federal Republic of Germany. Foodborne Listeriosis-Proceedings of a Symposium, Wiesbaden, West Germany. Sept. 7, pp. 52-70. Tompkin, R.B., L.N. Christiansen, A.B. Shaparis, R.L. Baker, and J.M. Schroeder. 1992. Control of Listeria monocytogenes in processed meats. Food Australia 44:370-376. U.S. Department of Health, Education and Welfare. 1972. Proceedings of the 1971 Conference on Food Protection. U.S. Government Printing Office, Washington, DC. U.S. Food and Drug Administration and Milk Industry Foundation-International Ice Cream Association. 1988. Recommended guidelines for controlling environmental contamination in dairy plants. Dairy Food Sanit. 852-56. U.S. Food and Drug Administration. 1995. Procedures for the safe and sanitary processing and importing of fish and fishery products. Final Rule 2 1 CFR 123 and 1240. Federal Register 60 CFR, pp. 65095-65202. Venables, L.J. 1989. Listeria rnonocytogenes in dairy products-the Victorian experience. Food .4ustralia 4 1 :942-943. Wagner, L.R. Van. 1989. FDA takes action to combat seafood contamination. Food Proc. 50(2):8-12. Walker, R.L., L.H. Jensen, H. Kinde, A.V. Alexander, and L.S. Owens. 199 1. Environmental survey for Listeria species in frozen milk products plants in California. J. Food Prot. 54: 178- 182. Wimpfeimer, L., N.S. Altman, and J.H. Hotchkiss. 1990. Growth of Listeria rnonocytogenes Scott A, serotype 4 and competitive spoilage organisms in raw chicken packaged under modified atmospheres and in air. Intern. J. Food Microbiol. 1 I :205-214. Woodhurn, M. 1989. Myth: wash poultry before cooking. Dairy Food Environ. Sanit. 9:65-66.

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Appendix Media to Isolate and Cultivate Listeria monocytogenes and Lisferia spp.

FLUID MEDIA FOR ENRICHMENT OF LISTERIA SPP. Demi-Fraser broth* Proteose peptone Tryptone Lab-Lemco powder Yeast Extract NaCl KHZPO, Na2HP04 Esculin Ferric ammonium citrate Lithium chloride Nalidixic acid Acrifl avine Distilled H 2 0

5.0 g 5.0 g 5.0 g 5.0 g 20.0 g 12.0 g 12.0 g 1.0 g 0.5 g 3.0 g 10.0 mg 12.5 mg 1000 ml

FDA enrichment broth* Trypticase Soy Broth Yeast Acrifl avine Nalidixic acid Cycloheximide Distilled H 2 0

30 g 6g 15 mg 40 mg 50 mg 1000 ml

Fraser broth Proteose peptone Tryptone Lab-Lemco powder Yeast extract NaCl KH2I’O, 71 1

Appendix

7 12

Na2HP04 Esculin Nalidixic acid Lithium chloride Acriflavine Ferric ammonium citrate Distilled H 2 0

12 g 1g 20 mg 3g 25 mg 0.5 g 1000 ml

IDF pre-enrichment broth Peptone NaCl Na2HP04. 12 H 2 0 K2HP04 Distilled H 2 0

10 g 5g 9g 1.5 g 1000 ml

IDF enrichment broth* Tryptone soy broth Yeast extract Acriflavine . HCl Nalidixic acid Cycloheximide Distilled H 2 0

30 g 6g 10 mg 40 mg 50 mg 1000 ml

Listeria repair broth [LRB] Trypticase soy broth Yeast Extract Glucose Magnesium sulfate Ferrous sulfate Sodium pyruvate 3-N-morpholinepropanesulfonic acid-free acid 3-N-morpholinepropanesulfonicacid-sodium salt Distilled H 2 0

30.0 g 6.0 g 5.0 g 2.46 g 0.3 g 10.0 g 8.5 g 13.7 g 1000 ml

LRB with selective agents LRB After 3-4 h of enrichment at 30°C add: Acriflavine Cycloheximide Nalidixic acid L-PALCAMY broth Special peptone (Oxoid) Yeast extract Lab-Lemco powder Peptonized milk (Oxoid) NaCl D-Mannitol Esculin

225 ml 3.4 mg 12.5 mg 9.0 mg

713

Appendix

Ferric ammonium citrate Phenol red Polyniyxin I3 Acriflavine . HCl Lithium chloride Ceftazidime, Latamoxef, or Moxalactam Egg yolk emulsion Distilled H 2 0

0.5 g 80 mg 100,000 IU 5 mg 10 g 30 mg 25 ml 1000 ml

Rodriguez enrichment medium 1 Peptone Neopeptone Lab-L,emco powder Yeast extract Glucose NaCl Disodium phosphate-2-hydrate Potassium phosphate, monobasic Nalidixic acid Polymyxin B Trypan blue Distilled H 2 0

5 g 5 g 50 g 53.22 g 1.35 g 50 mg 8 X 105IU 80 mg 1000 ml

Rodriguez enrichment medium 2 Peptone Neopeptone Lab-Lemco powder Rhamnose NaCl Disodium phosphate-2-hydrate Potassium phosphate, monobasic Nalidixic acid Trypan blue Distilled H 2 0

5 g 5g 10 g 2g 50 g 53.22 g 1.35 g 50 mg 80 mg 1000 ml

Rodriguez enrichment medium 3 Protease peptone Tryptone Lab-Lemco powder Yeast extract Esculin NaCl Disodium phosphate-2-hydrate Potassium phosphate, monobasic Ferric ammonium citrate Nalidixic acid Trypan blue Agar Distilled H 2 0

5g 5g 5 g 5g 1g 20 g 24 g 1.35 g 1g 30 mg 40 mg 3g 1000 ml

5 g

5 g

10 g

714

Appendix

USDA Listeria enrichment broth I UVM broth Containing nalidixic acid

1000 ml 20 mg

USDA Listeria enrichment broth 11" UVM broth Containing nalidixic acid

1000 ml 12.5 mg

UVM

Proteose peptone Tryptone Lab-Lemco powder Yeast extract NaCl Disodium phosphate-7-hydrate Potassium phosphate, monobasic Esculin Nalidixic acid Acriflavine - HC1 Distilled H 2 0

5 g 5g 5 g 5 g 20 g 12 8 1.35 g 1g 40 mg 12 mg 1000 ml

* Commercially available from Difco Laboratories, Detroit, MI; BBL, Cockeysville, MD; Oxoid Ltd., Basingstoke, Hampshire, England; Merck, Darmstadt, Germany.

SOLID MEDIA TO ISOLATE OF L/ST€R/A SPP. AC agar Columbia agar base Acriflavine Ceftazidime Distilled H 2 0

39.0 g 10 mg 50 mg 1000 ml

ALPAMY agar Columbia blood agar base (Oxoid) Lithium chloride D-Mannitol 2-Phenylethanol Ferric ammonium citrate Esculin Acriflavine Phenyl red Egg yolk emulsion (Oxoid) Distilled H 2 0

39 g 15 g 10 g 2.5 g 0.5 g 0.5 g 10 mg 80 mg 25 ml 1000 ml

ARS-modified MMLA Phenylethanol agar (Difco) Lithium chloride Glycine anhydride Cycloheximide Nalidixic acid

35.5 g 0.5 g 10 g 0.2 g 50 mg

715

Appendix

Moxalactam Bacitracin Distilled H 2 0

5 mg 20 mg 1000 ml

FDA-modified McBride Listeria agar (FDA-MMLA)* Phenylethanol agar Glycine anhydride Lithium chloride Cycloheximide Distilled H 2 0

35.5 g 10 g 0.5 g 0.2 g 1000 ml

Gum base nalidixic acid medium" Tryptone broth (Oxoid) Nalidixic acid MgCI2 6 H20 Hydrocolloid gum (Merck-Gellan Gum KA40) Distilled H 2 0

1000 ml

Lithium chloride-ceftazidime agar Brain-heart infusion agar Lithium chloride Glycine anhydride Ceftazidime pentahydrate Distilled H 2 0

52 g 5 g 10 g 2.5 ml 1000 ml

LPM agar" Phenylethanol agar Glycine anhydride Lithium chloride Moxalactam Distilled H 2 0

35.5 g 10.0 g 5.0 g 20 mg 1000 ml

McBride Listeria agar Phenylethanol agar Glycine Lithium chloride Sheep blood Distilled H 2 0

35.5 g 10.0 g 0.5 g 50 ml 1000 ml

Modified McBride Listeria agar* Phen ylethanol agar Glycine anhydride Lithium chloride Distilled H 2 0

35.5 g 10.0 g 0.5 g 1000 ml

Modified LPM agar Brain-heart infusion agar Lithium chloride Glycine anhydride Ceftazidime Distilled H 2 0

52 g 5g 10 g 50 mg 1000 ml

'

*

10 g 50 mg 0.7 g 8g

776

Modified Oxford agar* Columbia blood agar base Esculin Ferric ammonium citrate Lithium chloride 1% Colistin solution 1% Moxalactam solution Agar Distilled H 2 0

Appendix

39 g 1g 0.5 g 15 g 1 ml 1 ml 2g 1000 ml

Modified Vogel-Johnson agar Vogel-Johnson agar base Nalidixic acid Bacitracin Moxalactam 1% Potassium tellurite solution Distilled H 2 0

60 g 50 mg 20 mg 5 mg 20 ml 980 ml

Oxford Listeria agar* Columbia agar base Esculin Ferric ammonium citrate Lithium chloride Cycloheximide Colistin sulfate Acriflavine Cefotetan Fosfomycin Distilled H 2 0

39 g 1g 0.5 g 15 g 400 mg 20 mg 5 mg 2 mg 10 mg 1000 ml

PALCAM agar* Columbia agar base D-Glucose D-Mannitol Esculin Ferric ammonium citrate Phenol red Polymyxin B Acriflavine . HCI Lithium chloride Ceftazidime, Latamoxef, or Moxalactam Distilled H 2 0 RAPAMY agar Columbia blood agar base D-Mannitol 2-Phenylethanol D-Glucose Ferric ammonium citrate Esculin

39 g 0.5 g 10 8 0.8 g 0.5 g 80 mg 100,000IU 5 mg 15 g 20 mg 1000 ml 39 g 10 g 2.5 g 1g 0.5 g 0.5 g

Appendix

Nalidixic acid Acriflavine Phenol red Egg yolk emulsion (Oxoid) Distilled H 2 0 Rodriguez isolation medium I Peptone Neopeptone Lab-Lemco powder Glucose NaCl Disodium phosphate-2-hydrate Potassium phosphate, monobasic Nalidixic acid Acriflavine . HC1 Defibrinated sheep blood Agar Distilled H 2 0 Rodriguez isolation medium I1 Peptone Neopeptone Lab-Lemco powder Yeast extract Glucose NaCl Disodium phosphate-2-hydrate Potassium phosphate, monobasic Nalidixic acid Polymyxin B Acriflavine - HC1 Defibrinated sheep blood Agar Distilled H 2 0 Rodriguez isolation medium I11 Peptone Neopeptone Proteose peptone Esculin NaCl Disodium phosphate-2-hydrate Ferric ammonium citrate Nalidixic acid Acrifavine . HCI Defibrinated sheep blood Agar Distilled H 2 0

717

40 mg 10 mg 80 mg 25 ml 1000 ml 5g 5g 7g l g 5g 11.83 g 1.35 g 40 mg 12 mg 50 ml 15 g 1000 ml

5 g 5g

10 g 5g 5g 40 g 1.83 g 1.35 g 40 mg 3 x 1041u 18.7 mg 50 ml 15 g 1000 ml

718

Appendix

Trypaflavine nalidixic acid serum agar Peptone Lab-Lemco powder NaCl Inactivated bovine serum Trypaflavine Nalidixic acid Polymyxin B Agar Distilled H 2 0

10 g 10 g 5 g 50 ml 20 mg 40 mg 3 mg 15 g 1000 ml

* Commercially available from Difco Laboratories, Detroit, MI; BBL, Cockeysville, MD; Oxoid Ltd., Basingstoke, Hampshire, England; Merck, Darmstadt, Germany.

Index

Acanthumoeba, 100 Acetic acid, 158, 159, 160, 161, 164, 165, 167, 172, 389, 469, 479,480,482, 537, 550, 592, 642, 645 Acid adaptation, 151 Acid anionic sanitizer, 683, 685 Acid tolerance, 478 Acidifying agents, 157 (see also individual acids) acetic acid, 158, 159, 160, 162, 163, 164 benzoic acid, 164 citricacid, 158, 159, 161, 162, 163, 164 formic acid, 164, 165 hydrochloric acid, 158, 161 lactic acid, 157, 158, 159, 161, 162, 163, 164 malic acid, 158 propionic acid, 161, 164 sorbic acid, 162 tartaricacid, 161, 162, 163 Acidophilus milk, 445 Aciduric properties, 151 Acquired immunodeficiency syndrome (AIDS), 75, 79, 80, 303, 3 11, 321, 325 Acridine dye, 23 1 Acriflavin, 229, 231, 232, 233, 236, 240, 242, 250 Actin (filaments), 98, 102, 103, 108, 109, 110,291

Aeromenas hydrophila, 133, 188, 189, 615, 685, 695 Alcoholism, 79, 321 Alfalfa tablets, 346, 65 1 Algin, 550 Alkaline phosphatase, 316, 379, 413, 424 ALTA, 587, 620 p-Aminobenzoic acid, 164, 165 Aminoglycosides, 89 Ampicillin, 89 Anari cheese, 324, 325,417,421, 475, 479 Anionic acid sanitizer, 197, 198, 203 Anise, 174 Annato, 451, 651 Anthocyanins, 392 Anthotyros cheese, 479 Antibodies, 262, 263 Antibody-based detection systems, 270 Antigens, 280 Antimicrobial susceptibility testing, 282 Antioxidants, 171 Antiseptic soaps, 202 Aplastic anemia, 79 Arrhenius equation, 529 Arthritis, septic, 81 Arzua cheese, 484 Aseptic processing, 594 Asiago cheese, 428 Asparagus, 642, 643 Aspergillus niger, 202

719

720 ATPase, 108 Avidin, 590 Avocado, 648

Bacillus, 4, 108 Bacillus cereus, 347, 632 Bacillus stearothermophilus, 108 Bacillus subtilis, 106, 265 Bacitracin, 230, 232, 236 Bacterial surface-ripened cheeses, 459 Bacteriocin, 182, 183, 186, 187, 281, 447, 462, 469, 478, 482, 528, 549, 552, 553, 554, 555 Bacteriocin typing, 28 1 Bacteriophage typing, 281, 284, 289, 290, 292 Bacterium monocytogenes, 2, 565 Bacterium monocytogenes hominis, 2 Baker’s cheese, 475 Bakery products, 651 Bamboo shoots, 638 Bean cakes, 638 Bean sprouts, 636, 638, 644 Beef, 507, 508, 5 14, 5 15, 5 16, 517, 5 19, 525, 528, 529, 530, 54 1, 546, 547, 551, 552, 555, 566, 638, 647, 664, 667, 679, 692, 693 Beef jerky, 508, 539, 540, 664 Beef sausage, 535, 542 Beet pigment, 632, 651 Beet pulp, 341 Beets, 642, 650, 651 Behavior in cheese, 450 Behavior in fermented milks, 440 Behavior in meat products, 521 Behavior in unfermented dairy products, 382 autoclaved fluid products, 386 butter, 399 evaporated milk, 393 growth in mixed cultures, 394 ice cream, 398 intensively pasteurized milk, 385 non-fluid dairy products, 397 pasteurized milk, 385 raw milk, 382 sweetened condensed milk, 393 ultra-filtered milk, 394 Be1 Paese cheese, 459 Benzoic acid, 164, 167 Benzoic acid derivatives, 164 Beta-hemolysis, 238, 264 Beta vulgaris, 651 Biofilms, 169, 195, 196, 197, 201, 658 Biopreservation, 182

Index Bixia orellana, 451 Bleu de Bresse cheese, 436 Blueberries, 341, 648 Blue (Bleu) cheese, 150, 315, 321, 322, 324, 417,421, 426, 429, 432, 436, 440, 453, 454, 457, 458, 459, 482, 483, 485, 486 Blue cheese factory, 673, 674 Blue Costello cheese, 439 Blue Lymeswold cheese, 484 Blue Stilton cheese, 484 Bockwurst, 535 Bologna, 535, 536, 538, 542 Bonbel cheese, 414 Bordetella pertussis, 118 Bratwurst, 535, 536, 538 Braunschweiger, 535 Breakfast sausage, 535 Brevibacterium linens, 459, 460 Brick cheese, 150, 315,417,451,454, 459, 460, 482, 483,486, 692 Brie cheese, 365, 378, 411, 412, 413, 414, 4 18, 4 19, 420, 432, 436, 453, 455, 457, 484, 485, 648, 672, 692,704 Brie de Meaux cheese, 308, 322, 323, 360, 455 Brine, 330, 487, 488, 617, 622, 624, 672, 673 Broccoli, 345, 632, 634, 635, 642, 643 Brochothrix, 3, 4, 5 Brochothrix thermosphacta, 3, 4, 532, 533 Brocchio cheese, 479 Broiler carcasses, 568, 571 Brucella, 703 Brucella abortus, 484 Brucellosis, 484 Brussels sprouts, 645 Buffalo meat, 515, 516 Bulgarian buttermilk, 445 Bulgarian white-pickled cheese, 472 Bulk starter cultures, 442, 443 Biindnerfleisch, 520 Butter, 307, 369, 370, 37 1, 372, 373, 376, 377,379,399,400,453 Butterine, 376 Buttermilk, 399, 691 Butylated hydroxyanisol, 166, 167, 169, 171 Butylated hydroxytoluene, 171 Butyric acid, 165 Cabbage, 344,633,634,635,636,637,638, 639, 640, 642, 643, 644, 645, 646, 67 1,697

Index Cabbagejuice, 591,639,640,641,644,645, 649 Caciocavalle cheese, 46 1 Caciotta cheese, 434 Cadherin, 100, 101, 103, 104 Caffeine, 175, 176, 393 Calamari, 606,607 Calcium lactate, 478 Cambazola cheese, 484 Camel meat, 5 15, 5 16 Camembert cheese, 1 50,3 15,413,414,41 8, 432,436,440,453,454, 455,456, 457,458,460,482,483,484,485, 486,692,704 CAMP test, 10, 11, 264 Campylobacter coli, 370,4 17 Campylobacterjejuni, 182, 370,4 17 Campylobacter spp., 550 Cancer, 79, 80,3 11,321, 337,342, 567,695 Candida albicans, 202 Candy, 453,487 Cane sugar, 390,391,392,393,398 Capric acid, 168 Caproic acid, 165 Caprylic acid, 165 Carbon dioxide, 188, 189,479, 530, 545, 547, 548,549,577,578,643 Carnitine, 134, 156, 157 Carnobacterium,3, 4 Carnobacteriumpiscicola, 186, 187, 553 Carrageenan, 390,391,392,550,586 Carrots, 633, 634, 635, 636,637,638,642, 645,646,647,648 Carrot juice, 455,648 Caryophanon,3 Casein, 360, 371, 372, 378, 379, 381, 393, 400 Catalase, 113, 114, 146, 648 Catfish, 6 16 Cattle, 40, 47-51,319, 341, 522 Cauliflower, 345, 632, 634, 635, 642, 643 Caviar, 606,607 Cefotetan, 235 Ceftazidime, 229,233,235 Celery 343, 633, 636, 639, 642 Cell lines, 97, 105, 107, 114 Cell-to-cell spread, 108 Cervelat, 5 19, 520. 54 1 Cerviche, 613 Chachcaval cheese, 385 Cheddar cheese, 150,314,3 15,324,4 14, 417,423,438,443,451,454,462, 464,465,466,467,479,480,482, 483,485,486,692

721 Cheese certification program, 4 11 Cheese composition, 482, 483 Cheese food, 151,414,415,480, 481, 482, 483,484,487 Cheese sauce, 478 Cheese spread, 414,415, 484, 524 Chemokine, 115 Chemotaxonomy,4 Cherries, 648 Chicken breaded fillets, 580 breasts, 570, 575, 583, 584, 585, 586, 588 broiler carcasses, 568, 571 broth, 581,582, 583 carcasses, 572, 573, 587 casserole, 580 drumstick, 570 frozen, 574 gravy, 58 1, 582 legs, 568, 569, 570, 575 liver, 568, 569, 570, 575, 576 loaf, 579 meat, 333, 334, 346, 532, 566, 586, 647 mechanically deboned, 587 nuggets, 580 parts, 568 patties, 567 raw, growth of L. monocytogenes, 577 salad, 567 sandwiches, 577 skin, 587 slaughterhouse, 667,668 sliced, 579, 580 spread, 567 summer sausage, 581 thighs, 567 wings, 568,569,570,575,587 Chinchilla, 57, 341 Chinese cabbage, 638,642 Chinese medicinal plants, 64 1 Chitin, 615 Chloramphenicol, 89,282 Chlorine, 621, 637, 639,644, 645, 683, 685 Chlorine compounds, 198, 199,200 Chlorine dioxide, 199, 645 Chlortetracycline,60 Chocolate, 651, 673, 674 Chocolate factory, 673 Chocolate milk, 306, 307, 360, 371, 372, 373, 377, 379, 381, 382, 386, 387, 388, 389,390,393,398,418 Cholesterol, 105 Chromosomal DNA restriction endonuclease analysis, 283, 290, 292

722 Cichorium endivia, 64 1 Cinnamon, 173 Cirrhosis, 79 Citric acid, 158, 159, 161, 164, 165, 166,469, 479,591,651 Clams, 606,607,610,612,697 Clean-in-place systems, 685 Clostridium, 4, 176, 183, 188 Clostridium botulinum, 183, 189, 695, 703 Clostridiumperfringens, 182, 525, 527,632, 703 Cloves, 173, 174, 175 Coagulants, 450 Cockles, 6 12 Cocoa, 390,391,392,393 Coconut, 633 Cod, 622,623 Code of Hygienic Practices, 4 18 Colby cheese, 150,315,417,424,451,454, 462,463,464,467,483,485,486 Cold enrichment, 133, 144, 225, 226,227, 228,240, 241, 243, 249, 250,432, 465, 474,476,488, 523, 550, 574, 62 1 Cold shock proteins, 227 Coleslaw, 149,301,305,319,324,341,343, 344, 345, 378, 631, 636, 638,639, 658,671,697,705 Coliforms, 438,439,448 Colistin sulfate, 234 Collagen vascular diseases, 79 Colorants, 45 1,453,650,651 Commercial rapid test systems, 273 Confectionery products, 67 1 Cooked meat specialty items, 539 Cooling system fluids, 205 Combined treatments, 193 Control in food-processingfacilities, 68 1 cleaning and sanitizing, 683,684,685 factory environment, 682 factory design, 682 guidelines, 681 personnel cleanliness, 686 traffic patterns, 686 Conventional subtyping methods, 280 Cooked and ready-to-eat meat products, 508, 509,518,531 Cooked sausage, 508 Cooked smoked sausage, 536 Coriander, 174 Corn, 642,645,646 Corn sweetener, 398 Corned beef, 506,507,5 18,519,521, 528, 532,533,566,695

Index Corticosteroid therapy, 80 Corynebacteriaceae, 3 Corynebacterium diphtheriae, 182 Corynebacterium infantisepticum, 2, 302 Corynebacteriumpawulum, 2 Coryneform bacteria, 460 Cotijacheese, 310, 313, 314, 315,414,485 Cottage cheese, 150,151,227,302,309,316, 371,412,418,425,426,429,433, 440,443, 453, 454, 475, 476, 477, 478,479,482,484,485,660 Coxiella burnetti, 145 Crab, 601,606, 607,608,612,615,616, 617, 621,624,695,697 Crabmeat, 602,603,604,605,609,6 14,620, 622,670,696,697 Crawfish, 605,607,621,622 Cream, 140,302,303,307,308,370,371, 372, 379, 381, 382, 386, 412, 475, 480 Cream cheese, 4 15,475,479,484,485 Cr8me de Bleu cheese, 436 Creosote, 537 Crescenza cheese, 429 Cucumbers, 634,635,636,638,697 Cultured buttermilk, 308,418,443,444,445, 448 Cultured cream, 4 12,44 1,445,691 Cultured milks, 44 1, 69 1 Curcuma longa, 45 1 Curing salts, 146 Cutaneous infections, 8 1 Cutting boards, 521 Cycloheximide,235 Cytochalasins, 103 Cytochromes, 7 Cytokine, 114, 115 Cytolysin, 105 Cytoplasm, 97,98, 103, 105, 106, 108, 110, 115,116, 118, 159 Dairy industry clarifiers and separators, 687 farm environment, 687 fermented dairy products, 69 1 filling and packaging, 690 frozen dairy products, 691 pasteurization, 687 pipeline and cross connections, 689 reclaimed and reworked product, 690 Dairy plant environmental problems, 660, 662, 663, 673, 675, 679 Dairy processing facilities, 659, 660, 661, 662, 663, 664, 678, 680

Index Dairy Safety Initiative Program, 370, 418, 658, 659 Decontamination treatments, 552 Diabetes, 79, 80, 321 Diacetyl, 182, 482 Dill, 619, 620, 624 Dimethyl sulfide, 460 Diplococcin, 482 Direct fluorescens microscopy , 271 Direct plating, 225, 226, 238, 432, 584, 589 DNA macrorestriction analysis, 287, 305, 344 DNA restriction endonuclease profile, 3 19 DNA sequence-based subtyping, 2 91 Dogs, 40, 58, 303 Domestic Soft Cheese Surveillance Program, 412,413,416,417 Domiati (Damietta) cheese, 431, 469, 471, 472,489 D o m spp., 610, 612 Dried beef, 5 18 Dry fermented sausage, 542 Dry heat, 583, 584, 585, 586 Dry milk processing facilities, 662 Dry sausage, 334 DTH gene, 264, 266 Duck, 565, 566, 571, 572, 573 D-value, 132, 144, 147, 163, 179, 181, 190, 191, 192,461,464,467, 550, 552, 587, 593, 594, 595, 621, 622, 623, 624, 644 Edam cheese, 176,412,417,462 Eel, 606, 607 Egg industry, 695 Eggnog, 592 Egg processing facilities, 670 Eggs, 566 albumen, 589,590 boiled, 589, 590 broken, 588 dried and reconstituted, 590, 591 fried, 593 frozen, 592 growth of L. monocytogenes, 589 heat in activation of L. rnonocytogenes, 593 liquid, 590, 591 liquid whole, 588, 589, 590, 591, 592, 593, 594,595 powdered, 592 products, 338, 339, 588 raw, 590 reduced cholesterol liquid whole, 59 1 salted liquid whole, 593, 594

723 [Eggs1 scrambled, 592 shells, 588 sugared yolk, 589 whole, 588, 589, 592 yolk, 588, 589, 590, 593, 594 Electromorphs, 282 Electrophoretic enzyme type, 320, 325, 334, 344,567 Ellagic acid, 172 Emmentaler cheese, 462,467 Endive, 641,642,644 Endocarditis, 80 Endophthalmitis, 8 1 Endothelial cells, 100, 1 17 Enrichment media, 240 Enterobacter aerogenes, 444,448 Enterobacter cloacae, 685 Enterobacteriacea, 642 Enterococci, 228,233, 235, 460, 474 Enterococcus, 4, 187 Enterococcusfaecalis, 102, 178,232,235, 456,525,527 Enterococcusfaecium, 147,235,620 Enterocytes, 101 Enzyme-linked immunosorbent assay, 272, 2 73 Epidemiology, 279, 280, 281, 282, 283,284, 287, 301, 303, 304, 306, 307, 31 1, 316, 318, 320, 321, 323, 329, 330, 333,337, 339, 340, 342, 346, 359, 522,537 Epithelia1 cells, 100, 101, 104, 115 Ergo, 449 Erysipelothrix, 3 Erysipelothrix monocytogenes, 2 Escherichia coli, 1 14, 141, 15 1, 164, 172, 182, 184, 191, 202,228,232,284, 347,413, 417,418, 419,422,434, 438,439,444,448, 524, 525, 526, 527,540,603,604,685,695 Escherichia coli 0 157:H7, 182, 192, 550, 568 Esculin, 235, 242, 243 Esrom cheese, 42 1,439 Essential oils, 174, 647 Estilo Casero cheese, 3 14 Ethanol, 147, 151, 177, 194 Ethylenediaminetetraaceticacid, 167, 169, 177, 178, 182,535,536,645,646,647 Eugenol, 647 Evaporated milk, 393,394 Farm-house cheese, 436 Fatty acid monoesters, 166

724 Fatty acids and related compounds, 165 Fecal material, 24, 46, 50, 53, 54, 56, 302, 319, 326, 335, 338, 339, 344, 345, 368, 522, 565, 568, 572, 614,616, 632,633,639,667,670,696,697 Fennel, 636,642 Fermented sausage, 54 1, 554 Ferric ammonium citrate, 235, 243 Feta cheese, 150, 3 15, 324, 325,436,454, 469,470,471,473,479,482,483, 485,486,488,489 Fibroblasts, 100, 101, 102, 104, 106, 110, 114,115 Fimbriae, 280 Finnish sausage, 545, 546 Fish (fin), 58, 59,601,610,611,612,616 dried, 61 1 pickled, 611,613 processing facilities, 695-697 salad, 61 1,613 salted, 605,608,611 smoked 605,607,608,611,613,617,618, 624 steamed, 602,614 thermal death time of L. monocytogenes, 622 Fish and seafood industry, 695 Flagella, 280, 291, 523 Flavobacterium, 394, 396 Floor drains, 196 Flow cytometry, 273,364 Fontina cheese, 42 1,473 Food contact surfaces, 195, 658 Food, Drug and Cosmetic Act, 372 Food industry, 90 Foods of plant origin, 341 (see also individual foods) alfalfa tables, 346 blueberries, 34 1 broccoli, 345 cabbage, 344 cauliflower, 345 celery, 343 coleslaw, 341, 347 lettuce, 343,346,347 mushrooms, 345 nectarines, 34 1 potato salad, 347 rice salad, 347 strawberries, 34 1 tomatoes, 343 Formaldehyde, 537 Formic acid, 164, 165,482

Index Fosfomycin, 234, 235 Fourme de Bresse cheese, 436 Fowl-domestic and wild, 40, 53-56 Frankfurters, 332, 333, 334, 335, 346, 506, 509, 510, 512, 513, 517, 518, 520, 534, 535, 536, 537, 538, 539, 542, 547,551,554,648,694,695 Free fatty acids, 165, 458, 466 Fresh sausage, 535 Fromage des Burons (cheese), 420 Frozen foods, 136 Frozen yogurt, 377,418 Fruit and vegetable industry, 697 Fruit juice bars, 649 Fruit processing facilities, 67 1, 672 Fruit salad, 636 F-value, 550, 594 Gamma irradiation, 190, 191,455, 587, 588 Gammelost cheese, 484 Gel electrophoresis, 267, 274 Gelatida products, 373 Gemella, 3 Gene expression, 116, 117 Generation time, 387, 392, 398, 475, 486, 489, 528, 589, 590, 592, 616 Genistein, 103 Genoa salami, 541 Genomic groups, 6 Geotrichum candidum, 460 Gjetost cheese, 324, 385, 479 Glacee, 374 Glass, 195 Gluconic acid, 475, 476, 477 Glucono-delta-lactone, 475, 624 Glucose oxidase, 179, 180, 390 Gluteraldehyde, 204, 205 Glycerol rnonolaurate, 165, 56 1 Glycine, 234, 244 Glycine betaine, 134, 156, 157 Goat meat, 515, 516 Goat milk cheese, 415, 417, 428, 430, 433, 436,438,473,474,475 Goats, 40, 45, 46, 319, 324 Good manufacturing practices, 412, 485, 621, 658, 659, 675, 679, 681, 691, 692,698,700,703,705 Goose, 565, 576 Gorgonzola cheese, 415, 429, 434, 453, 457, 459 Gouda cheese, 176, 412,417, 436, 462, 463, 467, 486 Granulocytes, 115

Index Gravy, 533 Green beans, 633, 642, 645, 646 Green peppers, 633 Ground beef, 508, 510, 513, 514, 515, 516, 526, 527, 528, 530, 542, 543, 550, 551, 553 Ground meat, 332, 334, 335, 514, 516, 553, 57 1 Ground pork, 513, 514, 515, 551 Growth in mixed cultures, 394 Growth temperatures, 132, 146 Gruyke cheese, 436, 462, 467 Gudbrandsdalsost cheese, 479 Gulls, 616

Hafnia,228 Half and half, 371, 372, 373, 400,418,478 Halloumi cheese, 324,325,417,421,438, 475,489 Ham, 332,337,508,510,512,514,518,519, 520, 521, 528, 531, 532, 534, 535, 538,542,550,551,664 Ham salad, 509,5 10 Hard cheese, 321,322,324 Hazard Analysis Critical Control Point, 226, 244, 615, 658, 695, 699, 703, 704, 705 conduct hazard analysis, 701 determine CCPs, 701 establish corrective actions, 702 establish critical limits, 701 establish monitoring procedures, 702 establish record-keeping and documentation procedures, 702 establish verification procedures, 702 prerequisite program, 700 Head cheese, 535 Heart disease, 80 Heat inactivation, 136,644 Heat resistance, 305, 593 Heat shock, 144, 145, 146, 147, 148,551 Heat-shock protein, 1 16, 145 Heat-treated milk, 4 12 Heating meats, 549 Hemolysin, 11, 105, 264, 265,266,291 Hemolysis, 10, 11, 105, 106, 107, 638 Henry’s technique, 237 Heparan-sulfate proteglycan (receptor), 102 Hepatitis A virus, 703 Hepatocytes, 100, 101, 104 Hexanoic acid, 165, 166 Hispanic cheeses, 468,484 Homemade cheese, 325

725 Homogenization, 144, 145, 193 Horsemeat, 5 14, 5 16 Horses, 40, 56 Hot-boned beef, 528,529 Hot dogs (see Frankfurters) Household kitchens, 679,68 1 Hummus, 633 Hurdle concept, 193, 194 Hybridization assay, 263 Hybridoma technology, 27 1 Hybridomas, 272 Hydrochloric acid, 147, 158, 161, 162, 163, 188,476,477,649 Hydrogen peroxide, 114, 146, 147, 151, 178, 179, 180, 181, 182, 194,482,490, 642 Hydrostatic pressures, high, 183, 189, 192 2-Hydroxy isocaproic acid, 390 Hypochlorite, 197, 199, 200, 201, 204, 645 Hypochlorite ion, 199 Hypothiocyanate, 179 Hypothiocyanous acid, 179 Ice cream, 308,360,369,370,371,372,373, 374, 375, 376, 377, 379, 381, 398, 418,453,487,660,674,679,691 Ice cream factory, 673, 674, 675, 676, 677 Ice cream mix, 140, 373, 374, 398,418 Ice milk, 370, 371, 372, 373, 375, 376 Ice milk mix, 373,418 Ice milk shake mix, 373,374,418 Imitation crab meat, 340, 341 Immunoassays, 262 Immunosuppressive therapy, 79 Incidence in meat products, 506, 5 1 1, 5 13 Incidence in pasteurized milk and other unfermented dairy products, 369 Incidence in raw meats, 5 14 Incidence in sausage and ready-to-eat meats, 517,518,519 Incidence in unfermented dairy products, 360, 380 butter, 38 1 casein, 381 cream, 38 1 dry infant formula, 38 1 ice cream, 38 1 milk, 380 nonfat dry milk, 38 1 raw cow’s milk, 360, 361, 362, 363, 364, 365,366,367 raw ewe’s and goat’s milk, 369 Incubation conditions, 240

lndex Industry-specificequipment, processing methods and equipment, 687 Infectious mononucleosis,2 Injury, cellular, 135, 146, 163, 188, 226, 230, 249, 250,251,253, 262, 274, 372, 379, 400, 460, 476, 477, 482, 486, 550,584,657,696 Interferon, 114 Interleukin, 114 Internal pH-controlled starter media, 442,443 Internalin, 100, 101, 103, 104 Interstate milk shipment plant, 659 Intracellular growth, 104 Intracellular motility, 108 Invasion assay, 100 Invasion-associated protein, 264, 266, 29 1 Invasion mechanism, 103 Iodine monochloride, 203 Iodophors, 198,203,204,683,685 Iron overload, 79 Irradiation, 189, 533, 549,644 Isobutyric acid, 389 Isoeugenol, 173 Isolation media, 233 Isovaleric acid, 390 Italian cheese, 325,421,422,423, 461, 467, 482,483,674,675 Italian sausage, 535 Italic0 cheese, 434 Jarlsberg cheese, 42 1 Jellied pork tongue, 302, 322, 324, 326, 329, 330,334,5 11,705 Jocoque, 3 16 Jonesia denitrijicans, 7 (see also Listeria denitrijkans) Kachkaval cheese, 412,473 Kareish cheese, 43 1,469 Kashor cheese, 430 Kasseri cheese, 485 Ketchup, 642 Kidney, 523 Kielbasa, 535 Kiln, 624 Kimchi, 642,643 Klebsiella pneumoniae, I82 Koch Kaese (cheese), 484 Kurthia, 3 Labneh, 449 Lactate dehydrogenase, 100

Lactates, 159, 189 Lactic acid, 157, 158, 159, 161, 164, 165, 166, 170, 177, 201, 228, 389, 480, 481, 482, 524, 542, 555, 578, 587, 602 623,624,645 Lactic acid bacteria, 179, 182, 183,228,232, 240, 383, 441, 476, 479, 531, 537, 540, 541, 549, 552, 554, 620, 640, 649,691 Lactobacillaceae, 3, 4 Lactobacillus, 3,4, 541 Lactobacillus acidophilus, 447 Lactobacillus bararicus, 187, 553 Lactobacillus bulgaricus, 441, 445, 446, 447 Lactobacillus casei, 47 1, 553 Lactobacillus curvatus, 187 Lactobacillus delbrueckii subsp. bulgaricus, 233,441,447,449 (see also Lactobacillus bulgaricus) Lactobacillus paracasei, 456 Lactobacillus plantarum, 187, 460, 525, 527, 545,555 Lactobacillus saM, 549, 555 Lactobacillus salivarius, 187 Lactobacillus viridescens, 172 Lactocin, 553 Lactococcus lactis subsp. cremoris, 44 1,461, 489 (see also Streptococcus cremoris) Lactococcus lactis subsp. lactis, 183,441, 456,461,462,489 (see also Streptococcus lactis) Lactoferricin, 182 Lactoferrin, 181, 182 Lactoperoxidasesystem, 179, 180, 181, 383 Lamb meat, 514, 516, 517, 519, 530, 546, 547,548,566,667,679,692 Lamb patty, 5 13 Lambda gene (bacteriophage), 284 Langostino, 605, 61 1 Lantibiotics, 182 Lauric acid, 165, 168 Lebanon bologna, 541 Lecithinase, 106, 107, 108, 1 12 Leeks, 636 Legionella pneumophila, 118 Lettuce, 343,346,347,631,634,635,636, 637, 638, 639, 641, 642, 643, 645, 646,648 Lettuce juice, 64 1 Leucocytes, 305 Leuconostoc, 54 1 Leuconostoc carnosum, 187

Index Leuconostoc crernoris, 443 Leuconostoc dextranicum, 443 Leuconostoc gelidurn, 186 Leuconostoc rnesenteroides, 187 Leukemia, 321 Liederkranz cheese, 315,412,413,414,459 Ligase chain reaction, 265, 266 Limburger cheese, 412,415,421,436,459, 485,692 Linoleic acid, 165 Linolenic acid, 165 Lipase, 389,467 Lipoteichoic acid, 4, 7, 103, 280 Liquid smoke, 172, 173,537, 538, 539,625 Listerella, 2, 226 Listerella bovina, 2 Listerella cunniculi, 2 Listerella gallinaria, 2 Listerella heminis, 2 Listerella gerbilli, 2 Listerella hepatobytica ,2 Listerella monocytogenes, 2 Listeria biochemical characteristics, 9 chemotaxonomy,4 culture, 8 genomic groups, 6 growth temperature, 9 hemolysin, 11 hemolysis, 10 metabolism, 9 morphology, 8 multitest assays, commercial, 11 numerical taxonomy, 3 nutritional requirements, 9 phenotypic markers, 11 phylogentic position, 3 rRNA sequencing, 4 species identification, 10 taxonomy, 5 , 7 Listeria bulgarica, 5,6 Listeria denitrijkans, 7, 271, 575 (see also Jonesia denitrificans) Listeria grayi in cheese, 431 dairy plants, 680 dairy products, 371 growth temperature, 133 identification, 10 no antibody reaction, 271 pasteurized milk, 379 raw ewe’s milk, 369 raw milk, 363

727 [Listeria grgyi] sugar utilization, 389, 390 taxonomy, 6,7 Listeria innocua antibiotic resistance, 134 in beef, 524,555 beef carcasses, 552,553 in cheese, 413,424,425,433,434,435, 436,438 cheese factory, 672,676 on chicken meat, 568, 569, 570, 571, 574, 575,576 in chicken sandwiches, 577 chicken slaughterhouse, 667,668, 669 cottage cheese, 478 in crabmeat, 604, 609 dairy plants, 661,662,678,680 dairy products, 37 1 380, 38 1, 659 defeathering machine, 575 in egg products, 588,589 expression of ActA, 109 expression of inlA, 100 fatty acids, effects, 165 in fish, 6 13 food processing facilities 675, 676,678 frankfurters, 55 1 in frozen seafood, 604 genomic group, 6 growth in autoclaved milks, 389, growth in cottage cheese, 188 growth, low pH, 148 growth temperature, 133 high hydrostatic pressure, 192 household kitchens, 679 identification, 10 induction of NF-KB, 117 low inoculum effects, 134 in Mexican-style cheese, 3 16 monoclonal antibody reaction, 272 no cause of avian listeriosis, 55 not detected with antibodies, 271 in oysters, 616 pasteurized milk, 379, 380 potato processing plant, 672,676 poultry meat-related illness, 337 raw ewe’s milk, 369 raw milk, 361, 362, 363, 364,365, 366, 367 raw produce, 634,635,636,637,639,644 salt tolerance, 155 in shrimp, 6 12 sugar utilization, 389, 390 taxonomy, 5

Index [Listeria innocua] on turkey meat, 569, 571 turkey slaughterhouse, 668, 669 use in food processing tests, 133 Listeria ivanovii ActA-related protein, 110 avian listeriosis, 5 5 cattle listeriosis, 48 in cheese, 43 1,432 chlorine inactivation, 199,200 DTH gene, 264 genomic group, 6 growth in autoclaved milks, 389 growth, low pH, 148 growth prevented on medium, 235 hemolytic strains, 265 hybridizes, 263 264 identification, 10 intracellular life cycle, 118 irradiation, 190 monoclonal antibody reaction, 272 polymerizes F-actin, 110 prfA-like gene, 111 raw ewe’s and goat’s milk, 369 raw milk, 361 salt tolerance, 155 sanitizer inactivation, 203 selective agents, 229 sheep listeriosis, 45 sugar utilization, 389 taxonomy, 5,7 Listeria monocytogenes acidity, effects, 148 adhesion, 100 animal feed, 22,27 antimicrobial food components, 154 antioxidants, 17I apoptosis, 118 avian listeriosis, 53-56 behavior of in cheese, 450 in fish and seafood, 6 15 on food and nonfood contact surfaces, 195 in fruit juices, 649 in meat products, 521 in plant products, 650 in raw and cooked poultry products, 577 in unfermented dairy products, 382 on vegetables, 639 biopreservation, 182 catalase, 113 cattle listeriosis, 47-52

[Listeria monocytogenes] cell-to-cell spread, 108 in cheese composition, effects of, 482 chinchilla listeriosis, 57 cold enrichment, 226 combined treatments, 193 commercial rapid test systems, 273 control in food-processing facilities, 68 1 conventional subtyping methods, 280 cookedheady-to-eat poultry, 576 cultured cream, 445 cultured buttermilk, 443 dog listeriosis, 58 enrichment media, 240 environment affects virulence gene expression, 113 excretion, 25 fatty acids and related compounds, 165 fecal carriage, 76-79, 82 fecal material, 22, 24 fermented milks, 440,441 fish and crustaceans, 58, 59 food chain, 32 freezing, 135 gamma irradiation, 587 gene expression, 116 genomic group, 6 goat listeriosis, 45-47 growth in cookedheady-to-eat poultry products, 579 growth in mixed cultures, 394 growth and survival in seafood, 6 16 growth and survival in vegetables, 632 growth temperature, 132 heat inactivation in eggs, 593 hemolysin, 11 history, 1,2,3, 75 horse listeriosis, 56, 57 host cell respouses, 1 14 household kitchens, 679 human disease, 75-90 hydrogen perioxide, 178 identification of, 10 inactivation of in seafood, 620 incidence of in cheese, 426 in eggs, 588 in food processing facilities, 658, 672 on fruits, 648 in meat products, 506 in raw poultry, 567, 572 on raw vegetables, 632 in unfermented dairy products, 360

Index [Listeria monocytogenes] inhibition in seafood, 618 internalin proteins, I00 intracellular growth, 104 intracellular motility, 108 invasion, 100 mechanism of, 103 lactic starter cultures, 441 lactoferrin, 181 lactoperoxidasesystem, 179 liquid smoke, 172 llama listeriosis, 57 lysozyme, 176 modified atmosphere, 187 molecular subtyping methods, 282 monitoringherification program for poultry products, 566 nisin, 183 nonfermented dairy foods, 307 nonhemolytic mutants, 105 non-human primate listeriosis, 58 nonthermal processing, 189 nucleic acid-based methods, 265 official isolation methods, 244 organic acids and their salts, 157 other bacteriocins, 187 other names, 2 pasteurized milk, 304, 379 pediocin, 185 persistance in environment, 21, 22 phagocytes, invasion, 100-103 phagocytic vacuole, escape, 104 protein ActA, 102 protein p60, 101 rapid detection methods, 262 raw milk, 302,360-368 recalls domestic cheese, 4 12 imported cheese, 4 18 recovering injured cells, 249 regulation of virulence gene, 111 regulatory aspects, fish and seafood, 614 sampling plans, 703 sanitizers, 198 selective enrichment, 228 selective media, 233 sewage, 26 sheep listeriosis, 4 1-45 signal transduction pathways, 117 silage, 2 I, 27 sodium chloride, 154 sodium nitrite 169 soil, 22, 23

729 [L isteria m onocytogenesJ spices, herbs, and plant extracts, 173 status in foods, 704 stress adaptation, 145 subtyping methods compared, 29 1 sugar utilization, 389 superoxide dismutase, 113 surveys for in seafood, 602,609 surveys, non-U.S. cheese, 423 swine listeriosis, 52, 53 taxonomy, 5 , 7 thermal inactivation, 1367, 583 thermotolerance, 145 tissue tropism., 103 traditional fermented milks, 449 transmission, 30, 593,615 treatment animals, 59, 60 vegetation, 22 decayed, 21 virulence, 134, 147 water, 22, 26 water activity, 153 yogurt, 447 zoo-animal listeriosis, 57 Listeria murrayi in cheese, 431 on chicken carcasses, 575 growth temperature, 133 no antibody reaction, 271 raw goat’s milk, 369 sugar utilization, 389, 390 taxonomy, 6 Listeria seeligeri in cheese, 425,431,432,440 on chicken meat, 575,575 chicken sandwiches, 577 chlorine inactivation, 195,200 dairy plant, 678 dairy products, 37 1 food processing facilities, 675 genomic group, 6 growth in autoclaved milks, 389 growth, low pH, 148 growth prevented on medium, 235 hemolytic strain, 264,265 identification, 10 irradiation, 190 no cause of avian listeriosis, 55 potato processing facility, 672 raw ewe’s milk, 369 raw milk, 36 1, 363 raw produce, 634,635,637 salt tolerance, 155

730 [Listeria seeligeri] sanitizer inactivation, 203 selective agents, 229 sugar utilization, 389 taxonomy, 5 , 6 Listeria welshimeri in beef, 524 in cheese, 43 1 on chicken meat, 569,570,571,574,575 in crabmeat, 609 genomic group, 6 household kitchens, 679 identification, 10 monoclonal antibody reaction, 272 no cause of avian listeriosis, 55 pasteurized milk, 379, 380 poultry slaughterhouse,667,668,669 raw ewe’s milk, 369 raw milk, 361, 362, 363 raw produce, 634,636,637 taxonomy, 5,7 on turkey meat, 569,570,571 Listeriaphages, 198 Listeriolysin, 100, 105, 107, 112, 115, 117, 118, 148, 152, 153,264,265,266, 267 Listeriosis, animal avian, 40,53-56,336,565,572 cattle, 40,47-51 fish/shellfish, 58,59 goats, 40,45,46 incidence, 39 livestock losses, 40 minor species, 40,56,57 rabbits, 2 sheep, 28,3 1,40,4 1-44 swine, 40, 52, 53 transmission, 30 to humans, 40 treatment, 59,60 Listeriosis, avian, 40, 335,336, 565, 572 carriers, 54 chick embryo, 55 chickens, 53 feces, 54, 56 history, 572 incidence, 54, 55 meningioencephalitis,55 poultry products, 56 secondary infection, 54 septicemia, 54 transport stress, 56 wild and domestic fowl, 53, 54

Index Listeriosis, cattle, 40 abortion, 47,48,301,302,360,382 encephalitis, 47, 360 feces, 50 feed transmission, 47 incidence, 47 infected tissues, 5 1 Listeria ivanovii, 48 mastitis, 48, 301, 302, 360 milk, 48, 49, 301, 360, 368 pathology, 47 risk to humans, 51 seasonality-milk,50 septicemia, 47 silage, 47 stress-relate immunosuppression, 5 1 Listeriosis, foodborne, 30, 3 1, 32 Brie de Meaux cheese, 308,322 butter, 307 catered food, 83 celery, 84 cheeseborne, 324 (see also specilfc cheeses) chocolate milk, 82, 83 coleslaw, 83, 84 common source outbreaks, 300 eggs and egg products, 338 epidemic listeriosis, 75, 301 foods of plant origin, 34 1 goat cheese, 46 history, 299 hot dogs, 87 lettuce, 84 Mexican-style soft cheese, 85,302,305, 307,309,3 10, outbreaks, 84 pasteurized milk, 84,85,301 pat& 86,302, 305, 327 pork pat6 “rillettes”, 33 1 pork tongue in jelly, 86,302,329 poultry products, 87, 335 raw milk, 49, 88 seafood products, 339, 614 sporadic listeriosis, 75 Swiss soft cheese, 85,302, 308,3 17 turkey frankfurters, 88 undercooked chicken, 87 Listeriosis, goats, 40 abortion, 45 bacteremia, 45 cheese, 46 encephalitis, 45 feces, 46

Index

731

[Listeriosis, goats] intestinal tract, 45 mastitis, 369 meninogoencephalits,46 milk, 46, 303, 369 oral lesions, 46 septicemia, 45 silage, 46 vaccination, 46 Listeriosis, human abortion, 343 antibiotic treatment, 89 arthritis-septic, 8 1 breast milk, 303 cancer, 70, 567 conjunctivitis, 335, 336 cutaneous infection, 8 1 diagnosis, 88 dietary counseling, 8 1, 89, 90 donated blood, 88 in the elderly, 79 encephalitis, 3 17 endocarditis, 80 endophthalmitis, 8 1 epidemic, 75 epidemiology, 83-88 fecal carriage, 76, 77, 78, 79, 82, 326 fetal infection, 81 fish associated, 602,614 foodborne, 75,82-88,299-358 food industry, 90 high-risk groups, 75, 76 immunosuppresivetherapy, 79 incidence, 87 invasive diseasehonpregnant adults 79, 80, 81

meningitis, 82, 303, 304, 322, 324, 332, 335, 339, 342, 343, 567, 602, 614, 636 meningoencephalitis,3 17 miscarriage, 335 mortality rate, 75,76, 82, 304 neonatal disease-early onset, 8 1, 82 neonatal disease- late onset, 82 oral infective dose, 372 osteomyelitis, 8 1 peritonitis, 8 1 pleural infection, 8 1 poultry associated, 567 pregnancy, 81 prevention, 89,90 public health agencies, 90 septicemia, 303,304,3 17,322,338,343, 344,636 sexual transmission, 88

[Listeriosis, human] sporadic, 75 stillbirth, 302, 303, 335, 343 symptoms, 80,8 1,82 transplacental transmission, 82 treatment, 89 Listeriosis, minor species chinchilla, 57 dogs, 40,58 gazelle, 57 horses, 40, 56 llama, 57 non-human primates, 58 reindeer, 57 roe deer, 57 zoological animals, 57 Listeriosis, sheep, 28, 3 1, 40, 345 abortion, 4 1,42 clinical manifestations, 4 1 direct entry, 41 encephalitis, 41 ewe’s milk, 44, 303, 369 fetal infection, 42 infection, 4 1 ingestion, 4 1 mastitis, 369 meningoencephalitis,4 1 morbidity/mortality,42 outbreaks: L. ivanovii, 45 seasonality, 42,43 septicemia, 4 1, 42 silage quality, 43 stress factors, 43 vaccination, 44 Listeriosis, swine, 40 abortion, 52 age of animals, 52 carriers, 53 encephalitis, 52 feces, 53 husbandry practices, 53 pork, 53 seasonality, 52 septicemia, 52 silage, 53 symptoms, 52 tissues, 53 Lithium chloride, 229,230, 232,233,234, 235,236,243 Liver, 523, 524 Liver sausage, 535,55 1 Llama, 57 Lobster, 601, 603, 605, 607, 608, 610, 612, 615,617,621,622,624,695,697 Localization in tissues, 522

732 Low-fat milk, 371, 372, 374, 391 Low pH growth, 148 survival, 150 Lubricants, conveyor chain, 204, 205 Luncheon meats, 334,506,508,513,519, 521,531,532,536,694,695 Lung, 522 Lupus erythematosus, 337 Lymph nodes, 522,523 Lymphocytes, 141, 142 Lys Bleu cheese, 436 Lysozyme, 167, 176, 178, 182, 183, 193,383, 447, 455,478,487, 535, 536, 592, 639,644,645,646,647 Maasdam cheese, 462,463,367 Macrolides, 282 Macrophages, 97, 100, 103, 104, 105, 107, 113, 114, 115, 117, 141, 142,227, 265,384 Malic acid, 158, 469 Manchego cheese, 150,385,473 Manouri cheese, 479 MAP kinase phosphatase, 116 Margarine, 453 Marinated seafoods, 624 Mascarpone cheese, 434 Mayonnaise cholesterol-freereduced-calorie, 592 low calorie, 592 real, 592 reduced calorie, 592 Meatballs, 549, 550 Meat industry, 692 frankfurters and other link products, 693 luncheon meats, 694 roast beef, corned beef and other rebagged products, 693 Meat processing environmental problems, 664,665,666 Meat processing facilities, 664, 665,666,667 Meat products, 326, 507, 508,664 Meat salads, 508, 509, 5 17, 5 18 Meat slicers, 517 Meat spreads, 508, 509 Menaquinones, 7 Meningitis, 3 Mercury compounds, 203 Mesophilic starter cultures, 44 1, 462, 474 Metalloprotease, 100, 106, 107, 108, 110, 291 Methyl sulfide, 460 Methyl trisulfide, 460

Index Mettwutst, 5 18, 520, 535, 539, 555 Mexican-style cheese, 302,307,308,309, 310, 324, 325, 333, 344, 345, 359, 360, 370, 389, 390,411,412,413, 4 14, 4 15, 468,484, 566, 639, 658, 66 1 Microbacterium thermosphactum, 3 Microbial rennet, 345, 450, 451, 452 Microbiological Surveillance Program 370, 371,373 Micrococci, 236 Micrococcus spp., 235,459,525 Micro-Gard, 620 Microwave radiation, 583, 584, 585, 586 Milano salami, 541 Milk, 301, 314 Minas Frescal cheese, 43 1 Mint, 175 Modified atmosphere (packaging), 187, 188, 189,479, 531, 532, 545, 548, 549, 577, 578, 579, 617, 619, 639, 643, 644 Moist heat, 538, 584, 585, 586 Mold-ripened cheeses, 453 Monitoring programs, 506, 508, 566, 567 Monitoring sample, 506, 507, 567 Monoacylglycerols, 167, 168, 169 Monocaprin, 166, 168, 169 Monocaprylin, 166, 169 Monocin, 281, 282 Monoclonal antibody, 27 1,272,274, 327 Monoglycerides, 460 Monolaurin, 166, 167, 168, 169, 193, 551 Monolimolein, 166 Monomyristin, 166 Monoolein, 165 Monopalmitin, I66 Monostearin, 166 Monterey Jack cheese, 325,412,414, 417, 484,485 Morbier Rippoz cheese, 420,422 Mortality rate, 304, 317, 322, 329, 343 Moxalactam, 229,230,233,234,236 Mozzarella cheese, 150, 4 14, 4 17, 429,434, 443,445,46 1,462,479,482,484 Mucor miehei, 450 Muenster cheese, 4 12,419,436,45 1,484, 485 Multilocus enzyme electrophoresis, 282, 290, 292,313,318,321,330 Multitest assays, commercial, 11 Murein Rydrolase, 102 Murraya grayi, 7

Index Muscle tissue, 522, 523, 524, 530 Mushrooms, 345,632,634,635,636,637, 640,697 Mussels, 340, 606, 607, 608,610, 612, 622, 623,624 Mutants, 152 Mutschli cheese, 436 Mutton, 514,515,516 Mycella cheese, 484 Mycobacterium bovis, 145 Mycolic acid, 5 Myristic acid, 165, 168 Mysost cheese, 479 Myzithra cheese, 479 Nalidixic acid, 229,230,231,232, 235, 236, 242 National Conference on Interstate Milk Shipments, 370 Nectarines, 341,648 Neufchatel cheese, 475, 484 Nisin, 160, 170, 183, 184, 185, 186, 193, 398, 456,457,469,478,482, 524, 548, 549,555,591,620 Nonfat dry milk, 360, 369, 370, 371, 372, 378, 379, 381, 382, 388, 389, 400, 480,487 Nonfood contact surfaces, 195 Non-human primates, 58 Nucleic acid-based probes, 262, 263, 264, 265 Nucleic acid hybridization assay, 273 Nucleic acid sequence-based amplification, 267,269 Numerical taxonomy, 3 Nutmeg, 173, 174 Nuts, 636 Oblique lighting, 228, 230, 234,236,237 Octanoic acid, 165, 166 Official detection methods, 244-249 Old Heidelberg cheese, 4 14 Olives, 649 One-step enrichment, 243 Onions, 636,638 Oral infective dose, 372, 378, 622 Orange juice, 632,649 Orange serum, 632,649,650 Organic acids and salts, 157 Oscillating magnetic field, 189 Osmolytes, 134, 156 Osteomyelitis, 81 Ovoflavoprotein, 590 Ovotransferrin, 590

733 Oxolinic acid, 23 1 Oysters, 339, 340, 601, 606, 607, 610, 612, 614,616,617,621,695,697 Ozone, 201 Packaging (see also Modified atmosphere packaging), 665 Palmitic acid, I65 Parabens, 164, 166 Parmesan cheese, 150,417,445,454,462, 467,468,482,483,484 Parotid glands, 522 Pasta, 632 Pasteurization, 139, 140, 143, 144, 145, 301, 306, 307, 314, 315, 316, 370, 372, 394,413,484, 533, 550, 551, 552, 589, 594, 621, 645, 659, 661, 670, 687,697 Pasteurization processes, 688, 689 Pasteurized milk, 301, 302, 304, 305, 316, 331, 359, 360, 369, 370, 371, 372, 373, 378, 379, 380, 382, 385, 386, 387, 390, 398, 412,418,424, 425, 432, 435,436,439, 440,449,457, 460,461,463,466,467,475,477, 484,485,486,658,692 Pasteurized Milk Ordinance, 369, 690 P W , 302, 305, 319, 324, 326, 327, 328, 329, 330. 331, 334, 335, 337, 505, 51 1, 517, 518, 519, 520, 521, 528, 534, 540,54 1,574,576,577, 705 Pathogenesis adhesion, 100 apoptosis, I 18 catalase, 113 cell-to-cell spread, 108 environment affects virulence gene expression, 113 gene expression, 116 host cell responses, 1 14 internalin proteins, 100 intracellular growth, 104 intracellular motility, 108 invasion, 100 invasion mechanism, 103 phagocytes, invasion, 1100, 101, 102, 103 phagocytic vacuole, escape, 104 protein p60, 101 protein ActA, 102 regulation of virulence gene, 111 signal transduction pathways, 1 17 superoxide dismutase, 113 tissue tropism, 103 Peach, 648

734 Peanut sauce, 638 Pears, 648 Peas, 633 Pecorino Romano cheese, 422,429,473 Pediocin, 183, 185, 186, 187, 189, 554, 555 Pediococcus acidilactici, 185, 186, 187, 189, 478,54 1,544,554,58 1 Pediococcus cerevisiae, 54 1 Penicillin, 89 Penicillium camemberti, 453,455 Penicillium candidum, 457 Penicillium caseicolum, 453 Penicillium glaucum, 453, 457 Penicillium roqueforti, 453, 457, 458 Pepper, 174 Pepperoni, 541, 542, 544, 545 Pepsin-rennet extract, 450,45 1, 452 Peptidoglycan, 7, 645 Perfringolysin, 106 Peritonitis, 8 1 Perlac, 620 Permeate, 443 Peroxyautc acid, 204 Phage type, 305,307,313,317,318,319, 321,322,323,329,332,344,345 Phage typing, 281,284,289, 290,292, 327, 330,432,570 Phagocytes 97, 141, 142, 524 Phagocytic vacuole escape, 104 Phagolysosomes, 103 Phagosomal membrane, 106 Phagosome-lysosomefusion, 106 Phagosomes, 103, 107, 118 Pheasant, 565,566,571,572,573 Phenol, 203,204,537 Phenylethanol, 229, 230, 233, 235, 250 Phosphatidylcholine-specificphospholipase C, 100,106 Phosphatidylinositol-specificphospholipase C, 100, 106,291 Phosphoinositide-3-kinase,104 Phospholipase, 106, 107, 110, 117 Phospholipase B, 266 Phosphoribosyl-pyrophosphate synthetase, 100 Phytoalixim 6-methoxy mellein, 648 Pickled cheeses, 469 Pickled vegetables, 638 Pimento, 647 Pinene, 175 Pizza, 651 Planktonic cells, 197, 305 Plate pasteurizer, 139, 141, 142 Pleural infection, 8 1

Index Pneumolysin, 265 Polyclonal antibody, 270,27 1 Polyester-polyurethanebelt, 198 Polymerase chain reaction, 262, 263, 266, 267, 268, 270, 273, 286, 287,288, 289 Polymyxin By228,229,232,233,235,250 Polyphosphate, 160, 163, 170,489 Polypropylene, 195 Polysaccharides, 523 Ponderosa pine needles, 341 Pont Eveque cheese, 436 Pork, 332, 514, 515, 516, 517, 518, 519, 529, 530, 534, 541, 546, 547, 548, 549, 566,667,679,692,693 Pork piit6 “rilletes”, 326, 33 1, 334 Pork sausage, 332,334,335, 513, 515, 518, 520,535 Port du Salut cheese, 412,459 Post processing contiaminants, 657 Potassium lactate, 587 Potassium sorbate, 161, 162, 163, 166, 167, 177, 189,478,480,. 550, 579 Potassium tellurite, 228, 229, 230, 232 Potassium thiocyanate, 232 Potato juice, 642 Potato products, 674, 675 Potato salad, 347, 633,638 Potatoes, 633, 634, 635, 638, 697 Poultry industry, 694 Poultry processing facilities, 667 Poultry processing plant environmental problems, 667, 668, 669, 675, 678 Poultry products, 335, 566,679 back and neck testing program, 568 bologna, 567 chicken nuggets, 337 cook-chill products, 336 cooked, 337,566,664 diced, 567 frankfurters, 567 raw meat, 336, 566, 572, 577, 588 ready-to-eat, 517, 566, 567, 579 spread, 567, 664 turkey frankfurters, 337, 338 undercooked, 337 Poultry salads, 509, 664 Poultry sausage, 544,566 Poultry spreads, 509, 576 Practical approaches to food safety, 698 Prepackaged salads, 636 Prerequisite programs, 658,700 Prfa regulon, 1 12 Primary enrichment, 228

Index Problems in amplification methods, 268 Process cheese, 433,453,484,485 Proline, 156 Propionibacterium thoenii, 187 Propionic acid, 161, 164, 165 Propionic acid bacteria, 462 Propylgallate, 166, 167, 171, 172,648 Propylene glycol, 205, 453 Protein ActA, 102, 103, 108, 109, 110, 118 Protein p60, 10I , 104 Proteus, 228,233 Proteus vulgaris, 182 Provolone cheese, 434,461, 485 Pseudomenus, 204,228,233,394,620 Pseudomonus aeruginosa, 182,202,228,523 Pseudomonusfluorescens, 182,395,396, 397,525,526,527,685 Pseudornonas fragi, 185, 196,395,396,397, 548,549,579,581 Pseudomonas putrefaciens, 685 Pseudomonads, 179,398,642 Public health agencies, 90 Pulsed electric field, 183, 189 Pulsed-field gel electrophoresis, 284, 287, 288, 290, 292, 311, 318, 319, 321, 323,330,331 332,345,570,671 Pulsed light-high intensity, 189, 191, 192 Pyrolysis mass spectroscopy, 3 18,319 Quaternary ammonium sanitizer, 197, 198, 203, 204, 683, 685 Queen A m , 3 Quesito cheese, 314 Queso Anejo cheese, 314, 468 Queso Blanco cheese, 415, 468,469 Queso de Bola cheese, 468 Queso de Crema cheese, 468 Queso de 10s Ibores cheese, 468, 469 Queso de Prensa cheese, 468 Queso de Puna cheese, 468 Queso Fresco cheese, 310, 313, 314,414, 415, 429,468, 469, 485 Queso Panella cheese, 485 Queso Prensado cheese, 415 Queso Ranchero cheese, 485 Queso Sec0 cheese, 314 Rabbit meat, 332 Rabbits, 299, 333 Raclette cheese, 436 Radioimmunoassay, 27 1 Radishes, 633, 634, 635, 636, 638, 642, 697 Ranchero cheese, 3 14 Random amplification of polymorphic DNA, 288,290,292,313,318,344,346,671

Rapid detection methods, 262 Ravioli, 650, 651 Raw beef, 507, 523, 525, 550 Raw fish, 339, 340 Raw meat, 507, 512, 513, 514, 515, 516, 522, 524, 525, 529, 534, 550 Raw milk, 302, 303, 307, 314, 315, 316, 320, 323, 325, 333, 359, 360, 361, 362, 363, 364, 365, 366, 367, 371, 382, 383, 384, 385, 386, 387, 388, 412,413,417, 418, 424, 425,432, 435, 436, 441, 445, 449, 464, 471, 484, 487, 649, 661, 679 Raw milk cheese, 325, 435, 455, 484 Reblochon cheese, 436 Recall, 261, 310, 311, 316, 320, 322, 325, 331, 338, 341, 360, 373, 378, 379, 412, 413, 462, 479, 506, 507, 509, 511, 512, 623, 659, 670, 691 butter, 376, 377 crabmeat, 602, 609 defined, 373 domestic cheese, 412, 414, 415 fish and seafood, 602, 620 frozen yogurt, 376, 377 fruit juice bars, 649 ice cream products, 374, 375, 376, 377 imported cheese, 418, 420, 421 milk, 374 mussels, 612 plant products, 633 poultry products, 567 ready-to-eat meat products, 510, 512 seafood products, 608 sherbet, 376, 377 turkey frankfurters, 567 Recovering injured listeriae, 249 Red pepper, 633 Regulatory actions, 509, 511 Regulatory factor A, 111 Reindeer, 57 Renal disease-chronic, 79, 80 Renibacteriuni , 3 Rennet extract, 450, 45 1, 452 Repetitive element-based subtyping, 289 Restriction fragment length polymorphism analysis, 283, 367 Retentate, 443 Rhodococcus equi, 11 Ribotypes, 251, 252, 286, 313, 321, 344 Ribotyping, 283, 284, 289, 290, 291, 318, 323, 330, 367, 570 Rice salad, 347

Index Rictone cheese, 479 Ricotta cheese, 325, 379, 414, 417, 440, 443,479,484 rRNA sequencing, 4 Roast beef, 507, 510, 512, 513, 524, 528, 531, 547, 566, 695 Roast lamb, 513 Roast pork, 513, 524 Roe, 606, 607 Roe deer, 57 Rolls, 651 Romadur cheese, 436 Romano cheese, 445, 462 Roquefort cheese, 385, 453, 457, 473 Rosemary, 173, 175 Rubber, 195, 196, 197, 201 Ruditapes spp., 610, 612 Sage, 173 Sakacin, 549, 555 Salami, 151, 332, 334, 510, 518, 519, 520, 521,541,542,543,544,555 Salmide, 200 Salmon, 339 brines, 6 17,624 gravad, 619 heating, 623 packaging, 6 17 processing plants, 670, 671 smoked, 602,6 17,619 thermal death time of L. monocytogenes, 622 Salmonella, 104, 119, 192,270,281,347, 370, 392, 393, 417, 524, 552, 568, 587, 594, 667, 670, 679, 691, 694, 695 Salmonella enteritidis, 164, 175, 182, 192, 695 Salmonella typhi, 703 Salmonella typhimurium, 118, 147, 151, 179, 182, 184, 190, 192, 202,484, 540, 658 Salmonellosis, 370 Salvador-style white cheese, 4 14 Sampling plans, 703 Sandwich hybridization capture assay, 264 Sandwich spread, 592 Sandwiches, 509, 5 10, 5 12, 5 17, 567, 577, 65 1 Sapsago cheese, 484 Sacrcoidosis, 79 Sauerkraut, 645 Sausage, 505, 506, 512,513, 514,515, 516, 517, 518, 519, 520, 521, 534, 535,

[Sausage] 536, 537, 538, 539, 541, 542, 543, 544,545,547,550,664,693,695 Sausage casings, 335,514,516,621,693 Saveloy, 531 Scallops, 601, 606, 607, 610, 612, 624, 695 Scamorze cheese, 461,484 Seafood, 602,604,6 11,638 imitation, 606, 608 meusse, 606,607 pasta, 61 1 pfititc, 606, 607 salads, 605, 606, 607, 608, 613 smoked, 606 spread, 606,607,608 Seafood processing facilities, 670 Seafood processing plant environmental problems, 670, 67 1,696 Secondary enrichment, 228 Selective agents, 229 Selective enrichment, 225, 226, 228,241, 262,286 Selective media, 233 Semidry fermented sausage, 54 1 Semisoft and hard cheeses, 462 Serotypes, 305,307 Serotyping, 280,290,291,304, 570 Serratia, 204 Serratia marcescens, 191,685 Sewage, 26,59,615,616 Sheep, 40,41,42, 43, 44, 45, 319, 341, 344, 385,522,633,639 Sheep’s milk cheese, 426,428,430,433,438, 473 Shellfish, 58, 59, 339, 340 Sherbet, 373, 374, 376, 377,487 Shigella, 119, 695 Shigella dysenteriae, 703 Shigella Jexneri, 110, 1 18 Shredded cheese, 440 Shrimp, 340,60 1,602,603,604,605,607, 608, 609, 612, 614, 615, 616, 617, 621,623,624,671,695,696,697 Siderophores, 620 Silage, 2, 21, 27,29, 3 1, 40, 43,46,47, 53, 56,302,319,341,368,631 Skim milk, 371, 372, 387, 388, 389, 391, 393, 398,489,490 Skim milk cheese, 412 Sliced meat, 332 Smoked meat, 5 18 Smoked sausage, 535,536,539 Snails, 606, 607 Soaps, 202

Index Sodium acetate, 578, 620 Sodium ascorbate, 648 Sodium azide, 230 Sodium benzoate, 163,451 Sodium caseinate, 3 16 Sodium chloride, 146,151,154,155,1156,159, 160,161,167,169,170,184,193,250, 482,487,488,489,490,531,534,536, 539,540,542,543,545,546,549,550, 551,586,593,618,619,623,624,640, 641,645,648 Sodium diacetate, 160, 587, 620 Sodium dichloro-s-triazinetrione, 199 Sodium erythrobate, 540,550 Sodium hydroxide, 202 Sodium lactate, 478, 550, 55 1, 587,6 18, 6 19, 620 Sodium nitrite, 160, 163, 169, 170, 194, 53 1, 532, 534,536, 537,539, 540, 542, 543,544,545,546,6 18,619,620 Sodium phosphate, 550 Sodium propionate, 159, 161, 162, 163,451, 480,48 1,651 Sodium tripolyphosphate, 550, 551, 586 Soft cheese factory, 673,674 Soft chive cheese, 324 Soft unripened cheese, 475 Soil, 22, 23, 319, 368,633,634,650, 672 Somatic cells, 143 Sorbic acid, 162, 48 1 Sour cream, 308 Sour milk, 302, 307,412 Sour milk cheese, 432 Sous-vide, 147, 188, 587,620,623 Soybeans, 638,650 Soymilk, 632, 650 Spinach, 633 Spleen, 522, 523, 524 Spray drying, 372 Squid, 601,605,606,607,611 Staffordshire cheese, 325 Stainless steel, 195, 196, 197, 198, 201 Staphylococcus, 4,233,460 Staphylococcus aureus, 11, 169, 172, 178, 179, 182, 190, 192, 235,271, 347, 370,399,417,604,685,695 Staphylococcus carnosus, 545 Starter cultures, 44 1,479,482, 58 1 Starter distillate, 45 1, 453 Stearic acid, 165 St. Paulin cheese, 436,459 Strawberries, 34 1,648 Streptococci, 23 1, 236 Streptococcus, 3,4, 81, 82, 109

737 Streptococcus cremoris, 44 1,442,443,444, 475 Streptococcusfaecalis, 102,271 Streptococcus latis, 441,442,443,444,478 Streptococcus mutans, 182 Streptococcus alivarius ssp. thermophilus, 44 1,447,449 (see also Streptococcus thermophilus), Streptococcus thermophilius, 233,44 1,445, 446,447 Streptolysin-0, 105, 265 Stress adaptation, 145 Stress response protein, 116 String cheese, 485 Sublethal injury (see Injury, cellular) Subtyping methods compared, 291 Sucrose, 146, 593,618,623 Sucrose monolaurate, 169 Sudanese white-pickled cheese, 472, 488 Sultanas, 636 Summer sausage, 554, 555, 581 Superoxide dismutase, 113, 114, 146,648 Superoxide radicals, 114 Surfactants, 200 Surimi, 605,606,611, 613 Sweetened condensed milk, 393,394 Swine, 40, 52, 53, 522 Swiss cheese, 150,415,417, 445, 462,466, 467,479,482,485

Talleggio cheese, 429,434 Tartaric acid, 161 Taxonomy, 5,7 T-cell-mediated immunity, 114 Teflon, 196,201 Teichoic acid, 7 Teleme cheese, 469, 489 Tertiary butylhydroxyquinon, 166, 167, 171, 172 Tetracyclines, 60, 89, 282 Tetrahymena, 100 Thallous acetate, 232 Theobromine, 175, 176,393 Thermal death time, 62 1,622 Thermotolerance, 145, 146 Thermal inactivation, 136, 657 Thermus aquaticus, 266 Thiocyanate, 179, 180, 181 Thuringer, 535 Tilsiter cheese, 436,459,460, 479 Tissue tropism, 103, 104 Tomato juice, 642 Tomato sauce, 642

Index Tomatoes, 343,634,635,636,639,642,643, 644,648 Tongue, 518 Tourre de 1’Aubier cheese, 420 Traditional approaches to food safety, 698 Traditional fermented milk products, 449 Transmission, 30, 31, 32, 593, 615 Transposon mutagenesis, 97, 100, 105, 107, 108,264 Trappist cheese, 150,459,46 1 Trimethoprim-sulfamethoxazole,89 Tripe, 518, 520 Trisodium phosphate, 201, 587,620,645 Trout, 624 Trypaflavin, 229,23 1 Trypan blue, 242 Tumor necrosis factor, 115 Turkey, 565,566,584,638 bologna, 579 carcasses, 572, 573 frankfurter, 335, 337, 338, 505, 566,567, 586,68 1,695 ground, 572 legs, 569, 570, 571 liver, 570 meat emulsion, 586 parts, 571,572 sausage, 567 slaughterhouse, 667, 668 sliced, 579, 580 summer sausage, 581 tails, 569, 571 wings, 569,570,571 Turkish white-brined cheese, 470,47 1 Turmeric, 451 Tyrosine kinase, 103 Tyrosine phosphatase, 103 Ulcerative colitis, 79 Ultrafiltered milk, 394,443 Ultra-high temperature treated milk, 386,489 Ultrapasteurization, 594, 595 Ultraviolet irradiation, 190, 191 Uncooked smoked sausage, 539 Unfermented sausage, 534 Vacherin Mont d’Or cheese, 302,308,3 14, 317,318, 319,320,321,322,324, 333,344,345,359, 366,378, 412, 434,436,455,575,672,692 Valerianella olitoria, 641 Vanadate, 103 Veal, 5 16 Vegetable processing facilities, 67 1, 672 Vegetable rennet, 345

Vegetable salads, 634,637,641,642 Vegetation, 23, 368, 63 1 Vegetation, decayed, 2 1, 75 Verification sample, 506, 507, 566 Vibrio, 615, 695 Vibrio cholerae, 3 , 4 17, 604, 703 Vibrioparahaemolyticus, 4 17, 604 Vibrio vulnijkus, 604 Virulence, 152,589 Virulence determinants, 99 Virulence gene (cluster), 98, 100, 102, 107, 111,112,113,263,266,286,291 Virulence gene expression, 113 Warm enrichment, 144,228, 229, 241, 621 Water, 22, 26, 59, 75, 632, 675, 682 Water activity 153, 154, 394, 482, 534, 543, 544,593,619 Watercress, 636 Whey, 149,389,400,455,476,477,486, 487,489,490 Whey cheeses, 479 Whey powder, 398,479,480,487 Whey processing facilities, 662 Whipping cream, 373, 374, 377, 387, 388, 389,400,418 Whitefish, 6 16 White Lymeswold cheese, 484 White Stilton cheese, 484 White pickled cheese, 150, 430, 43 1,436, 469,470,471,472 Whole milk, 387, 388, 389, 390 Wieners (see Frankfurters) Wild animal meat, 5 13 Wiirstel, 5 18, 520 Xenopus, 108 X-ray irradiation, 455 Yersinia, 119, 524, 662 Yersinia enterocolitica, 133, 182, 188, 189, 190, 192,202, 370, 417, 548, 604, 650 Yersiniapestis, 3 Yersiniosis, 3 70 Yugoslavian white-brined cheese, 472 Yogurt, 151,308,324,325,377,412,444, 445,447,448,453,660,691

Zero tolerance, 261, 321, 341, 372, 378,484, 509,567,602,614,622,704 Zoological animals, 57, 571 2-value, 621, 622 Zygosaccharomyces bailii, 202