Listeria, Listeriosis and Food Safety 2nd ed. - E. Ryser, et. al., (Marcel Dekker, 1999) WW.pdf

l i steriu,

Listeriosis,

and

Food Safety

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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

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Preface to the Second Edition

Listeriosis and Listeria monocytogenes continue to be of worldwide interest to the food industry and regulatory agencies, 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 (730 240-3 1 I 201-271

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7 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], 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 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 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 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 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., 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 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 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 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

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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

24 1

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

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a

n

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Streak to

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II 35°C

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L Examine for Listeria-like colonies

FIGURE3 FDA procedure for isolating

I_.

monocytogenesfrom foods. (From Ref. 74.)

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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

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0 1 ml + I0 ml Fraser Broth

U 26 f 2h 35°C

I

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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

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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

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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

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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

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(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

253

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.

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112. Ortel, S. 197I . Ausscheidung von Listeria rnonocytogenes irn Stuhl gesunder Personen. Zentralbl. Bakteriol. 1 Abt. Orig. 217:41-46. 113. Ortel, S. 1972. Experience with nalidixic acid-trypaflavine agar. Acta Microbiol. Acad. Sci. Hung. 19:363-365. 114. Palumbo, S.A., and A.C. Williams. 1991. Resistance of Listeria rnonocytogenes to freezing in foods. Food Microbiol. 8:63-68. 115. Papageorgiou, D.K., and E.H. Marth. 1989. Fate of Listeria monocytogenes during the manufacture, ripening and storage of Feta cheese. J. Food Prot. 52:82-87. 116. Papageorgiou, D.K., and E.H. Marth. 1989. Fate of Listeria rnonocytogenes during the manufacture and ripening of blue cheese. J. Food Prot. 52:459-465. 117. Patterson, M. 1989. Sensitivity of Listeria rnonocytogenes to irradiation on poultry meat and in phosphate-buffered saline. Lett. Appl. Microbiol. 8: 181-- 184. 118. Pini, P.N., and R.J. Gilbert. 1988. A comparison of two procedures for the isolation of Listeria monocytogenes from raw chickens and soft cheeses. Int. J. Food Microbiol. 7:33 1-337. 119. Petran, R.I., and K.M.J. Swanson. 1993. Simultaneous growth of Listeria rnonocytogenes and Listeria innocua. J. Food Prot. 56:616-618. 120. Pritchard, T.J., and C.W. Donnelly. 1995. Combined secondary enrichment of UVM and LRB primary enrichment broths increases the sensitivity of Listeria detection. IFT Annual Meeting: Book of Abstracts. Abstr. 34-2, p. 96. 121. Pritchard, T.J., K.J. Flanders, and C.W. Donnelly. 1995. Comparison of the incidence of Listeria on equipment versus environmental sites within daisy processing plants. Int. J. Food Microbiol. 26:375-384. 122. Ralovich, B.S. 1975. Selective and enrichment media to isolate Listeria. In: M. Woodbine, ed. Problems of Listeriosis. Proceedings of the Sixth International Symposium. Leicester University Press, Leicester, UK, pp. 286-294. 123. Ralovich, B. 1984. Listeriosis Research-Present Situation and Perspective. Budapest: Akademiai Kiado. 124. Ralovich, B., A. Forray, E. Mero, and H. Malovics. 1970. Additional data on diagnosis and epidemiology of Listeria infections. Zentralbl. Bakteriol. 1 Abt. Orig. 214:23 1-235. 125. Ralovich, B., L. Emody, I. Malovics, E. Mero, and A. Forray. 1972. Methods to isolate Listeria rnonocytogenes from different materials. Acta Microbiol. Acad. Sci. Hung. 19:367369. 126. Ralovich, B., A. Forray, E. Mero, H. Malovics, and I. Szazados. 1971. New selective medium for isolation of L. rnonocytogenes. Zentralbl. Bakteriol. 1 Abt. Orig. 216:88-91. 127. Rodriguez, D.L., G.S. Fernandez, J.F.F. Garayzabal, and E.F.. Ferri. 1984. New methodology for the isolation of Listeria microorganisms from heavily contaminated environments. Appl. Environ. Microbiol. 47: 1 188- 1 190. 128. Rodriguez, D.L., J.F. Fernandez, V. Briones, J.L. Blanco, and G. Suarez. 1988. Assessment of different selective agar media for enumeration and isolation of Listeria from dairy products. J. Dairy Res. 55579-583. 129. Rodriguez, D.L., J.F.F. Garayzabel, J.A.V. Boland, E.R. Ferri, and G.S. Fernandez. 1985. Isolation de microorganisms de listeria a partir de lait cru destine a le consommation humaine. Can. J. Microbiol. 3 1 :938-941. 130. Roth. T.T., and C.W. Donnelly. 1995. Injury of Listeria rnonocytogenes by acetic and lactic acids: mechanisms of repair and sites of sublethal damage. IFT Annual Meeting, Book of Abstracts. Abstr. 81D-I, p. 246. 131. Ryser, ET., E.H. Marth, and M.P. Doyle. 1985. Survival of Listeria rnonocytogenes during manufacture and storage of cottage cheese. J. Food Prot. 50:7-13. 132. Ryser, E.T., and E.H. Marth. 1987. Behavior of Listeria rnonocytogenes during the manufacture and ripening of Cheddar cheese. J. Food Prot. 50:7-13. 133. Ryser, E.T., and E.H. Marth. 1987. Fate of Listeria monocytogenes during manufacturing and ripening of Camembert cheese. J. Food Prot. 50:372-378.

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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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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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Sample preparation is a subject of intense interest but few determined efforts. Several different approaches to sample preparation have been proposed including:

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 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

Subtyping L. monocytogenes

281

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

Subtyping L. (ionocytogenes I

2’83

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 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

Foodborne Listeriosis

333

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-

334

Ryser

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

Foodborne Listeriosis

335

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

Foodborne Listeriosis

337

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 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-

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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

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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

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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.)

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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 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 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'

Farber and Peterkin

536

t t

/

/

/

/

/

/

/

/

CI

/

4.......+ /

1

1

I

I

I

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 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 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

-

Listeria monocytogenes in Poultry and Egg Products

593

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

-

-

594

Cox et al.

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 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 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

670

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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

672

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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

673

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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674

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

Listeria in Food-Processing Facilities

675

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,

676

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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

677

Lister ia in Food- Processing Facilities

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

678

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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

Listeria in Food-Processing Facilities

679

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

680

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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

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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]:

Listeria in Food-Processing Facilities

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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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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