Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1992 Classification of enterococci and their roles in spoilage of pork products and as sanitary indicators in pork processing Linda Marie Knudtson Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Agriculture Commons, Food Microbiology Commons, and the Microbiology Commons Recommended Citation Knudtson, Linda Marie, "Classification of enterococci and their roles in spoilage of pork products and as sanitary indicators in pork processing " (1992). Retrospective Theses and Dissertations. 10124. https://lib.dr.iastate.edu/rtd/10124 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. 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Ann Arbor,MI 48106 Classification of enterococci and their roles in spoilage of pork products and as sanitary indicators in pork processing by Linda Marie Knudtson A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Microbiology, Immunology and Preventive Medicine Major: Microbiology Approved: Signature was redacted for privacy. In Charge of Major Work Signature was redacted for privacy. For the Major Signature was redacted for privacy. Major Department Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1992 il TABLE OF CONTENTS GENERAL INTRODUCTION 1 Dissertation Format 2 LITERATURE REVIEW 4 History of the Enterococci 4 Origin of the species of the genus Enterococcus 5 Importance of the Enterococci 12 Habitat of the enterococci 12 Enterococci as fecal indicators in food and water 14 Enterococci in Meats 18 Enterococci as a cause of food spoilage 18 Hazard analysis critical control points 19 Meat sampling techniques 21 Enterococci in Food Poisoning 23 Clinical Importance of the Enterococci 24 Isolation and Identification Methods for the Enterococci 30 Selective media 30 Rapid methods for identification 36 Molecular approaches to identification 42 PAPER 1. ROUTINE PROCEDURES FOR ISOLATION AND IDENTIFICATION OF ENTEROCOCCI AND FECAL STREPTOCOCCI 45 ABSTRACT 47 INTRODUCTION 48 MATERIALS AND METHODS 50 RESULTS AND DISCUSSION 52 ACKNOWLEDGMENTS 57 LITERATURE CITED 58 iii PAPER 2. ENTEROCOCCI IN PORK PROCESSING 76 ABSTRACT 78 INTRODUCTION 79 MATERIALS AND METHODS 81 RESULTS 84 DISCUSSION 88 ACKNOWLEDGMENTS 91 LITERATURE CITED 92 PAPER 3. COMPARISON OF fGTC AND KF AGARS TO ENUMERATE ENTEROCOCCI AND FECAL STREPTOCOCCI IN MEATS 105 ABSTRACT 107 INTRODUCTION 108 MATERIALS AND METHODS 110 RESULTS AND DISCUSSION 112 ACKNOWLEDGMENT 116 LITERATURE CITED 117 PAPER 4. ANTIBIOTIC RESISTANCE AMONG ENTEROCOCCAL ISOLATES FROM CLINICAL AND ENVIRONMENTAL SOURCES 124 ABSTRACT 126 INTRODUCTION 127 MATERIALS AND METHODS 129 RESULTS AND DISCUSSION 130 ACKNOWLEDGMENT 133 iv LITERATURE CITED 134 PAPER 5. COMPARISON OF FOUR LATEX AGGLUTINATION KITS TO RAPIDLY IDENTIFY LANCEFIELD GROUP D ENTEROCOCCI AND FECAL STREPTOCOCCI 141 ABSTRACT 142 LITERATURE CITED 148 GENERAL SUMMARY AND DISCUSSION 150 LITERATURE CITED 157 ACKNOWLEDGMENTS 171 APPENDIX A; MEAN COUNTS AND ENTEROCOCCAL IDENTIFICATIONS FROM PORK CARCASSES SAMPLED IN THREE PLANTS 172 APPENDIX B: MEAN COUNTS AND ENTEROCOCCAL IDENTIFICATIONS FROM TWO PORK PROCESSING PLANTS 176 APPENDIX C; MEAN COUNTS AND ENTEROCOCCAL IDENTIFICATIONS FROM RETAIL SAMPLES 179 APPENDIX D: MEAN COUNTS AND ENTEROCOCCAL IDENTIFICATIONS FROM WATER SAMPLES 181 APPENDIX E; SOURCES AND IDENTIFICATIONS OF CLINICAL ISOLATES FROM MERCY HOSPITAL MEDICAL CENTER, DES MOINES, IOWA 183 1 GENERAL INTRODUCTION The classification of the enterococci has undergone significant changes in recent years. In 1984, Schleifer and Kilpper-Balz (132) used DNA-DNA and DNA-rRNA hybridization studies to show that the enterococcus group of the streptococci was a separate genus, Enterococcus. Currently, 19 species belong to this genus; E. avium (26), E. casseliflavus (26), £ cecorum (152), E. columbae (39), E. dispar (27), E. durans (26), E. faecalis (132), E. faecium (132), E. flavescens (121), E. gallinarum (26), E. hirae (58), E. malodoratus (26), E. mundtii (25), E. pseudoavium (24), E. raffinosus (24), E. saccharolyticus, E. seriolicida (89), E. solitarius, (24) and E. sulfureus (102). Generally, the habitat of the enterococci is the intestinal tracts of warm blooded animals (111), including domestic (126) and wild animals (110). Enterococci also have been isolated from the feces of reptiles (110) and birds (37) as well as from insects (113). Some enterococci can establish an epiphytic relationship with plants (113) and they have been isolated from the soil, where they are considered contaminants (25, 26). Despite their ubiquity in nature, the natural habitat of the enterococci is the intestinal tracts of man and animals and their occurrence in food or water implies either indirect or direct fecal contamination (62). The relative resistance of these organisms to adverse conditions, such as tolerance to extremes in temperature, pH, and salinity is advantageous in the examination of sea water, soft drinks and dried, frozen and processed foods where conforms might not have survived (108). Although the enterococci are not entirely host specific, they do show some degree of host specificity (70). The proportions of enterococcal species differ in various animal and human feces. 2 Therefore, the identification of enterococci rather than their enumeration was proposed to pinpoint the origin of fecal contamination (124). The enterococci also cause spoilage of pork and other meat products (64), and they have been implicated as a cause of diarrhea in suckling rats (48) and possibly humans (71). Besides possible food poisoning, enterococci cause a number of other human infections, such as urinary tract infections, bacteremia, and endocarditis (114). In this study, a schema for differentiating 13 species of the genus Enterococcus and S. bovis and S. equinus by using rapid test kits and a minimum of supplemental tests was proposed. Using this schema, identifications of isolates from pork slaughtering plants and processed pork were made; counts also were compared. Two media selective for enterococci, fGTC and KF agars, were compared for recovery of enterococci from pork carcasses, and processed pork, beef, and poultry. Identities of isolates on both media also were compared. Antibiotic resistances of enterococci isolated from pork, water, and clinical infections were examined. Species comparisons of antibiotic resistance were made for 13 enterococcal species and S. bows and S. equinus. Finally, four Lancefield grouping kits were compared for their ability to accurately group the enterococcal isolates, most of which are Lancefield group D. Dissertation Format This dissertation includes five papers that are manuscripts. Paper 1 appeared in the September 1992 issue (Volume 9) of Applied and Environmental Microbiology. Paper 2 has been accepted for publication in the Journal of Food Protection. Paper 3 has been submitted to Applied and 3 Environmental Microbiology as a research note. Paper 4 has been submitted to the Journal of Food Protection. Paper 5 has been submitted to the Journal of Rapid Methods and Automation in Microbiology as a research note. All papers follow the style of the journal to which they were submitted. A general literature review precedes the first manuscript, and a general summary and discussion follows the fifth manuscript. There are separate lists of references for each