R1. Modern Food Microbiology_7th Edition.pdf

Preface The 7th edition of Modern Food Microbiology, like previous editions, focuses on the general biology of the microorganisms that are found in foods. All but one of the 31 chapters have been extensively revised and updated. The new material in this edition includes over 80 new bacterial and 10 new genera of fungi. This title is suitable for use in a second or subsequent course in a microbiology curriculum, or as a primary food microbiology course in a food science or food technology curriculum. Although organic chemistry is a desirable prerequisite, it is not necessary for one to get a good grasp of most of the topics covered. When used as a microbiology text, the following sequence may be used. A synopsis of the in- formation in Chapter 1 will provide students with a sense of the historical developments that have shaped this discipline and how it continues to evolve. Memorization of the many dates and events is not recommended since much of this information is presented again in the respective chapters. The material in Chapter 2 includes a synopsis of modern methods currently used to classify bacteria, taxonomic schemes for yeasts and molds, and brief information on the genera of bacteria and fungi encountered in foods. This material may be combined with the intrinsic and extrinsic parameters of growth in Chapter 3 as they exist in food products and as they affect the common foodborne organisms. Chapters 4 to 9 deal with speciﬁc food products, and they may be covered to the extent desired with appropriate reviews of the relevant topics in Chapter 3. Chapters 10 to 12 cover methods for culturing and identifying foodborne organisms and/or their products, and these topics may be dealt with in this sequence or just before foodborne pathogens. The food protection methods in Chapters 13 to 19 include some information that goes beyond the usual scope of a second course, but the principles that underlie each of these methods should be covered. Chapters 20 and 21 deal with food sanitation, indicator organisms, HACCP, and FSO systems; and coverage of these topics is suggested before dealing with the pathogens. Chapters 22 to 31 deal with the known (and suspected) foodborne pathogens including their biology and methods of control. Chapter 22 is intended to provide an overview of the chapters that follow. Some of it includes ways in which foodborne pathogens differ from nonpathogens, their behavior in bioﬁlms, and some information on the known roles of sigma factors and quorum sensing among foodborne organisms. The other material in this chapter that deals with the mechanisms of pathogenesis is probably best dealt with when the speciﬁc pathogens are covered in their respective chapters. The new Appendix section presents a simpliﬁed scheme for grouping foodborne and some general environmental bacterial genera by use of Gram, oxidase, and calalase reactions along with colony pigmentation. v vi Modern Food Microbiology For most semester courses with a 3-credit lecture and accompanying 2 or 3 credit laboratory, only about 65-70% of the material in this text is likely to be covered. The remainder is meant for reference puiposes. The following individuals assisted us by critiquing various parts or sections of this edition, and we extend special thanks to each: B. P. Hedlund, K. E. Kesterson, J. Q. Shen, and H. H. Wang. Those who assisted with the previous six editions are acknowledged in the respective editions. Contents Part I—HISTORICAL BACKGROUND............................................... 1 1—History of Microorganisms in Food................................................ 3 Historical Developments............................................................ 4 Food Preservation............................................................... 5 Food Spoilage................................................................... 6 Food Poisoning.................................................................. 7 Food Legislation................................................................. 8 Part II—HABITATS, TAXONOMY, AND GROWTH PARAMETERS................... 11 2—Taxonomy, Role, and Signiﬁcance of Microorganisms in Foods...................... 13 Bacterial Taxonomy................................................................ 14 rRNA Analyses.................................................................. 14 Analysis of DNA................................................................ 15 The Proteobacteria............................................................... 15 Primary Sources of Microorganisms Found in Foods.................................. 17 Synopsis of Common Foodborne Bacteria............................................ 20 Synopsis of Common Genera of Foodborne Molds.................................... 27 Synopsis of Common Genera of Foodborne Yeasts.................................... 31 3—Intrinsic and Extrinsic Parameters of Foods That Affect Microbial Growth.......... 39 Intrinsic Parameters................................................................ 39 pH............................................................................. 39 Moisture Content................................................................ 45 Oxidation–Reduction Potential.................................................... 49 Nutrient Content................................................................. 52 Antimicrobial Constituents....................................................... 53 Biological Structures............................................................. 54 Extrinsic Parameters............................................................... 54 Temperature of Storage........................................................... 54 Relative Humidity of Environment................................................ 56 Presence and Concentration of Gases in the Environment............................ 56 Presence and Activities of Other Microorganisms................................... 56 vii viii Modern Food Microbiology Part III—MICROORGANISMS IN FOODS........................................... 61 4—Fresh Meats and Poultry.......................................................... 63 Biochemical Events That Lead to Rigor Mortis....................................... 64 The Biota of Meats and Poultry...................................................... 64 Incidence/Prevalence of Microorganisms in Fresh Red Meats........................... 66 Bacteria........................................................................ 68 Soy-Extended Ground Meats..................................................... 73 Mechanically Deboned Meats..................................................... 74 Hot-Boned Meats................................................................ 75 Organ and Variety Meats......................................................... 77 Microbial Spoilage of Fresh Red Meats.............................................. 78 Mechanism..................................................................... 82 Spoilage of Fresh Livers............................................................ 87 Incidence/Prevalence of Microorganisms in Fresh Poultry.............................. 88 Microbial Spoilage of Poultry....................................................... 89 Carcass Sanitizing/Washing......................................................... 91 5—Processed Meats and Seafoods..................................................... 101 Processed Meats................................................................... 101 Curing.......................................................................... 101 Smoking........................................................................ 103 Sausage, Bacon, Bologna, and Related Products....................................... 103 Spoilage........................................................................ 104 Bacon and Cured Hams............................................................. 108 Safety.......................................................................... 108 Seafoods.......................................................................... 109 Fish and Shellﬁsh................................................................ 109 Microorganisms................................................................. 109 Spoilage of Fish and Shellﬁsh....................................................... 115 Fish............................................................................ 115 Shellﬁsh........................................................................ 118 6—Vegetable and Fruit Products...................................................... 125 Fresh and Frozen Vegetables........................................................ 125 Spoilage........................................................................ 128 Bacterial Agents................................................................. 128 Fungal Agents................................................................... 134 Spoilage of Fruits.................................................................. 137 Fresh-Cut Produce................................................................. 138 Microbial Load.................................................................. 138 Seed Sprouts.................................................................... 139 Pathogens....................................................................... 140 Internalization of Pathogens...................................................... 142 Disease Outbreaks............................................................... 143 Contents ix 7—Milk, Fermentation, and Fermented and Nonfermented Dairy Products............. 149 Fermentation...................................................................... 149 Background..................................................................... 149 Deﬁned and Characterized........................................................ 150 The Lactic Acid Bacteria......................................................... 150 Metabolic Pathways and Molar Growth Yields...................................... 154 Acetic Acid Bacteria............................................................... 155 Dairy Products..................................................................... 156 Milk............................................................................ 156 Processing...................................................................... 157 Pasteurization................................................................... 157 General Microbiota of Milk....................................................... 158 Milk-Borne Pathogens........................................................... 158 Spoilage........................................................................ 160 Probiotics and Prebiotics........................................................... 161 Lactose Intolerance................................................................ 162 Starter Cultures, Fermented Products................................................. 163 Fermented Products.............................................................. 164 Cheeses......................................................................... 168 Diseases caused by Lactic Acid Bacteria............................................. 169 8—Nondairy Fermented Foods and Products.......................................... 175 Meat Products..................................................................... 175 Fish Products...................................................................... 178 Breads............................................................................ 179 Plant Products..................................................................... 180 Sauerkraut...................................................................... 180 Olives.......................................................................... 180 Pickles......................................................................... 181 Beer, Ale, Wines, Cider, and Distilled Spirits......................................... 182 Beer and Ale.................................................................... 182 Wines.......................................................................... 184 Cider........................................................................... 185 Distilled Spirits.................................................................. 186 Miscellaneous Products............................................................. 188 9—Miscellaneous Food Products...................................................... 197 Delicatessen and Related Foods..................................................... 197 Eggs.............................................................................. 198 Mayonnaise and Salad Dressing..................................................... 202 Cereals, Flour, and Dough Products.................................................. 203 Bakery Products................................................................... 203 Frozen Meat Pies.................................................................. 204 Sugars, Candies, and Spices......................................................... 204 Nutmeats.......................................................................... 205 Dehydrated Foods.................................................................. 206 x Modern Food Microbiology Enteral Nutrient Solutions (Medical Foods)........................................... 206 Single-Cell Protein (SCP)........................................................... 207 Rationale for SCP Production..................................................... 207 Organisms and Fermentation Substrates............................................ 207 SCP Products................................................................... 209 Nutrition and Safety of SCP...................................................... 209 Bottled Water...................................................................... 210 Part IV—DETERMINING MICROORGANISMS AND/OR THEIR PRODUCTS IN FOODS............................................................................ 215 10—Culture, Microscopic, and Sampling Methods...................................... 217 Conventional Standard Plate Count.................................................. 217 Homogenization of Food Samples................................................. 218 The Spiral Plater................................................................. 219 Membrane Filters.................................................................. 220 Direct Epiﬂuorescent Filter Technique............................................. 221 Microcolony-DEFT.............................................................. 221 Hydrophobic Grid Membrane Filter (HGMF)....................................... 222 Microscope Colony Counts......................................................... 223 Agar Droplets..................................................................... 223 Dry Film and Related Methods...................................................... 223 Most Probable Numbers............................................................ 224 Dye Reduction..................................................................... 225 Roll Tubes........................................................................ 225 Direct Microscopic Count (DMC)................................................... 225 Howard Mold Counts............................................................ 226 Microbiological Examination of Surfaces............................................. 226 Swab/Swab-Rinse Methods....................................................... 227 Contact Plate.................................................................... 227 Agar Syringe/“Agar Sausage” Methods............................................ 228 Other Surface Methods........................................................... 228 Metabolically Injured Organisms.................................................... 229 Recovery/Repair................................................................. 231 Mechanism of Repair............................................................ 233 Viable but Nonculturable Organisms................................................. 233 11—Chemical, Biological, and Physical Methods........................................ 241 Chemical Methods................................................................. 241 Thermostable Nuclease........................................................... 241 Limulus Lysate for Endotoxins.................................................... 244 Adenosine Triphosphate Measurement............................................. 247 Radiometry..................................................................... 247 Fluorogenic and Chromogenic Substrates.......................................... 248 Immunological Methods............................................................ 250 Serotyping...................................................................... 250 Fluorescent Antibody............................................................ 251 Contents xi Enrichment Serology............................................................. 252 Salmonella 1–2 Test............................................................. 252 Radioimmunoassay.............................................................. 253 ELISA............................................................................ 253 Gel Diffusion...................................................................... 255 Immunomagnetic Separation........................................................ 255 Hemagglutination.................................................................. 256 Molecular Genetic Methods......................................................... 256 Nucleic Acid (DNA) Probes...................................................... 257 Polymerase Chain Reaction....................................................... 258 Lux Gene Luminescence......................................................... 261 Ice Nucleation Assay............................................................. 262 Fingerprinting Methods............................................................. 263 Bacteriophage Typing............................................................ 263 Ampliﬁed Fragment Length Polymorphism........................................ 265 Multilocus Enzyme Electrophoresis Typing........................................ 265 Restriction Enzyme Analysis..................................................... 266 Random Ampliﬁcation of Polymorphic DNA....................................... 266 Pulsed Field Gel Electrophoresis.................................................. 267 Restriction Fragment Length Polymorphism....................................... 267 Ribotyping...................................................................... 268 Microarrays..................................................................... 268 Physical Methods.................................................................. 269 Biosensors...................................................................... 269 Impedance..................................................................... 272 Microcalorimetry................................................................ 273 Flow Cytometry................................................................. 274 BioSys Instrument............................................................... 275 12—Bioassay and Related Methods.................................................... 285 Whole-Animal Assays.............................................................. 285 Mouse Lethality................................................................. 285 Suckling (Infant) Mouse.......................................................... 288 Rabbit and Mouse Diarrhea....................................................... 288 Monkey Feeding................................................................. 289 Kitten (Cat) Test................................................................. 289 Rabbit and Guinea Pig Skin Tests................................................. 289 Sereny and Anton Tests.......................................................... 290 Animal Models Requiring Surgical Procedures........................................ 290 Ligated Loop Techniques......................................................... 290 The RITARD Model............................................................. 291 Cell Culture Systems............................................................... 291 Human Mucosal Cells............................................................ 292 Human Fetal Intestine............................................................ 292 Human Ileal and Intestinal Cells................................................... 292 Guinea Pig Intestinal Cells........................................................ 292 HeLa Cells...................................................................... 294 xii Modern Food Microbiology Chinese Hamster Ovary Cells..................................................... 294 Vero Cells...................................................................... 295 Y-1 Adrenal Cell Assay.......................................................... 295 Other Assays.................................................................... 295 Part V—FOOD PROTECTION AND SOME PROPERTIES OF PSYCHROTROPHS, THERMOPHILES, AND RADIATION-RESISTANT BACTERIA....................... 299 13—Food Protection with Chemicals, and by Biocontrol................................. 301 Benzoic Acid and The Parabens..................................................... 301 Sorbic Acid....................................................................... 303 The Propionates................................................................... 305 Sulfur Dioxide and Sulﬁtes......................................................... 305 Nitrites and Nitrates................................................................ 306 Organisms Affected.............................................................. 307 The Perigo Factor................................................................ 308 Interaction with Cure Ingredients and Other Factors................................. 308 Nitrosamines.................................................................... 309 Nitrite–Sorbate and Other Nitrite Combinations..................................... 309 Mode of Action................................................................. 310 Summary of Nitrite Effects....................................................... 311 Food Sanitizers.................................................................... 312 Acidiﬁed Sodium Chlorite....................................................... 312 Electrolized oxidizing water...................................................... 312 Activated Lactoferrin (ALF, Activin).............................................. 314 Ozone (O3 )..................................................................... 314 Hydrogen Peroxide (H2 O2 )....................................................... 315 Chlorine and Other Agents....................................................... 317 NaCl and Sugars................................................................... 320 Indirect Antimicrobials............................................................. 321 Antioxidants.................................................................... 321 Flavoring Agents................................................................ 322 Spices and Essential Oils......................................................... 323 Phosphates...................................................................... 324 Medium-Chain Fatty Acids and Esters............................................. 324 Acetic and Lactic Acids............................................................ 326 Salts of Acetic and Lactic Acids................................................... 326 Antibiotics........................................................................ 327 Monensin....................................................................... 328 Natamycin...................................................................... 329 Tetracyclines.................................................................... 329 Subtilin......................................................................... 330 Tylosin......................................................................... 330 Antifungal Agents for Fruits........................................................ 330 Ethylene and Propylene Oxides...................................................... 331 Miscellaneous Chemical Preservatives............................................... 331 Chitosans....................................................................... 331 Dimethyl Dicarbonate............................................................ 332 Contents xiii Ethanol......................................................................... 332 Glucose Oxidase................................................................ 333 Polyamino Acids................................................................ 333 Biocontrol......................................................................... 333 Microbial Interference........................................................... 333 Nisin and Other Bacteriocins...................................................... 336 Other Bacteriocins............................................................... 339 Endolysins........................................................................ 339 Bacteriophages as Biocontrol Agents................................................ 340 The Hurdle Concept................................................................ 341 14—Food Protection with Modiﬁed Atmospheres....................................... 351 Deﬁnitions........................................................................ 351 Hypobaric (Low Pressure) Storage................................................ 351 Vacuum Packaging............................................................... 352 Modiﬁed Atmosphere Packaging.................................................. 353 Equilibrium-Modiﬁed Atmosphere................................................ 353 Controlled-Atmosphere Packaging or Storage...................................... 354 Primary Effects of CO2 on Microorganisms........................................... 354 Mode of Action................................................................. 354 Food Products................................................................... 356 Fresh and Processed Meats....................................................... 356 Poultry......................................................................... 358 Seafoods........................................................................ 358 The Safety of Map Foods........................................................... 359 Other Pathogens................................................................. 362 Spoilage of Map and Vacuum-packaged Meats....................................... 363 Volatile Components of Vacuum-Packaged Meats and Poultry........................ 365 15—Radiation Protection of Foods, and Nature of Microbial Radiation Resistance........ 371 Characteristics of Radiations of Interest in Food Preservation........................... 372 Ultraviolet Light................................................................. 372 Beta Rays....................................................................... 372 Gamma Rays.................................................................... 372 X-Rays......................................................................... 373 Microwaves..................................................................... 373 Principles Underlying The Destruction of Microorganisms by Irradiation.................................................................... 373 Types of Organisms.............................................................. 373 Numbers of Organisms........................................................... 374 Composition of Suspending Menstruum (Food)..................................... 374 Presence or Absence of Oxygen................................................... 374 Physical State of Food........................................................... 375 Age of Organisms............................................................... 375 Processing of Foods for Irradiation.................................................. 375 Selection of Foods............................................................... 375 Cleaning of Foods............................................................... 375 Packaging....................................................................... 375 xiv Modern Food Microbiology Blanching or Heat Treatment..................................................... 375 Application of Radiation............................................................ 376 Gamma Radiation............................................................... 376 Electron Beams/Accelerated Electrons............................................. 377 Radappertization, Radicidation, and Radurization of Foods............................. 377 Deﬁnitions...................................................................... 377 Radappertization................................................................ 378 Radicidation.................................................................... 382 Seed sprouts and other vegetables................................................. 383 Radurization.................................................................... 383 Legal Status of Food Irradiation..................................................... 384 Effect of Irradiation on Food Quality................................................. 385 Storage Stability of Irradiated Foods................................................. 387 Nature of Radiation Resistance of Microorganisms.................................... 387 Biology of Extremely Resistant Species............................................ 388 Apparent Mechanisms of Resistance............................................... 390 16—Protection of Foods with Low-Temperatures....................................... 395 Deﬁnitions........................................................................ 395 Temperature Growth Minima....................................................... 396 Preparation of Foods for Freezing................................................... 396 Freezing of Foods and Freezing Effects.............................................. 399 Storage Stability of Frozen Foods.................................................... 399 Effect of Freezing on Microorganisms............................................... 401 Effect of Thawing............................................................... 403 Some Characteristics of Psychrotrophs and Psychrophiles.............................. 404 The Effect of Low Temperatures on Microbial Physiologic Mechanisms......................................................... 406 Nature of The Low Heat Resistance of Psychrotrophs/Psychrophiles................................................... 409 17—Food Protection with High Temperatures.......................................... 415 Factors Affecting Heat Resistance of Microorganisms................................. 416 Water........................................................................... 416 Fat............................................................................. 416 Salts............................................................................ 417 Carbohydrates................................................................... 418 pH............................................................................. 418 Proteins and Other Substances.................................................... 419 Numbers of Organisms........................................................... 419 Age of Organisms............................................................... 420 Growth Temperature............................................................. 421 Inhibitory Compounds........................................................... 421 Time and Temperature........................................................... 421 Effect of Ultrasonics............................................................. 422 Relative Heat Resistance of Microorganisms.......................................... 422 Spore Resistance................................................................ 422 Contents xv Thermal Destruction of Microorganisms............................................. 423 Thermal Death Time............................................................. 424 D Value......................................................................... 425 z Value......................................................................... 426 F Value......................................................................... 428 Thermal Death Time Curve....................................................... 428 12-D Concept................................................................... 429 Some Characteristics of Thermophiles............................................... 429 Enzymes........................................................................ 430 Ribosomes...................................................................... 432 Flagella......................................................................... 432 Other Characteristics of Thermophilic Microorganisms................................ 432 Nutrient Requirements........................................................... 432 Oxygen Tension................................................................. 433 Cellular Lipids.................................................................. 433 Cellular Membranes............................................................. 434 Effect of Temperature............................................................ 434 Genetics........................................................................ 435 Canned Food Spoilage.............................................................. 435 Low Acid (pH > 4.6)............................................................ 435 Acid (pH 3.7–4.0 to 4.6)......................................................... 435 High Acid (pH < 4.0–3.7)........................................................ 436 18—Protection of Foods by Drying..................................................... 443 Preparation and Drying of Low-Moisture Foods....................................... 443 Effect of Drying on Microorganisms................................................. 445 Storage Stability of Dried Foods..................................................... 447 Intermediate-Moisture Foods........................................................ 447 Preparation of IMF.............................................................. 448 Microbial Aspects of IMF........................................................ 452 Storage Stability of IMF.......................................................... 453 IMF and Glass Transition......................................................... 454 19—Other Food Protection Methods................................................... 457 High Hydrostatic Pressures (HHP, HPP).............................................. 457 Some Principles and Effects of HHP on Foods and Organisms........................ 458 Effects of HHP on Speciﬁc Foodborne Organisms................................... 459 Pulsed Electric Fields.............................................................. 463 Aseptic Packaging................................................................. 466 Manothermosonication (Thermoultrasonication)...................................... 467 Part VI—INDICATORS OF FOOD SAFETY AND QUALITY, PRINCIPLES OF QUALITY CONTROL, AND MICROBIOLOGICAL CRITERIA................... 471 20—Indicators of Food Microbial Quality and Safety................................... 473 Some Indicators of Product Quality.................................................. 473 Indicators of Food Safety........................................................... 475 xvi Modern Food Microbiology Coliforms....................................................................... 476 Enterococci..................................................................... 481 Biﬁdobacteria................................................................... 485 Coliphages/Enteroviruses......................................................... 487 The Possible Overuse of Fecal Indicator Organisms................................... 489 Predictive Microbiology/Microbial Modeling......................................... 491 21—The HACCP and FSO Systems for Food Safety..................................... 497 Hazard Analysis Critical Control Point (HACCP) System.............................. 497 Prerequisite Programs............................................................ 498 Deﬁnitions...................................................................... 498 HACCP Principles............................................................... 499 Flow Diagrams.................................................................. 503 Application of HACCP Principles................................................. 503 Some Limitations of HACCP..................................................... 506 Food Safety Objective (FSO)........................................................ 506 Microbiological Criteria............................................................ 506 Deﬁnitions...................................................................... 507 Sampling Plans.................................................................. 508 Microbiological Criteria and Food Safety.......................................... 509 Microbiological Criteria for Various Products....................................... 511 Other Criteria/Guidelines......................................................... 512 Part VII—FOODBORNE DISEASES.................................................. 517 22—Introduction to Foodborne Pathogens.............................................. 519 Introduction....................................................................... 519 Foodborne Illness Cases in the United States....................................... 519 The Fecal–Oral Transmission of Foodborne Pathogens.............................. 522 Host Invasion...................................................................... 522 “Universal” Requirements........................................................ 522 Attachment Sites................................................................ 524 Quorum Sensing................................................................... 524 Bioﬁlms.......................................................................... 527 Apparent Role of Quorum Sensing................................................ 529 Sigma (δ) Factors.................................................................. 529 Alternative Sigma Factors........................................................ 529 Pathogenesis...................................................................... 532 Gram-Positive Bacteria........................................................... 532 Gram-Negative Bacteria.......................................................... 533 Summary......................................................................... 538 23—Staphylococcal Gastroenteritis..................................................... 545 Species of Concern in Foods........................................................ 545 Habitat and Distribution............................................................ 547 Incidence in Foods................................................................. 548 Nutritional Requirements for Growth................................................ 548 Contents xvii Temperature Growth Range......................................................... 548 Effect of Salts and Other Chemicals.................................................. 548 Effect of pH, Water Activity, and Other Parameters.................................... 549 NaCl and pH.................................................................... 549 pH, aw , and Temperature......................................................... 549 NaNO2 , Eh, pH, and Temperature of Growth....................................... 550 Staphylococcal Enterotoxins: Types and Incidence.................................... 550 Chemical and Physical Properties................................................. 552 Production...................................................................... 554 Mode of Action................................................................. 557 The Gastroenteritis Syndrome....................................................... 558 Incidence and Vehicle Foods........................................................ 559 Ecology of S. aureus Growth........................................................ 560 Prevention of Staphylococcal and Other Food-Poisoning Syndromes.................... 560 24—Food Poisoning Caused by Gram-Positive Sporeforming Bacteria................... 567 Clostridium Perfringens Food Poisoning............................................. 567 Distribution of C. perfringens..................................................... 568 Characteristics of the Organism................................................... 568 The Enterotoxin................................................................. 570 Vehicle Foods and Symptoms..................................................... 571 Prevention...................................................................... 572 Botulism.......................................................................... 573 Distribution of C. botulinum...................................................... 574 Growth of C. botulinum Strains................................................... 576 Ecology of C. botulinum Growth.................................................. 578 Concerns for Sous Vide and Related Food Products................................. 579 Nature of the Botulinal Neurotoxins............................................... 580 The Adult Botulism Syndrome: Incidence and Vehicle Foods........................ 581 Infant Botulism.................................................................. 582 Bacillus Cereus Gastroenteritis...................................................... 583 B. cereus Toxins................................................................. 583 Diarrheal Syndrome............................................................. 584 Emetic Syndrome................................................................ 585 25—Foodborne Listeriosis............................................................. 591 Taxonomy of Listeria.............................................................. 591 Serotypes....................................................................... 594 Subspecies Typing............................................................... 594 Growth........................................................................... 595 Effect of pH..................................................................... 595 Combined Effect of pH and NaCl................................................. 596 Effect of Temperature............................................................ 597 Effect of aw..................................................................... 598 Distribution....................................................................... 598 The Environment................................................................ 598 Foods and Humans.............................................................. 598 xviii Modern Food Microbiology Prevalence...................................................................... 600 Thermal Properties................................................................. 600 Dairy Products.................................................................. 601 Nondairy Products............................................................... 602 Effect of Sublethal Heating on Thermotolerance.................................... 603 Virulence Properties............................................................... 603 Listeriolysin O and Ivanolysin O.................................................. 603 Intracellular Invasion............................................................. 604 Monocytosis-Producing Activity.................................................. 604 Sphingomyelinase............................................................... 605 Animal Models and Infectious Dose................................................. 605 Incidence and Nature of The Listeriosis Syndromes................................... 606 Incidence....................................................................... 606 Source of Pathogens............................................................. 607 Syndromes...................................................................... 609 Resistance to Listeriosis............................................................ 609 Persistence of L. monocytogenes in Foods............................................ 610 Regulatory Status of L. monocytogenes in Foods...................................... 611 26—Foodborne Gastroenteritis Caused by Salmonella and Shigella...................... 619 Salmonellosis..................................................................... 619 Serotyping of Salmonella......................................................... 620 Distribution..................................................................... 620 Growth and Destruction of Salmonellae............................................ 623 The Salmonella Food-Poisoning Syndrome......................................... 625 Salmonella Virulence Properties................................................... 625 Incidence and Vehicle Foods...................................................... 625 Prevention and Control of Salmonellosis........................................... 629 Competitive Exclusion to Reduce Salmonellae Carriage in Poultry.................... 629 Shigellosis........................................................................ 631 Foodborne Cases................................................................ 634 Virulence Properties............................................................. 634 27—Foodborne Gastroenteritis Caused by Escherichia coli.............................. 637 Serological Classiﬁcation........................................................... 637 The Recognized Virulence Groups................................................... 637 Enteroaggregative E. coli (EAggEC)............................................... 637 Enterohemorrhagic E. coli (EHEC)................................................ 639 Enteroinvasive E. coli (EIEC)..................................................... 647 Enteropathogenic E. coli (EPEC).................................................. 648 Enterotoxigenic E. coli (ETEC)................................................... 648 Prevention........................................................................ 650 Travelers’ Diarrhea................................................................ 650 28—Foodborne Gastroenteritis Caused by Vibrio, Yersinia, and Campylobacter Species.... 657 Vibriosis (Vibrio parahaemolyticus)................................................. 657 Growth Conditions............................................................... 657 Contents xix Virulence Properties............................................................. 659 Gastroenteritis Syndrome and Vehicle Foods....................................... 660 Other Vibrios...................................................................... 661 Vibrio cholerae.................................................................. 661 Vibrio vulniﬁcus................................................................. 663 Vibrio alginolyticus and V. hollisae................................................ 664 Yersiniosis (Yersinia enterocolitica)................................................. 664 Growth Requirements............................................................ 665 Distribution..................................................................... 666 Serovars and Biovars............................................................. 666 Virulence Factors................................................................ 667 Incidence of Y. enterocolitica in Foods............................................. 668 Gastroenteritis Syndrome and Incidence........................................... 668 Campylobacteriosis (Campylobacter jejuni).......................................... 668 Distribution..................................................................... 669 Virulence Properties............................................................. 670 Enteritis Syndrome and Prevalence................................................ 671 Prevention........................................................................ 671 29—Foodborne Animal Parasites....................................................... 679 Protozoa.......................................................................... 679 Giardiasis....................................................................... 680 Amebiasis...................................................................... 682 Toxoplasmosis.................................................................. 683 Distribution of T. gondii.......................................................... 684 Sarcocystosis.................................................................... 686 Cryptosporidiosis................................................................ 687 Cyclosporiasis................................................................... 689 Flatworms......................................................................... 690 Fascioliasis..................................................................... 691 Fasciolopsiasis.................................................................. 691 Paragonimiasis.................................................................. 692 Clonorchiasis................................................................... 692 Diphyllobothriasis............................................................... 693 Cysticercosis/Taeniasis........................................................... 695 Roundworms...................................................................... 696 Trichinosis...................................................................... 697 Anisakiasis..................................................................... 702 30—Mycotoxins....................................................................... 709 Aﬂatoxins......................................................................... 709 Requirements for Growth and Toxin Production.................................... 710 Production and Occurrence in Foods............................................... 711 Relative Toxicity and Mode of Action............................................. 713 Degradation..................................................................... 714 Alternaria Toxins.................................................................. 715 Citrinin........................................................................... 715 xx Modern Food Microbiology Ochratoxins....................................................................... 716 Patulin............................................................................ 716 Penicillic Acid..................................................................... 717 Sterigmatocystin................................................................... 717 Fumonisins........................................................................ 718 Growth and Production........................................................... 718 Prevalence in Corn and Feeds..................................................... 719 Physical/Chemical Properties of FB1 and FB2...................................... 719 Pathology....................................................................... 720 Sambutoxin....................................................................... 721 Zearalenone....................................................................... 722 Control of Production.............................................................. 722 31—Viruses and Some Other Proven and Suspected Foodborne Biohazards.............. 727 Viruses........................................................................... 727 Incidence in Foods and the Environment........................................... 728 Destruction in Foods............................................................. 728 Hepatitis A Virus................................................................ 729 Noroviruses..................................................................... 730 Rotaviruses..................................................................... 731 Bacteria........................................................................... 732 Enterobacter sakazakii........................................................... 732 Histamine-Associated (Scombroid) Poisoning...................................... 732 Prion Diseases..................................................................... 737 Bovine spongiform encephalopathy (BSE)......................................... 737 Creutzfeldt-Jakob Diseases (CJD, vCJD)........................................... 738 Chronic wasting disease (CWD).................................................. 739 Toxigenic Phytoplanktons.......................................................... 739 Paralytic Shellﬁsh Poisoning...................................................... 739 Ciguatera Poisoning............................................................. 740 Domoic Acid.................................................................... 740 Appendix............................................................................. 747 Index................................................................................. 751 Chapter 1 History of Microorganisms in Food Although it is extremely difﬁcult to pinpoint the precise beginning of human awareness of the presence and role of microorganisms in foods, the available evidence indicates that this knowledge preceded the establishment of bacteriology or microbiology as a science. The era prior to the es- tablishment of bacteriology as a science may be designated the prescientiﬁc era. This era may be further divided into what has been called the food-gathering period and the food-producing period. The former covers the time from human origin over 1 million years ago up to 8,000 years ago. During this period, humans were presumably carnivorous, with plant foods coming into their diet later in this period. It is also during this period that foods were ﬁrst cooked. The food-producing period dates from about 8,000 to 10,000 years ago and, of course, includes the present time. It is presumed that the problems of spoilage and food poisoning were encountered early in this period. With the advent of prepared foods, the problems of disease transmission by foods and of faster spoilage caused by improper storage made their appearance. Spoilage of prepared foods apparently dates from around 6000 bc. The practice of making pottery was brought to Western Europe about 5000 bc from the Near East. The ﬁrst boiler pots are thought to have originated in the Near East about 8,000 years ago.11 The arts of cereal cookery, brewing, and food storage, were either started at about this time or stimulated by this new development.10 The ﬁrst evidence of beer manufacture has been traced to ancient Babylonia as far back as 7000 bc.8 The Sumerians of about 3000 bc are believed to have been the ﬁrst great livestock breeders and dairymen and were among the ﬁrst to make butter. Salted meats, ﬁsh, fat, dried skins, wheat, and barley are also known to have been associated with this culture. Milk, butter, and cheese were used by the Egyptians as early as 3000 bc. Between 3000 bc and 1200 bc, the Jews used salt from the Dead Sea in the preservation of various foods.2 The Chinese and Greeks used salted ﬁsh in their diet, and the Greeks are credited with passing this practice on to the Romans, whose diet included pickled meats. Mummiﬁcation and preservation of foods were related technologies that seem to have inﬂuenced each other’s development. Wines are known to have been prepared by the Assyrians by 3500 bc. Fermented sausages were prepared and consumed by the ancient Babylonians and the people of ancient China as far back as 1500 bc.8 Another method of food preservation that apparently arose during this time was the use of oils such as olive and sesame. Jensen6 has pointed out that the use of oils leads to high incidences of staphylococcal food poisoning. The Romans excelled in the preservation of meats other than beef by around 1000 bc and are known to have used snow to pack prawns and other perishables, according to Seneca. The practice of smoking meats as a form of preservation is presumed to have emerged sometime during this period, as did the making of cheese and wines. It is doubtful whether people 3 4 Modern Food Microbiology at this time understood the nature of these newly found preservation techniques. It is also doubtful whether the role of foods in the transmission of disease or the danger of eating meat from infected animals was recognized. Few advances were apparently made toward understanding the nature of food poisoning and food spoilage between the time of the birth of Christ and ad 1100. Ergot poisoning (caused by Claviceps purpurea, a fungus that grows on rye and other grains) caused many deaths during the Middle Ages. Over 40,000 deaths due to ergot poisoning were recorded in France alone in ad 943, but it was not known that the toxin of this disease was produced by a fungus.12 Meat butchers are mentioned for the ﬁrst time in 1156, and by 1248 the Swiss were concerned with marketable and nonmarketable meats. In 1276, a compulsory slaughter and inspection order was issued for public abattoirs in Augsburg. Although people were aware of quality attributes in meats by the thirteenth century, it is doubtful whether there was any knowledge of the causal relationship between meat quality and microorganisms. Perhaps the ﬁrst person to suggest the role of microorganisms in spoiling foods was A. Kircher, a monk, who as early as 1658 examined decaying bodies, meat, milk, and other substances and saw what he referred to as “worms” invisible to the naked eye. Kircher’s descriptions lacked precision, however, and his observations did not receive wide acceptance. In 1765, L. Spallanzani showed that beef broth that had been boiled for an hour and sealed remained sterile and did not spoil. Spallanzani performed this experiment to disprove the doctrine of the spontaneous generation of life. However, he did not convince the proponents of the theory because they believed that his treatment excluded oxygen, which they felt was vital to spontaneous generation. In 1837, Schwann showed that heated infusions remained sterile in the presence of air, which he supplied by passing it through heated coils into the infusion.9 Although both of these men demonstrated the idea of the heat preservation of foods, neither took advantage of his ﬁndings with respect to application. The same may be said of D. Papin and G. Leibniz, who hinted at the heat preservation of foods at the turn of the eighteenth century. The history of thermal canning necessitates a brief biography of Nicolas Appert (1749–1841). This Frenchman worked in his father’s wine cellar early on, and he and two brothers established a brewery in 1778. In 1784, he opened a confectioner’s store in Paris that was later transformed into a wholesale business. His discovery of a food preservation process occurred between 1789 and 1793. He established a cannery in 1802 and exported his products to other countries. The French navy began testing his preservation method in 1802, and in 1809 a French ministry ofﬁcial encouraged him to promote his invention. In 1810, he published his method and was awarded the sum of 12,000 francs.7 This, of course, was the beginning of canning as it is known and practiced today.5 This event occurred some 50 years before L. Pasteur demonstrated the role of microorganisms in the spoilage of French wines, a development that gave rise to the rediscovery of bacteria. A. Leeuwenhoek in the Netherlands had examined bacteria through a microscope and described them in 1683, but it is unlikely that Appert was aware of this development and Leeuwenhoek’s report was not available in French. The ﬁrst person to appreciate and understand the presence and role of microorganisms in food was Pasteur. In 1837, he showed that the souring of milk was caused by microorganisms, and in about 1860 he used heat for the ﬁrst time to destroy undesirable organisms in wine and beer. This process is now known as pasteurization. HISTORICAL DEVELOPMENTS Some of the more signiﬁcant dates and events in the history of food preservation, food spoilage, food poisoning, and food legislation are listed below. The latter pertains primarily to the United States. History of Microorganisms in Food 5 Food Preservation 1782—Canning of vinegar was introduced by a Swedish chemist. 1810—Preservation of food by canning was patented by Appert in France. —Peter Durand was issued a British patent to preserve food in “glass, pottery, tin, or other metals, or ﬁt materials.” The patent was later acquired by Hall, Gamble, and Donkin, possibly from Appert.1,4 1813—Donkin, Hall, and Gamble introduced the practice of postprocessing incubation of canned foods. —Use of SO2 as a meat preservative is thought to have originated around this time. 1825—T. Kensett and E. Daggett were granted a U.S. patent for preserving food in tin cans. 1835—A patent was granted to Newton in England for making condensed milk. 1837—Winslow was the ﬁrst to can corn from the cob. 1839—Tin cans came into wide use in the United States.3 —L.A. Fastier was given a French patent for the use of brine bath to raise the boiling temperature of water. 1840—Fish and fruit were ﬁrst canned. 1841—S. Goldner and J. Wertheimer were issued British patents for brine baths based on Fastier’s method. 1842—A patent was issued to H. Benjamin in England for freezing foods by immersion in an ice and salt brine. 1843—Sterilization by steam was ﬁrst attempted by I. Winslow in Maine. 1845—S. Elliott introduced canning to Australia. 1853—R. Chevallier-Appert obtained a patent for sterilization of food by autoclaving. 1854—Pasteur began wine investigations. Heating to remove undesirable organisms was introduced commercially in 1867–1868. 1855—Grimwade in England was the ﬁrst to produce powdered milk. 1856—A patent for the manufacture of unsweetened condensed milk was granted to Gail Borden in the United States. 1861—I. Solomon introduced the use of brine baths to the United States. 1865—The artiﬁcial freezing of ﬁsh on a commercial scale was begun in the United States. Eggs followed in 1889. 1874—The ﬁrst extensive use of ice in transporting meat at sea was begun. —Steam pressure cookers or retorts were introduced. 1878—The ﬁrst successful cargo of frozen meat went from Australia to England. The ﬁrst from New Zealand to England was sent in 1882. 1880—The pasteurization of milk was begun in Germany. 1882—Krukowitsch was the ﬁrst to note the destructive effects of ozone on spoilage bacteria. 1886—A mechanical process of drying fruits and vegetables was carried out by an American, A.F. Spawn. 1890—The commercial pasteurization of milk was begun in the United States. —Mechanical refrigeration for fruit storage was begun in Chicago. 1893—The Certiﬁed Milk movement was begun by H.L. Coit in New Jersey. 1895—The ﬁrst bacteriological study of canning was made by Russell. 1907—E. Metchnikoff and co-workers isolated and named one of the yogurt bacteria, Lactobacillus delbrueckii subsp. bulgaricus. —The role of acetic acid bacteria in cider production was noted by B.T.P. Barker. 6 Modern Food Microbiology 1908—Sodium benzoate was given ofﬁcial sanction by the United States as a preservative in certain foods. 1916—The quick freezing of foods was achieved in Germany by R. Plank, E. Ehrenbaum, and K. Reuter. 1917—Clarence Birdseye in the United States began work on the freezing of foods for the retail trade. —Franks was issued a patent for preserving fruits and vegetables under CO2. 1920—Bigelow and Esty published the ﬁrst systematic study of spore heat resistance above 212◦ F. The “general method” for calculating thermal processes was published by Bigelow, Bohart, Richardson, and Ball; the method was simpliﬁed by C.O. Ball in 1923. 1922—Esty and Meyer established z = 18◦ F for Clostridium botulinum spores in phosphate buffer. 1928—The ﬁrst commercial use of controlled-atmosphere storage of apples was made in Europe (ﬁrst used in New York in 1940). 1929—A patent issued in France proposed the use of high-energy radiation for the processing of foods. —Birdseye frozen foods were placed in retail markets. 1943—B.E. Proctor in the United States was the ﬁrst to employ the use of ionizing radiation to preserve hamburger meat. 1950—The D value concept came into general use. 1954—The antibiotic nisin was patented in England for use in certain processed cheeses to control clostridial defects, 1955—Sorbic acid was approved for use as a food preservative. —The antibiotic chlortetracycline was approved for use in fresh poultry (oxytetracycline followed a year later). Approval was rescinded in 1966. 1967—The ﬁrst commercial facility designed to irradiate foods was planned and designed in the United States. The second became operational in 1992 in Florida. 1988—Nisin was accorded GRAS (generally regarded as safe) status in the United States. 1990—Irradiation of poultry was approved in the United States. 1997—The irradiation of fresh beef up to a maximum level of 4.5 kGy and frozen beef up to 7.0 kGy was approved in the United States. 1997—Ozone was declared GRAS by the U.S. Food and Drug Administration for food use. Food Spoilage 1659—Kircher demonstrated the occurrence of bacteria in milk; Bondeau did the same in 1847. 1680—Leeuwenhoek was the ﬁrst to observe yeast cells. 1780—Scheele identiﬁed lactic acid as the principal acid in sour milk. 1836—Latour discovered the existence of yeasts. 1839—Kircher examined slimy beet juice and found organisms that formed slime when grown in sucrose solutions. 1857—Pasteur showed that the souring of milk was caused by the growth of organisms in it. 1866—L. Pasteur’s Étude sur le Vin was published. 1867—Martin advanced the theory that cheese ripening was similar to alcoholic, lactic, and butyric, fermentations. 1873—The ﬁrst reported study on the microbial deterioration of eggs was carried out by Gayon. —Lister was ﬁrst to isolate Lactococcus lactis in pure culture. 1876—Tyndall observed that bacteria in decomposing substances were always traceable to air, sub- stances, or containers. History of Microorganisms in Food 7 1878—Cienkowski reported the ﬁrst microbiological study of sugar slimes and isolated Leuconostoc mesenteroides from them. 1887—Forster was the ﬁrst to demonstrate the ability of pure cultures of bacteria to grow at 0◦ C. 1888—Miquel was the ﬁrst to study thermophilic bacteria. 1895—The ﬁrst records on the determination of numbers of bacteria in milk were those of Von Geuns in Amsterdam. —S.C. Prescott and W. Underwood traced the spoilage of canned corn to improper heat processing for the ﬁrst time. 1902—The term psychrophile was ﬁrst used by Schmidt-Nielsen for microorganisms that grow at 0◦ C. 1912—The term osmophile was coined by Richter to describe yeasts that grow well in an environment of high osmotic pressure. 1915—Bacillus coagulans was ﬁrst isolated from coagulated milk by B.W. Hammer. 1917—Geobacillus stearothermophilus was ﬁrst isolated from cream-style corn by P.J. Donk. 1933—Oliver and Smith in England observed spoilage by Byssochlamys fulva; ﬁrst described in the United States in 1964 by D. Maunder. Food Poisoning 1820—The German poet Justinus Kerner described “sausage poisoning” (which in all probability was botulism) and its high fatality rate. 1857—Milk was incriminated as a transmitter of typhoid fever by W. Taylor of Penrith, England. 1870—Francesco Selmi advanced his theory of ptomaine poisoning to explain illness contracted by eating certain foods. 1888—Gaertner ﬁrst isolated Salmonella enteritidis from meat that had caused 57 cases of food poisoning. 1894—T. Denys was the ﬁrst to associate staphylococci with food poisoning. 1896—Van Ermengem ﬁrst discovered Clostridium botulinum. 1904—Type A strain of C. botulinum was identiﬁed by G. Landman. 1906—Bacillus cereus food poisoning was recognized. The ﬁrst case of diphyllobothriasis was recognized. 1926—The ﬁrst report of food poisoning by streptococci was made by Linden, Turner, and Thom. 1937—Type E strain of C. botulinum was identiﬁed by L. Bier and E. Hazen. 1937—Paralytic shellﬁsh poisoning was recognized. 1938—Outbreaks of Campylobacter enteritis were traced to milk in Illinois. 1939—Gastroenteritis caused by Yersinia enterocolitica was ﬁrst recognized by Schleifstein and Coleman. 1945—McClung was the ﬁrst to prove the etiologic status of Clostridium perfringens (welchii) in food poisoning. 1951—Vibrio parahaemolyticus was shown to be an agent of food poisoning by T. Fujino of Japan. 1955—Similarities between cholera and Escherichia coli gastroenteritis in infants were noted by S. Thompson. —Scombroid (histamine-associated) poisoning was recognized. —The ﬁrst documented case of anisakiasis occurred in the United States. 1960—Type F strain of C. botulinum identiﬁed by Moller and Scheibel. —The production of aﬂatoxins by Aspergillus ﬂavus was ﬁrst reported. 8 Modern Food Microbiology 1965—Foodborne giardiasis was recognized. 1969—C. perfringens enterotoxin was demonstrated by C.L. Duncan and D.H. Strong. —C. botulinum type G was ﬁrst isolated in Argentina by Gimenez and Ciccarelli. 1971—First U.S. foodborne outbreak of Vibrio parahaemolyticus gastroenteritis occurred in Maryland. —First documented outbreak of E. coli foodborne gastroenteritis occurred in the United States. 1975—Salmonella enterotoxin was demonstrated by L.R. Koupal and R.H. Deibel. 1976—First U.S. foodborne outbreak of Yersinia enterocolitica gastroenteritis occurred in New York. —Infant botulism was ﬁrst recognized in California. 1977—The ﬁrst documented outbreak of cyclosporiasis occurred in Papua, New Guinea; ﬁrst in United States in 1990. 1978—Documented foodborne outbreak of gastroenteritis caused by the Norwalk virus occurred in Australia. 1979—Foodborne gastroenteritis caused by non-01 Vibrio cholerae occurred in Florida. Earlier out- breaks occurred in Czechoslovakia (1965) and Australia (1973). 1981—Foodborne listeriosis outbreak was recognized in the United States. 1982—The ﬁrst outbreaks of foodborne hemorrhagic colitis occurred in the United States. 1983—Campylobacter jejuni enterotoxin was described by Ruiz-Palacios et al. 1985—The irradiation of pork to 0.3 to 1.0 kGy to control Trichinella spiralis was approved in the United States. 1986—Bovine spongiform encephalopathy (BSE) was ﬁrst diagnosed in cattle in the United Kingdom. Food Legislation 1890—The ﬁrst national meat inspection law was enacted. It required the inspection of meats for export only. 1895—The previous meat inspection act was amended to strengthen its provisions. 1906—The U.S. Federal Food and Drug Act was passed by Congress. 1910—The New York City Board of Health issued an order requiring the pasteurization of milk. 1939—The new Food, Drug, and Cosmetic Act became law. 1954—The Miller Pesticide Chemicals Amendment to the Food, Drug, and Cosmetic Act was passed by Congress. 1957—The U.S. Compulsory Poultry and Poultry Products law was enacted. 1958—The Food Additives Amendment to the Food Drug, and Cosmetics Act was passed. 1962—The Talmadge-Aiken Act (allowing for federal meat inspection by states) was enacted into law. 1963—The U.S. Food and Drug Administration approved the use of irradiation for the preservation of bacon. 1967—The U.S. Wholesome Meat Act was passed by Congress and enacted into law on December 15. 1968—The Food and Drug Administration withdrew its 1963 approval of irradiated bacon. —The Poultry Inspection Bill was signed into law. 1969—The U.S. Food and Drug Administration established an allowable level of 20 ppb of aﬂatoxin for edible grains and nuts. 1973—The state of Oregon adopted microbial standards for fresh and processed retail meat. They were repealed in 1977. History of Microorganisms in Food 9 REFERENCES 1. Bishop, P.W. 1978. Who introduced the tin can? Nicolas Appert? Peter Durand? Bryan Donkin? Food Technol. 32:60–67. 2. Brandly, P.J., G. Migaki, and K.E. Taylor. 1966. Meat Hygiene, 3rd ed., chap. 1. Philadelphia: Lea & Febiger. 3. Cowell, N.D. 1995. Who introduced the tin can?—A new candidate. Food Technol. 49:61–64. 4. Farrer, K.T.H. 1979. Who invented the brine bath?—The Isaac Solomon myth. Food Technol. 33:75–77. 5. Goldblith, S.A. 1971. A condensed history of the science and technology of thermal processing. Food Technol. 25:44–50. 6. Jensen, L.B. 1953. Man’s Foods, chaps. 1, 4, 12. Champaign, IL: Garrard Press. 7. Livingston, G.E., and J.P. Barbier. 1999. The life and work of Nicolas Appert, 1749–1841. Abstract # 7-1, p. 10, Institute of Food Technol. Proceedings. 8. Pederson, C.S. 1971. Microbiology of Food Fermentations. Westport, CT: AVI. 9. Schormüller, J. 1966. Die Erhaltung der Lebensmittel. Stuttgart: Ferdinand Enke Verlag. 10. Stewart, G.F., and M.A. Amerine. 1973. Introduction to Food Science and Technology, chap. 1. New York: Academic Press. 11. Tanner, F.W. 1944. The Microbiology of Foods, 2nd ed. Champaign, IL: Garrard Press. 12. Tanner, F.W., and L.P. Tanner. 1953. Food-Borne Infections and Intoxications, 2nd ed. Champaign, IL: Garrard Press. Chapter 2 Taxonomy, Role, and Signiﬁcance of Microorganisms in Foods Because human food sources are of plant and animal origin, it is important to understand the biological principles of the microbial biota associated with plants and animals in their natural habitats and respective roles. Although it sometimes appears that microorganisms are trying to ruin our food sources by infecting and destroying plants and animals, including humans, this is by no means their primary role in nature. In our present view of life on this planet, the primary function of microorganisms in nature is self-perpetuation. During this process, the heterotrophs and autotrophs carry out the following general reaction: All organic matter (carbohydrates, proteins, lipids, etc.) ↓ Energy + Inorganic compounds (nitrates, sulfates, etc.) This, of course, is essentially nothing more than the operation of the nitrogen cycle and the cycle of other elements. The microbial spoilage of foods may be viewed simply as an attempt by the food biota to carry out what appears to be their primary role in nature. This should not be taken in the teleological sense. In spite of their simplicity when compared to higher forms, microorganisms are capable of carrying out many complex chemical reactions essential to their perpetuation. To do this, they must obtain nutrients from organic matter, some of which constitutes our food supply. If one considers the types of microorganisms associated with plant and animal foods in their natural states, one can then predict the general types of microorganisms to be expected on this particular food product at some later stage in its history. Results from many laboratories show that untreated foods may be expected to contain varying numbers of bacteria, molds, or yeasts, and the question often arises as to the safety of a given food product based on total microbial numbers. The question should be twofold: What is the total number of microorganisms present per gram or milliliter and what types of organisms are represented in this number? It is necessary to know which organisms are associated with a particular food in its natural state and which of the organisms present are not normal for that particular food. It is, therefore, of value to know the general distribution of bacteria in nature and the general types of organisms normally present under given conditions where foods are grown and handled. 13 14 Modern Food Microbiology BACTERIAL TAXONOMY Many changes have taken place in the classiﬁcation or taxonomy of bacteria in the past two decades. Many of the new taxa have been created as a result of the employment of molecular genetic methods, alone or in combination with some of the more traditional methods: 1. DNA homology and mol% G + C content of DNA 2. 23S, 16S, and 5S rRNA sequence similarities 3. Oligonucleotide cataloging 4. Numerical taxonomic analysis of total soluble proteins or of a battery of morphological and biochemical characteristics 5. Cell wall analysis 6. Serological proﬁles 7. Cellular fatty acid proﬁles Although some of these have been employed for many years (e.g., cell wall analysis and serological proﬁles) others (e.g., ribosomal RNA [rRNA] sequence similarity) came into wide use only during the 1980s. The methods that are the most powerful as bacterial taxonomic tools are outlined and brieﬂy discussed below. rRNA Analyses Taxonomic information can be obtained from RNA in the production of nucleotide catalogs and the determination of RNA sequence similarities. First, the prokaryotic ribosome is a 70S (Svedberg) unit, which is composed of two separate functional subunits: 50S and 30S. The 50S subunit is composed of 23S and 5S RNA in addition to about 34 proteins, whereas the 30S subunit is composed of 16S RNA plus about 21 proteins. The 16S subunit is highly conserved and is considered to be an excellent chronometer of bacteria over time.53 Using reverse transcriptase, 16S rRNA can be sequenced to produce long stretches (about 95% of the total sequence) to allow for the determination of precise phylogenetic relationships.31 Alternatively, the 16S rDNA may be sequenced after ampliﬁcation of speciﬁc regions by polymerase chain reaction (PCR)-based methods. To sequence 16S rRNA, a single-stranded DNA copy is made by use of reverse transcriptase with the RNA as template. When the single-stranded DNA is made in the presence of dideoxynucleotides, Taxonomy, Role, and Signiﬁcance of Microorganisms in Foods 15 DNA fragments of various sizes result that can be sequenced by the Sanger method. From the DNA sequences, the template 16S rRNA sequence can be deduced. It was through studies of 16S rRNA sequences that led Woese and his associates to propose the establishment of three kingdoms of life forms: Eukaryotes, Archaebacteria, and Prokaryotes. The last include the cyanobacteria and the eu- bacteria, with the bacteria of importance in foods being eubacteria. Sequence similarities of 16S rRNA are widely employed, and some of the new foodborne taxa were created primarily by its use along with other information. It appears that the sequencing of 23S rDNA will become more widely used in bacterial taxonomy. Nucleotide catalogs of 16S rRNA have been prepared for a number of organisms, and extensive libraries exist. By this method, 16S rRNA is subjected to digestion by RNase T1, which cleaves the molecule at G(uanine) residues. Sequences (-mers) of 6–20 bases are produced and separated, and similarities SAB (Dice-type coefﬁcient) between organisms can be compared. Although the relationship between SAB and percentage similarity is not good below SAB value of 0.40, the information derived is useful at the phylum level. The sequencing of 16S rRNA by reverse transcriptase is preferred to oligonucleotide cataloging, as longer stretches of rRNA can be sequenced. Analysis of DNA The mol% G + C of bacterial DNA has been employed in bacterial taxonomy for several decades, and its use in combination with 16S and 5S rRNA sequence data makes it even more meaningful. By 16S rRNA analysis, the Gram-positive eubacteria fall into two groups at the phylum level: one group with mol% G + C >55, and the other

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