Food Microbiology: Fundamentals and Frontiers, Third Edition

Food microbiologists must understand microbiology and food systems and be able to integrate them to solve problems in complex food ecosystems. This chapter addresses this in three parts by (i) examining foods as ecosystems and discussing intrinsic and extrinsic environmental factors that control bacterial growth, (ii) explaining first-order or pseudo-first-order kinetics which govern the log phase of microbial growth and many types of lethality, and (iii) focusing on physiology and metabolism of foodborne microbes. Growth of Clostridium botulinum in foods such as potatoes and sauteed onions exposed to air has caused botulism outbreaks. Bacteria are classified as psychrophiles, psychrotrophs, mesophiles, and thermophiles according to the way in which temperature influences their growth. Additional barriers to microbial growth should be incorporated into refrigerated foods containing no other inhibitors. Food microbiology is concerned with all four phases of microbial growth. Growth curves showing the lag, exponential logarithmic or log, stationary, and death phases of a culture are normally plotted as the number of cells on a logarithmic scale or log10 cell number versus time. These plots represent the states of microbial populations rather than individual microbes. Thus, both the lag phase and stationary phase of growth represent periods when the growth rate equals the death rate to produce no net change in cell numbers. Food microbiologists frequently use doubling times (td) to describe growth rates of foodborne microbes. Developments in molecular biology and microbial ecology will change or deepen the perspective about the growth of microbes in foods.

Antibiotics are used in food production to treat infected animals, prevent infection of potentially exposed animals, and promote growth. Many argue that the widespread use of antibiotics at subtherapeutic levels exacerbates the selective pressure that enriches microbial populations for antibiotic-resistant bacteria and suggest that growth promotion and disease prevention could be achieved through nonantibiotic alternatives and novel management practices. Many food antimicrobials, such as nisin, lysozyme, lactoferrin, essential oils, and organic acids, are derived from natural sources, but even if their source is microbial, they are not classified as antibiotics. Antimicrobial resistance is useful to distinguish among antibiotic resistance, food antimicrobial resistance, and resistance to sanitizers and disinfectants. Human fecal contamination, either on the farm or during food preparation, is the root cause of shigellosis. The infection is classified as a foodborne illness because food is often the vehicle for Shigella. An understanding of mechanisms that result in resistance and adaptation can enable food microbiologists to develop effective intervention strategies to improve the overall safety of foods. A mechanistic understanding may also identify opportunities of collateral sensitivity, where the cellular changes resulting in resistance leave the cell more vulnerable to other types of antimicrobial agents. The main preventive measure food microbiologists can take to counter antibiotic-resistant organisms is to continue to develop and implement effective interventions to improve the overall safety of foods.

This chapter describes the fundamental basis of sporulation and problems that spores present to the food industry. The first obvious morphological event in sporulation is an unequal cell division. One purpose of the chapter is to highlight the state of knowledge of molecular mechanisms of sporulation, spore resistance and dormancy, and spore germination and outgrowth, and hopefully, provide a counterpoint to more applied aspects of this system. The chapter focuses on molecular mechanisms, most of which have been examined in Bacillus subtilis. The sporulating bacteria discussed in the chapter form heat-resistant endospores that contain dipicolinic acid (DPA) and are refractile or phase bright under phase-contrast microscopy. Most studies on sporulation, spores, and spore germination have been carried out with species of either the aerobic bacilli or the anaerobic clostridia. The discussion of gene expression control mechanisms has been simplified and concentrates on major regulatory gene products. As detailed mechanistic data is available for B. subtilis, the discussion on spore resistance is concentrated on B. subtilis. The most effective way to kill pressure-germinated spores is by heat, and thus pressure treatments are often carried out at elevated temperatures. In the anaerobic growth environment of clostridia, transition metals would be expected to have important roles in the sporulation and resistance properties of spores. The scientific investigation of sporeformers has greatly contributed to the development of microbiology for the enhancement of food safety and quality.

Microbiological criteria provide the food industry and regulatory agencies with guidelines for control of food processing systems and are an underlying component of any critical control point that addresses a microbiological hazard in Hazard Analysis and Critical Control Points (HACCP) systems. Aerobic plate count (APC) or standard plate count (SPC) is commonly used to determine “total” numbers of microorganisms in a food product. Depending on the pathogen, low levels of the microorganism in the food product may or may not be of concern. Some microorganisms have such a low infective dose that their mere presence in a food presents a significant public health risk. For such microbes, the concern is not whether the pathogen is able to grow in the food but that the microorganism could survive for any length of time in the food. Food products frequently subject to contamination by harmful microorganisms, such as shellfish, may benefit from the application of microbiological criteria. Enterococci counts have few useful applications in microbiological criteria for food safety. Some microbiological criteria related to safety rely on tests for metabolites to indicate a potential hazard rather than direct tests for pathogenic or indicator microorganisms. The current acceptable limits are based on the criterion that there is an 80% probability that the plant is actually exceeding the target value if it exceeds the acceptable limit.

This chapter examines the risk of intentional food contamination and provides an overview of current methods for evaluating risk and defining appropriate interventions. The microbial pathogens and toxins of most concern are listed, along with some of the potential chemical agents of concern provided for comparison; several references are available that go into significant detail on the agents of concern and illness progression and food protection more broadly. The chapter focuses specifically on biological contaminants, although a number of chemicals can also be of concern. A composite score is compiled for the food system or for the section of the food system that is under consideration. An overview of technologies currently available for use in analyzing food items for potential contamination can be found in recent articles. The overall food system risk management system that has developed over the last few decades serves as a sound and necessary foundation for approaches that will protect the food system from intentional abuse, but it is not sufficient. While similar to the continual improvement approach dictated by food safety systems to prevent unintentional microbiological contamination of food products, food system biosecurity represents an additional set of challenges. Ensuring that the biosecurity of the food system is not compromised will require that all aspects are addressed, with food microbiology being a necessary, but not the only, element.

A muscle food, including meat, poultry, and seafood, is described as spoiled when it is considered unacceptable by consumers based on its sensory characteristics. Economic losses as well as the food wasted and loss of consumer confidence due to spoilage, however, are of major significance. Among bacteria, genera in the family Enterobacteriaceae, Photobacterium phosphoreum, Shewanella (Alteromonas) putrefaciens, Brochothrix thermosphacta, Pseudomonas spp., Aeromonas spp., and lactic acid bacteria have been found to be major contributors to muscle food spoilage, depending on the product type and the conditions surrounding the product. Chemical (chlorine, organic acids, inorganic phosphates, proteins, oxidizers, etc.) or physical (knife-trimming, cold or hot water, vacuum, and/or steam) agents and combinations of two or more agents simultaneously or in sequence are applied as carcass decontamination/sanitization treatments in the United States. Acidic decontamination of meat may lead to changes in the dominating microbial association and if combined with long-term storage may lead to development of yeasts. The microbial associations developing on muscle tissues stored aerobically at cold temperatures are characterized by an oxidative metabolism. The contribution of specific nutrients to bacterial behavior has been shown in single-culture or coculture studies. Glucose is the preferred energy substrate and the first to be used by various microorganisms growing on muscle foods. Glycolytic enzymes indigenous to muscle tissues participate in the postmortem glycolysis that ceases when the ultimate postmortem pH reaches 5.4 to 5.5. Freshness and safety of muscle food products are generally considered the most important contributors to quality.

This chapter describes the interactions of microorganisms with dairy foods that lead to commonly encountered product defects. The major microbial inhibitors in raw milk are lactoferrin and the lactoperoxidase system. Fluid milk, cheese, and cultured milks are the major dairy products susceptible to spoilage by non-spore-forming fermentative bacteria. Non-spore-forming bacteria responsible for fermentative spoilage of dairy products are mostly in either the lactic acid-producing or coliform group. The most common fermentative defect in fluid milk products is souring caused by the growth of lactic acid bacteria. The defect in noncultured fluid milk products is usually caused by growth of specific strains of lactococci. Spore-forming bacteria that spoil dairy products usually originate in the raw milk. The defect in milk products is described as sweet curdling, since it first appears as coagulation without significant acid or off flavor being formed. The major heat-resistant species in milk is Geobacillus stearothermophilus (formerly Bacillus stearothermophilus). A practical means to prevent sporeformers from spoiling nonfermented liquid dairy products given sub-ultrahigh-temperature (UHT) heat treatments has not been developed. The most common yeasts present in dairy products are Kluyveromyces marxianus and Debaryomyces hansenii (the teleomorph) and their asporogenous counterparts (the anamorph), Candida species, and Zygosaccharomyces microellipsoides. Yeasts and molds that spoil dairy products can usually be isolated in the processing plant on packaging equipment, in the air, in salt brine, on manufacturing equipment, and in the general environment (floors, walls, ventilation ducts, etc.).

This chapter focuses on the origin, description, and control of bacterial and fungal spoilage of fruits and vegetables. Plant tissues in fruits and vegetables consist of an assemblage of cells surrounded by a pectic and cellulosic cell wall organized in a network, and a middle lamella rich in pectin cementing together cell walls. Specific factors associated with virulence of microorganisms are the primary mechanisms at the origin of post-harvest spoilage of fruits and vegetables. Postharvest control of temperature, relative humidity, and composition of the gaseous atmosphere aims at reducing the physiological activity of fruits and vegetables by delaying ripening and senescence, consequently prolonging the shelf life. The most suitable temperature, relative humidity, and modified atmosphere for preserving the quality of most fruits and vegetables are now relatively well established. Modified atmospheres reduce microbial spoilage of fruits and vegetables in many instances, although some spoilage microorganisms are not directly inhibited. Decontamination aims at reducing the number of microbial contaminants on the surface of fruits and vegetables, thereby prolonging the time required to develop spoilage. Chlorine at concentrations that will cause several decimal reductions of pathogens in pure water is often slightly more efficient than washing produce with pure water. Chitosan, a compound derived from chitin, has antimicrobial properties when used, for instance, to coat fresh fruits and vegetables for the purpose of regulating gas and moisture exchange.

This chapter presents an overview of the behavior of microorganisms on nuts, cereals, and products, with particular emphasis on describing conditions that permit or inhibit growth and treatments that can be used for their control or elimination. Seed coats or pellicles are present on all nuts and develop from tissues originally surrounding the ovule. Worldwide, production, harvesting, and processing techniques for nuts range from highly mechanized to labor-intensive, and methods vary significantly for the various types of nuts. Water activity values of less than 0.70 essentially eliminate bacterial and fungal growth in nuts. Aflatoxins, produced by A. flavus, Aspergillus parasiticus, and Aspergillus nomius, are the most common mycotoxins found in nuts and nut products. Soils are partially removed during harvest by forced air or sifting, but significant amounts may be brought into storage and processing facilities and can contaminate equipment and nuts during processing. Long-term storage temperatures range from 4°C to ambient, but after processing and packaging, nuts are generally distributed and displayed at the retail level at ambient temperature.

Salmonella spp. are facultatively anaerobic gram-negative rod-shaped bacteria belonging to the family Enterobacteriaceae. The biochemical identification of foodborne and clinical Salmonella isolates is generally coupled to serological confirmation, a complex and labor-intensive technique involving the agglutination of bacterial surface antigens with Salmonella-specific antibodies. National epidemiologic registries continue to underscore the importance of Salmonella spp. as a leading cause of foodborne bacterial illnesses in humans, where reported incidents of foodborne salmonellosis tend to dwarf those associated with other foodborne bacterial pathogens. The antibiotic resistance genes currently present in bacterial pathogens may originate from antibiotic-producing bacteria present in soil. The major global impact of typhoid and paratyphoid salmonellae on human health led to the early development of parenteral vaccines consisting of heat-, alcohol-, or acetone-killed cells. Salmonella invasion genes are organized into contiguous and functionally related loci, Salmonella pathogenicity islands (SPI1), located within a 40 to 50-kb segment of chromosomal DNA (i.e., pathogenicity island) that encodes determinant factors for the facilitated entry of salmonellae into host cells. The presence of virulence plasmids within the Salmonella genus is limited and has been confirmed for serovars Typhimurium, Dublin, Gallinarum-Pullorum, Enteritidis, Choleraesuis, and Abortusovis. This chapter provides a brief overview of the operating characteristics and sensitivity of polymerase chain reaction (PCR) technology, which is rapidly gaining application in food microbiology.

Campylobacter jejuni is susceptible to a variety of environmental conditions that make it unlikely to survive for long periods of time outside the host. Campylobacter spp. produce a number of enzymes that inactivate reactive oxygen intermediates, such as superoxide dismutase, alkyl hydroperoxide reductase, and catalase, and the enzymes protect the bacteria from the products of oxygen exposure. C. jejuni is susceptible to low pH, and hence, the gastric environment is sufficient to kill most campylobacters. C. jejuni can cause an enterotoxigenic-like illness with loose or watery diarrhea or an inflammatory colitis with fever and the presence of fecal blood and leukocytes and occasionally bacteremia that suggests an invasive mechanism of disease. Motility and flagella are important determinants for the invasion-translocation process. Motility mutants are frequently isolated in mutagenesis studies and point to the importance of this factor in virulence. Flagellin is an important immunogen during Campylobacter infection, and antibodies against this protein correlate to some degree with protective immunity. Several researchers are working toward developing a vaccine for Campylobacter infection. Campylobacter has received renewed attention by governmental agencies, which has already led to infusion of resources for research in the future.

Diarrheagenic Escherichia coli isolates are categorized into specific groups (pathotypes) based on virulence properties, mechanisms of pathogenicity, clinical syndromes, and distinct O:H serotypes. This chapter focuses on enterohemorrhagic E. coli (EHEC), which among the E. coli strains that cause foodborne illness in the United States, is the most significant group based on frequency and severity of illness. Since E. coli O157:H7 is the most common serotype of the EHEC and because more is known about this serotype than other serotypes of EHEC, the chapter focuses on E. coli O157:H7. E. coli O157:H7 strains isolated from humans, animals, and food have developed resistance to multiple antibiotics, with streptomycin-sulfisoxazoletetracycline being the most common resistance profile. Details of many reported foodborne and waterborne outbreaks of EHEC infections are provided in the chapter. The nomenclature of the Shiga toxins (Stx) family and their important characteristics are listed. Stx and Stx1 production is negatively regulated at the transcriptional level by an iron-Fur protein corepressor complex which binds at the stx1 promoter but is unaffected by temperature. The severity of the illness it causes combined with its apparent low infectious dose qualifies E. coli O157:H7 to be among the most serious of known foodborne pathogens. E. coli O157: H7 is still by far the most important serotype of Shiga toxin-producing E. coli (STEC) in North America. Isolation of non-O157:H7 STEC requires techniques not generally used in clinical laboratories; hence, these bacteria are infrequently sought or detected in routine practice.

This chapter provides a review of the basic biology, ecology, pathogenicity, and epidemiology of Enterobacter sakazakii known as an emerging opportunistic foodborne pathogen. E. sakazakii produces colonies with distinct morphologies. The chapter describes susceptibility of E. sakazakii to physical and chemical treatments. The major food product linked to cases of E. sakazakii infection is powdered infant formula (PIF). The natural habitat of E. sakazakii remains unknown. In recent years, efforts have focused on detecting this organism in a wide variety of environments. E. sakazakii has been isolated from environmental samples such as water, dust, soil, plant materials, mud, and even household vacuum cleaner bags, indicating that its ecological niche is quite diverse. One of the most commonly used methods for isolation of E. sakazakii from PIF was described by the U.S. Food and Drug Administration (FDA). The FDA method was developed for the isolation and enumeration of E. sakazakii from PIF. E. sakazakii has been associated mainly with necrotizing enterocolitis, septicemia, and meningitis. Most of the E. sakazakii infections that have been reported have occurred in developed nations. The chapter focuses on virulence factors and pathogenicity of E. sakazakii. Several studies have described the resistance of Enterobacter isolates to quinolones, ß-lactams, and trimethoprim-sulfamethoxazole. E. sakazakii has been reported as being more sensitive than other Enterobacter spp. to some antibiotics including the aminoglycosides, ureidopenicillins, ampicillin, and carboxypenicillins. E. sakazakii has become a growing concern for government regulatory agencies, health care providers, and PIF manufacturers.

Yersinia enterocolitica is an invasive enteric pathogen whose virulence determinants have been the subject of intensive investigation, but not all strains of Y. enterocolitica are equally virulent. Schemes for subtyping Yersinia species include bacteriophage typing, multienzyme electrophoresis, multilocus sequence typing, and the demonstration of restriction fragment length polymorphisms of chromosomal and plasmid DNA. As the mechanisms by which biotype 1A strains cause disease are largely unknown, a section of this chapter focuses chiefly on the virulence determinants of the classical pathogenic, i.e., pYV-bearing, highly invasive strains of Y. enterocolitica. As with other enterobacteria, Y. enterocolitica can be classified as smooth or rough depending on the amount of O side chain polysaccharide attached to the inner core region of the cell wall lipopolysaccharide (LPS). The major receptor for ferri-yersiniabactin complex is a 65-kDa outer membrane protein named FyuA, which also serves as a receptor for pesticin, a bacteriocin produced by Y. pestis. The effector, Yops, achieve these outcomes mostly by disrupting the proinflammatory signaling pathways that are activated in response to stimulation by invasin, YadA, and LPS. Although much remains to be learned about Y. enterocolitica, investigations into the pathogenesis of yersiniosis to date have provided fascinating new insights into bacterial pathogenesis as a whole and into its genetic control.

This chapter talks about the members of the Shigella species and the disease they cause. Modes of transmission and examples of recent foodborne outbreaks are also presented in the chapter. It also discusses the current understanding of the genetics of Shigella pathogenesis, the genes involved in causing disease, and how they are regulated. Common foods that have been implicated in outbreaks caused by shigellae include potato salad, chicken salad, tossed salad, and shellfish. The locations where these types of Shigella-contaminated foods were served ranged from the home to restaurants, camps, picnics, schools, airlines, sorority houses, and military mess halls. The adverse effect of foodborne pathogenesis on public health is reflected by its notable morbidity and mortality. Reactive arthritis is a postinfection sequela to shigellosis that is strongly associated with individuals of the HLA-B27 histocompatibility group. The syndrome is comprised of three symptoms, urethritis, conjunctivitis, and arthritis, with the last being the most dominant symptom. The single most effective means of preventing secondary transmission is handwashing. Observation of Shigella mutants established intracellular spread as the fourth hallmark of Shigella virulence.

A number of reviews on the pathogenic vibrios have appeared over the years, although with the exception of those of Vibrio cholerae and V. parahaemolyticus, relatively little is known of the virulence mechanisms they employ. One of the most consistent features of human vibrio infections is a recent history of seafood consumption. A survey of frozen raw shrimp imported from Mexico, China, and Ecuador found over 63% to harbor Vibrio species, including V. vulnificus and V. parahaemolyticus. By employing sucrose as a differentiating trait, 11 of the 12 human pathogenic vibrios can be separated on thiosulfate-citrate-bile salts-sucrose (TCBS) into 6 species which are generally sucrose positive and 5 species which are generally sucrose negative. With the exception of those of V. cholerae, V. parahaemolyticus, and V. vulnificus, relatively little is known of the susceptibilities of vibrios to various food preservation methods. As with that of other Vibrio species, the reservoir of V. mimicus is the aquatic environment. Indeed, studies from other laboratories using arbitrarily primed polymerase chain reaction (PCR), ribotyping, pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphisms all indicate that no two V. vulnificus isolates have the same chromosomal arrangement. In addition to the role of capsule, iron, and endotoxin in the pathogenesis of V. vulnificus infections, V. vulnificus produces a large number of extracellular compounds, including hemolysin, protease, elastase, collagenase, DNase, lipase, phospholipase, mucinase, chondroitin sulfatase, hyaluronidase, and fibrinolysin.

Recent studies have identified previously unknown Aeromonas virulence mechanisms that may contribute significantly to human pathogenicity. This chapter reviews existing knowledge of Aeromonas virulence factors and host responses and future directions that may provide a more detailed understanding of Aeromonas-associated human diseases. Aeromonas extraintestinal diseases (peritonitis, endocarditis, pneumonia, conjunctivitis, and urinary tract infections) are more common and varied than those caused by Plesiomonas shigelloides (cellulitis, arthritis, endophthalmitis, and cholecystitis). Aeromonas spp., therefore, may produce different types of cytotonic enterotoxins that are functionally similar. It is plausible that the expression of various virulence factors of Aeromonas may be controlled by quorum sensing. The major signal molecule synthesized by the ahyI locus in A. hydrophila was N-(butanoyl)-L-homoserine lactone (BHL), also referred to as C4-HSL, with N-acylhomoserine lactone (AHL) synthesized in relatively smaller amounts. At present, it is not possible to identify the disease-causing strains because of our incomplete understanding of Aeromonas virulence mechanisms. Some of the confusion surrounding Aeromonas enterotoxins has been resolved, and several new virulence factors have been described. The case for Plesiomonas’s being an enteric pathogen is less convincing than that for Aeromonas spp. Although Plesiomonas species has been isolated from diarrheal patients and has been incriminated in several large water- and foodborne outbreaks, no definite virulence mechanism has been identified in most strains associated with gastrointestinal infections.

Botulism is a neuroparalytic disease in humans and animals, resulting from the actions of neurotoxins produced by Clostridium botulinum and rare strains of Clostridium butyricum and Clostridium baratii. This chapter talks about historical features of C. botulinum and botulism, and biochemistry and pharmacology of botulinal neurotoxins. The hallmark clinical symptoms of botulism are a bilateral and descending weakening and paralysis of skeletal muscles. Currently there is no treatment for botulism except for passive administration of antibodies at early stages in the disease before botulinal neurotoxin has begun internalization into nerves. The major treatment of botulism is supportive nursing care, with specific attention given to respiratory ability and the need for mechanical ventilation. The chapter discusses epidemiology of foodborne botulism. The defining feature of botulinogenic clostridia is that they produce botulinal neurotoxin. Prevention of botulinal neurotoxin formation in foods can be achieved by avoiding contamination of foods by spores; inactivating spores that are present in foods; preventing spores from germination and vegetative cell growth resulting in botulinal neurotoxin formation; and inactivation of botulinal neurotoxins in food. The primary factors that control growth of C. botulinum in foods are temperature, pH and acidity, water activity, redox potential, nutrient sufficiency, the presence of antimicrobials, and competitive microflora. The chapter describes the use of predictive modeling and challenge studies for evaluation of C. botulinum neurotoxin formation, and genomics of C. botulinum. Remarkable advances have been achieved during the past decade in elucidating the biochemistry, structure, and pharmacological mechanisms of botulinum neurotoxins.

Clostridium perfringens was initially recognized as an important cause of foodborne disease in the 1940s and 50s. It later became apparent that C. perfringens causes two quite different human foodborne diseases, i.e., C. perfringens type A food poisoning and necrotic enteritis. Since foodborne necrotic enteritis is rare in industrialized societies, this chapter focuses mainly on C. perfringens type A food poisoning. Compared to most other anaerobes, C. perfringens requires only relatively modest reductions in oxidation-reduction potential (Eh) for growth. A distinct toxin type is associated with each of the two foodborne diseases caused by C. perfringens. Necrotic enteritis, a life-threatening illness, is usually caused by type C isolates, with ß-toxin being considered the primary virulence factor responsible for this illness. C. perfringens enterotoxin (CPE) is classified as an enterotoxin because it induces fluid and electrolyte losses from the gastrointestinal (GI) tract of many mammalian species. The cytotoxic action of CPE is responsible for the tissue damage that initiates CPE-induced intestinal fluid and electrolyte alterations. Interestingly, both CPE-induced apoptosis and oncosis involve the cytoplasmic proteins calmodulin and calpain. Effective immunity against C. perfringens type A food poisoning may require a secretory immunoglobulin A response in the intestinal lumen.

Bacillus cereus is widespread in nature and frequently isolated from soil and growing plants . In addition to rice, pasta, and spices, dairy products are among the most common food vehicles for B. cereus. There are two types of B. cereus foodborne illness. The first type, which is caused by an emetic toxin, results in vomiting, whereas the second type, which is caused by enterotoxin(s), results in diarrhea. Little is known about susceptible populations, but the more severe types of the illness have occasionally involved young athletes (19 years) or the elderly (60 years). The emetic toxin, causing vomiting, had been isolated and characterized, whereas the diarrheal disease is caused by one or more enterotoxins. The spore of B. cereus is an important factor in foodborne illness. First, the B. cereus spore is more hydrophobic than any other Bacillus spp. spores, which enables it to adhere to several types of surfaces. Neither of the two commercial immunoassays available for enterotoxin detection can quantify the toxicity of B. cereus enterotoxins. Desserts, meat dishes, and dairy products are most frequently associated with diarrheal illness, whereas rice and pasta are the most common vehicles of emetic illness. Three types of B. cereus enterotoxins involved in outbreaks of foodborne illness have been identified. Two of these enterotoxins possess three components and are related, whereas the third is a one-component protein (CytK).

Listeriosis is an atypical foodborne illness of major public health concern because of the severity of the disease (meningitis, septicemia, and abortion), a high case fatality rate (approximately 20 to 30% of cases), a long incubation time, and a predilection for individuals who have an underlying condition that leads to impairment of T-cell-mediated immunity. Certain ready-to-eat (RTE) processed foods are high-risk vehicles for transmitting listeriosis for susceptible populations as determined by active surveillance for sporadic listeriosis and epidemiologic investigation of listeriosis outbreaks. The risk assessment model was used to estimate the likely impact of control strategies by changing one or two input parameters and measuring the change in the model outputs. Most thermal-inactivation studies of Listeria monocytogenes in milk have shown that cells of L. monocytogenes suspended in milk were effectively inactivated under high temperature-short time pasteurization (HTST) conditions (71°C for 15s or equivalent). The LisRK two-component signal transduction system is implicated in virulence, acid and ethanol tolerance, and oxidative stress. The major heat shock chaperones, GroES and GroEL, are induced at high temperature, at low pH, and during cell infection. Research during the past 25 years has led to the (i) identification of the internalin receptor of mammalian cells; (ii) elucidation of the role of internalin multigene functions; (iii) understanding of the function of actA; (iv) understanding of prfA and the global regulation of virulence; and (v) the modulation of host cell signaling by the pathogen.

This chapter addresses primarily staphylococcal food poisoning (SFP); however, in regard to the staphylococcal enterotoxins (SEs), there is significant overlap in the natural histories of both diseases. Hence, toxic shock syndrome (TSS) is also discussed in the chapter in which this overlap is most relevant. The chapter discusses the nomenclature and evolution of the SE family of toxins. SEC expression is affected by glucose through at least two different mechanisms. First, the metabolism of glucose indirectly influences SEC production through agr by reducing pH. Glucose also reduces sec expression in agr mutant strains. This observation suggests the existence of a second glucose-dependent mechanism for reduction of SE expression, independent of agr and apparently not involving pH. The chapter talks about toxic dose and susceptible populations. The study of purified SEs has provided useful comparative information and has important research applications, but its direct relevance to SFP is uncertain because potential stabilization of unpurified SEs by food is an important consideration. The chapter discusses virulence factors and mechanisms of pathogenicity. Although the increased number of identified SEs and putative SEs is beginning to make immunological detection of SEs obsolete, several commercial reagents relying on this technique are still widely used. However, detection based on antigenicity is gradually being replaced by molecular techniques, especially multiplex PCR. Progress has been made toward understanding the molecular aspects relevant to SFP.

This chapter provides an introduction to epidemiology and epidemiologic methods as they are applied to problems of foodborne diseases. The concepts and methods of epidemiology can be used to examine the relationships between disease and all levels of food safety, from production and distribution to preparation and consumption. In discussing the epidemiology of foodborne diseases it is important to keep in mind the chain of infection. This includes the agent, the reservoir that contains the agent, a means of escape from the reservoir, a mode of transmission to a susceptible host, and a means of entry into the host. These elements define the agent-host environment that results in the occurrence of illness. Many of potential hazards, either in terms of specific agents, specific food ingredients, or various agent-food interactions, were originally determined as a result of foodborne illness surveillance. As food sources and foodborne disease agents are constantly changing, hazard analysis is an ongoing process that requires continuous support from public health surveillance of foodborne diseases. Public health surveillance of foodborne disease is critical to the performance of food safety systems that are based on hazard analysis and critical control point (HACCP) systems plans. Surveillance is required to identify new hazards. It also provides the ultimate feedback on the efficacy of HACCP plans. Although this chapter focuses on experiences in the United States, the same methods of observation and analysis should form the basis of foodborne disease surveillance in developed and developing countries throughout the world.

Aspergillus species occur in foods as spoilage or biodeterioration fungi. The most important toxigenic Aspergillus species in foods are the aflatoxigenic molds, A. flavus and A. parasiticus, along with a recently described but much less common species, A. nomius, all of which are classified in Aspergillus section Flavi. The most effective medium for rapid detection of aflatoxigenic molds is A. flavus and parasiticus agar, a medium formulated specifically for this purpose. The combination of characteristics most useful in differentiation among the three aflatoxigenic species is summarized. A. flavus is widely distributed in nature, but A. parasiticus is probably less widespread, determination of the actual extent of its occurrence being complicated by the tendency for both species to be reported indiscriminately as A. flavus. Aflatoxins are one of the few mycotoxins covered by legislation. A. ochraceus is the most commonly occurring species in what was known as the A. ochraceus group by Raper and Fennell, now correctly known as Aspergillus section Circumdati. Aspergillus clavatus is the most common member of the section Clavati, subgenus Clavati, and is easily recognizable by its large, blue-green clavate (club-shaped) heads. The genus Eurotium is an ascomycete genus characterized by the formation of bright yellow cleistothecia, often enmeshed in yellow, orange, or red hyphae, overlayed by the gray-green (glaucous) Aspergillus heads of the anamorphic state. Aspergillus is one of the most important genera in the spoilage of foods and animal feeds, particularly in warm-temperate climates and the tropics.

The discovery of penicillin in 1929 gave impetus to a search for other Penicillium metabolites with antibiotic properties and, ultimately, to the recognition of citrinin, patulin, and griseofulvin as “toxic antibiotics” or, later, mycotoxins. In a comprehensive review of the literature on fungal metabolites, about 120 common mold species were found to be demonstrably toxic to higher animals. The majority of important toxigenic and food spoilage species are found in the subgenus Penicillium. The major source of ochratoxin A in foods is bread made from barley or wheat in which P. verrucosum has grown. It has been suggested that ochratoxin A is a causal agent of Balkan endemic nephropathy, a kidney disease with a high mortality rate in certain areas of Bulgaria, Yugoslavia, and Romania. Several tremorgenic mycotoxins are produced by Penicillium species, with the most important being the highly toxic penitrem A. Verruculogen, which is equally toxic, is not produced by species of common occurrence in foods. Secalonic acid D, the only secalonic acid produced by Penicillium species, has significant animal toxicity. Basic procedures for mycotoxin assays are sampling and subsampling, extraction and cleanup, and detection, quantification, and confirmation. The newest approach to rapid assays involves the use of antibodies developed to specific toxins. Immunoassays using either spot or minicolumn tests have been developed for several major toxins. Much more research is needed to improve detection methods, to understand the ecology of toxigenic Penicillium species, and to evaluate the significance of Penicillium toxins in human health.

Toxigenic molds in genera other than Aspergillus and Penicillium are most often found as contaminants of plant-derived foods, especially cereal grains. The most important group of mycotoxigenic molds other than Aspergillus and Penicillium species are species of the genus Fusarium. A very severe human disease that occurred in the former Soviet Union during World War II, known as alimentary toxic aleukia, is believed to be caused by T-2 and HT-2 toxins produced by Fusarium sporotrichioides and F. poae of the Sporotrichiella section of Fusarium. Dietary deoxynivalenol has been shown to stimulate immunoglobin production, causing elevated immunoglobin A levels in mice. Among the harmful effects of this stimulation is kidney damage very similar to a common human kidney condition known as glomerulonephritis or immunoglobulin A nephropathy. The most distinctive feature of F. poae is its production of abundant globose to oval, almost pyriform (pear-shaped) microconidia, with few macroconidia. The major mycotoxins produced by Fusarium graminearum are deoxynivalenol and zearalenone. The main human disease associated with F. verticillioides is esophageal cancer. The first problem encountered in the analysis of grains for Fusarium toxins is the same as that for other mycotoxins, i.e., sampling. Deoxynivalenol is the most common trichothecene found in commodity grains; therefore, the greatest potential exists for it to occur in finished foods. Other potentially toxic molds, aside from Aspergillus, Penicillium, and Fusarium species, that may contaminate foods include species of the genera Acremonium, Alternaria, Byssochlamys, Chaetomium, Cladosporium, Claviceps, Myrothecium, Neosartorya, Phomopis, Rhizoctonia, and Rhizopus.

Human enteric viruses have properties that are unique from those of bacterial foodborne pathogens. From an epidemiologic perspective, the noroviruses (NoVs) and hepatitis A virus (HAV) are the two most important enteric virus groups transmitted by foodborne routes. Human isolates of HAV comprise a single serotype, and monoclonal antibodies raised to different isolates fail to distinguish the isolates from one another. The rotaviruses usually are transmitted by either waterborne or person-to-person routes, but they can be food borne. This virus group is the leading cause of infantile diarrhea worldwide and is responsible for up to 130 million illnesses and 600,000 to 870,000 deaths per year, with the vast majority of rotavirus associated deaths occurring in developing countries. The contamination of produce items usually occurs before the product reaches food service establishments. The most commonly implicated bivalves are oysters, followed by clams, and it is now estimated that human enteric viruses are the most common disease agents transmitted by molluscan shellfish. Adherence to strict hygienic practices when handling and preparing foods is critical to control viral contamination of ready-to-eat (RTE) food products. A potential control strategy, albeit in developmental stages, would be the detection of viral contamination in foods. Improved and more widespread reporting and investigation of foodborne viral disease outbreaks, and targeted epidemiologic studies to identify the risk factors for viral gastroenteritis, would improve the understanding of attribution.

Human transmissible spongiform encephalopathies (TSEs) include the prototypic disease kuru, which is limited to Papua New Guinea and is now virtually extinct. In the event that infectivity is definitively found to be present in muscle from cattle infected with Bovine Spongiform Encephalopathy (BSE), available scientific and epidemiological evidence to date suggests that it does not appear to exist in amounts sufficient to increase the risk to human health in countries with adequate prevention and control measures. The continuance of BSE cases due to cross contamination and cross feeding of ruminant meat and bone meal (MBM) has resulted in extended feed bans which have prohibited feeding of all mammalian or animal meat and bone meal (MBM) to any animals used for human food. Humans most likely became infected with the agent that causes BSE through the consumption of beef products contaminated by central nervous systems (CNS) tissue, such as mechanically recovered meat that was pressure extracted from carcasses and often contained spinal cord tissue and paraspinal ganglia in addition to residual muscle shards. The combination of psychiatric and sensory symptoms in an adolescent or young adult is sufficient to raise a suspicion of Creutzfeldt-Jakob disease (CJD) together with its variant form (vCJD) in patients who reside or have resided in countries in which BSE has occurred.

Foodborne parasites have undoubtedly had an impact on human health throughout history. There are four meat-borne helminths of medical significance: Trichinella spp., Taenia solium, and Taenia asiatica, which occur primarily in pork, and Taenia saginata, which is found in beef. Encapsulated species of Trichinella have been found only in mammalian hosts. All species of Trichinella complete their life cycle within one host; no intermediate hosts or extrinsic development is required. Infection in humans typically represents a dead end in the parasite’s life cycle. Trichinellosis, human disease resulting from infection with species of the genus Trichinella, is considered a zoonosis because infection occurs as a result of ingestion of raw or poorly cooked meat from infected animals. Damage caused by Trichinella infection varies with the intensity of the infection and the tissues invaded. Classical symptoms of trichinellosis include fever, myalgia, and periorbital edema, and these symptoms may vary depending on the species of Trichinella. Mebendazole is also believed to be active against developing and encysted larvae but at dosages higher than those used against adult worms. Taeniasis results when raw or inadequately cooked beef containing tissue cysts is consumed. Chronic persistence of and periodic increases in human T. saginata infections correlate with factors that govern infections in cattle.

A variety of human helminthic infections can be acquired through the consumption of food products from infected animals and plants, through the accidental ingestion of infected invertebrates in foodstuffs or drinking water, or through inadvertent fecal contamination by humans or animals. Prevention of biohelminth infections can be accomplished by avoiding the intermediate hosts or by adequately cooking foods. In contrast, helminths with eggs or free-living stages that can survive a certain length of time in the external environment, termed geohelminths, are typically transmitted via contaminated water or foods and are best controlled by improved sanitation. Several related nematodes of the genera Anisakis, Pseudoterranova, and Contracaecum may be acquired by eating raw fish or squid in seafood dishes such as sushi, sashimi, seviche, and lomi-lomi. Infection may also be acquired from shellfish juices used in food dishes or folk remedies, from food prepared by using contaminated utensils or chopping blocks, or from drinking water contaminated with metacercariae released from dead or injured crustaceans. Raw foods, particularly fish and chicken, should be avoided in areas where infection is endemic, and drinking water should be filtered before consumption. Human infection with Dicrocoelium is explained by the accidental ingestion of ants on vegetation, and Angiostrongylus costaricensis is thought to be acquired by eating raw fruits and vegetables on which snails have left larvae in mucus deposits or by accidentally ingesting infected snails on unwashed vegetation. Preventive measures include protection of grains and foodstuffs from insects and rodent control.

Protozoan parasites have long been associated with foodborne and waterborne outbreaks of disease in humans. A major characteristic of apicomplexan parasites is that a vertebrate host is required to complete the complex life cycle and produce infectious cysts. Of this group, Cryptosporidium species, Cyclospora cayetanensis, and Isospora belli inhabit the intestinal mucosa and produce diarrheal illnesses in humans. Microsporidia, once considered to be protozoan parasites, have been reclassified as fungi based on recent phylogenetic analyses. Cryptosporidium was first isolated in 1910 from the intestines of mice. Various species of Cryptosporidium capable of infecting animals have since been described. Genotype differentiation is based on the sequencing of the thrombospondin-related adhesive protein (TRAP-C2). Cyclospora was probably first reported to occur in humans in 1979 by Ashford, who described it as an Isospora-like coccidian affecting humans in Papua New Guinea. In Nepal, the prevalence of Cyclospora infection is highest in adult expatriates, whereas in areas of Peru, where infection is endemic, children under 10 years of age are the most susceptible to infection, though most are asymptomatic. Isospora is a coccidian parasite which infects humans. Isospora can be acquired by ingestion of contaminated food or water. Toxoplasmosis can be acquired by ingestion of lamb, poultry, horse, and wild game animals. Giardia infection can be diagnosed by finding cysts or, less commonly, trophozoites in fecal specimens. Fulminant colitis is characterized by severe bloody diarrhea, fever, and abdominal tenderness due to transmural necrosis of the bowel.

Physical methods of food preservation are those that utilize physical treatments to inhibit, destroy, or remove undesirable microorganisms without involving antimicrobial additives or products of microbial metabolism as preservative factors. Microorganisms can be destroyed by established physical microbicide treatments such as heating (including microwave heat treatment), UV or ionizing radiation, and emerging methods of new nonthermal treatments, such as the use of high hydrostatic pressure, pulsed electric fields (PEFs), oscillating magnetic fields, photodynamic effects, and a combination of physical processes such as heat irradiation, dehydroirradiation, and manothermosonication. Mechanical removal of microorganisms from food may be accomplished by membrane filtration of food liquids. This chapter discusses the microbiological fundamentals of the physical preservation methods outlined above, with the exception of mechanical removal. One of the oldest methods for preserving food is dehydration, and water is one of the most important factors controlling the rate of deterioration of food, by either microbial or nonmicrobial effects. The most effective and most widely used method for destroying microorganisms and inactivating enzymes is heat treatment. Sensitization of microorganisms with the aim of lowering the dose can be obtained by heating and chemical means. During recent years, several other physical treatments such as the use of high hydrostatic pressure and electric and magnetic fields have attracted much interest as promising tools for food processing and preservation and for the creation of new types of food products.

Antimicrobials are classified as traditional when they (i) have been used for many years, (ii) are approved by many countries for inclusion as antimicrobials in foods (e.g., lysozyme and lactoferrin, which are naturally occurring but regulatory-agency approved), or (iii) are produced by synthetic means (as opposed to natural extracts). Many organic acids are used as food additives, but not all have antimicrobial activity. Research suggests that the most active are acetic, lactic, propionic, sorbic, and benzoic acids. Acetic acid was the most effective antimicrobial in ground roasted beef slurries against Escherichia coli O157:H7 growth in comparison with citric or lactic acid. Sorbate is applied to foods by direct addition, dipping, spraying, dusting, or incorporation into packaging. The mechanism by which dimethyl dicarbonate (DMDC) acts is most likely related to inactivation of enzymes. A related compound, diethyl dicarbonate, reacts with imidazole groups, amines, or thiols of proteins. Lysozyme is most active against gram-positive bacteria, most likely because the peptidoglycan of the cell wall is more exposed. The primary use for sodium nitrite as an antimicrobial is to inhibit Clostridium botulinum growth and toxin production in cured meats. Sulfites may be used to inhibit acetic acid-producing bacteria, lactic acid bacteria, and spoilage bacteria in meat products. In the future, traditional food antimicrobials will continue to play an important role in food preservation.

This chapter provides an overview of the biologically based preservation technologies termed “biopreservation.” Acid production by lactic acid bacteria (LAB) in temperature-abused foods (controlled acidification) is covered in the chapter. Some LAB produce antimicrobial proteins, called bacteriocins, which inhibit spoilage and pathogenic bacteria without changing (e.g., through acidification, protein denaturation, and other processes) the physicochemical nature of the food. While organic acids are usually added to foods, LAB can produce lactic acid in situ. The controlled production of acid in situ is an important form of biopreservation. There are many different ways to use bacteriocins in foods. The first is to add bacteriocins directly to the food for the purpose of inhibiting spoilage or pathogenic bacteria. The second way to use bacteriocins is to add bacteriocinogenic cultures to the food or use them as starter cultures that produce the bacteriocin in situ. A third way to use bacteriocins is to facilitate the use of defined starter cultures in fermented foods. Emulsifiers such as Tween 80 or the entrapment of the pediocin in multilamellar vesicles increases pediocin effectiveness in fatty foods. Nisin is the only bacteriocin approved internationally for use in foods. The biological methods of food preservation covered here mark only the beginning of the biopreservation era in the food industry.

The primary microflora used in the production of fermented milk products are the homofermentative lactic acid bacteria (LAB). Additionally, yeasts, molds, and several other species of bacteria, including heterofermentative LAB, may be added to specific products; however, their purpose is not for acid development but for the production of flavor components or carbon dioxide. In some fermented dairy products, additional bacteria, often referred to as secondary microflora (but essential to flavor development), are added to influence flavor and alter texture of the final product. The use of nonstarter LAB, especially lactobacilli, as adjunct flavor cultures is a burgeoning research area and is practiced commercially. Proteolytic systems in LAB contribute to their ability to grow in milk and are necessary for the development of flavor in ripened cheeses. The production of high-quality fermented dairy products is dependent on the proteolytic systems of LAB. Bacteriophage infection may lead to a decrease or complete inhibition of lactic acid production by the starter culture. This has a major impact on the manufacture of fermented dairy products, as lactic acid synthesis is required to produce these products. Researchers have begun to characterize host components required for bacteriophage adsorption. It is now thought that bacteriophages initially interact reversibly with cell envelope-associated polysaccharide and then interact irreversibly with cell membrane protein(s).

Vegetable fermentation by lactic acid bacteria (LAB) in a salt brine began as a way to preserve foods for out-of-season use and for long journeys, especially by sea. Prior to the 1920s, research in the United States on pickled vegetables was primarily focused on product surveys and descriptions of brining methods. Reports on the microbiology and biochemistry of vegetable fermentations appeared in the literature between 1918 and 1920. Current research on pickled vegetables includes the genomics of LAB, mathematical modeling of bacterial growth and competition, the molecular ecology of vegetable fermentations, closed-tank fermentation technology to reduce salt waste, the use of clays to filter brines for recycling, sensory perception of pickled vegetable products, and the safety of acidified foods. There is continuing research interest in fermentation and storage of vegetables, particularly cucumbers, with reduced salt. The most notable effect of fermentation on cucumber volatiles was the inhibition of production of (E, Z)-2,6-nonadienal and 2-nonenal, the two most important odor impact compounds in fresh cucumbers. Among Lactococcus lactis genome sequences are those belonging to the predominant bacteria present in fermented vegetables, Lactobacillus plantarum, Lactobacillus mesenteroides, Pediococcus pentosaceus, and Lactobacillus brevis. As expected, none of the predominant LAB in fermented vegetables have predicted genes for a complete citric acid cycle. The development of low-salt fermentations and storage of fermented vegetables for commercial use present significant technological hurdles, including the potential need for starter cultures (and the impact of bacteriophage on starter cultures) and for new product handling equipment.

Understanding the technological, microbiological, and biochemical processes that occur during meat, poultry, and fish fermentation is essential for ensuring safe, palatable products. Dry and semidry sausages represent the largest category of fermented meat products, with many present-day processing practices having their origin in the Mediterranean region. Micrococci and staphylococci reduce nitrate to nitrite to generate nitric oxide, which reacts with myoglobin to produce the characteristic cured color of fermented meats. Slightly larger amounts of fermentable carbohydrate should be used in formulas containing higher-pH meats, such as poultry. Mechanically deboned poultry meat is an acceptable meat source for fermented dry sausages when limited to 10% of the meat block, as sausages tend to become soft when larger amounts are used. Thus, the combined effects of low pH, increased acidity, concomitant loss of moisture during drying, reduction of aw, concentration of curing salts, such as sodium chloride and sodium nitrite, bacterial inhibition of spoilage or pathogenic microorganisms, and heat processing (if applied) preserve fermented meat and poultry products against spoilage by inactivating indigenous tissue and bacterial enzymes. Pyruvate-formate lyase generates formate and acetyl Coenzyme A (CoA) from pyruvate and CoA, and the acetyl CoA can be used either as a precursor for substrate-level phosphorylation, for direct reduction to ethanol, or both. This alteration in pyruvate metabolism has been demonstrated in studies where lactobacilli have been shown to shift fermentation patterns and, concomitantly, the amount of potential ATP formed as the growth rate changes.

A variety of fermented foods can be found widespread over the world. Some of them are described in this chapter, mainly to illustrate the complexity of biochemical, nutritional, and sensorial changes that result from an array of microbial activities in a range of raw materials. Recently, experiments with oncom-miso made from soybeans and oncom demonstrated increased anti-oxidative and antimutagenic activity associated with the enzymatic release of isoflavone-aglycones. Actinomucor elegans and A. taiwanensis are used as pure-culture starters in the manufacture of Chinese fu-ru, or sufu. The major functions of the fermentation of idli include the leavening of batter and improvement of flavor and nutritional value. There are two specific fermentation stages involved in soy sauce production, the first being an aerobic koji fermentation. Seed (tane) koji is produced by culturing single or mixed strains of Aspergillus oryzae or Aspergillus sojae on either steamed, polished rice or a mixture of wheat bran and soybean flour. Seed koji is added to a soybean-wheat mixture at a concentration of 0.1 to 0.2% and fermented into what is then simply called koji. The second stage is an anaerobic moromi or salt mash which undergoes lactic acid bacteria (LAB) and yeast (zygosaccharomyces rouxii) fermentations. Of interest to food microbiologists and sanitarians is the possibility of microorganisms' producing toxic substances or of pathogenic microorganisms' surviving during fermentation or storage of indigenous fermented foods.

The majority of the world's cocoa is fermented on drying platforms, in heaps covered with banana leaves, in baskets, or in an assortment of wooden boxes. Wet cocoa beans are spread directly onto drying platforms where they ferment and dry during the day and are heaped into piles each night to conserve heat and retard the growth of surface molds. The initial microbial population is variable in number and type; however, the key groups active during fermentation are yeasts, lactic acid bacteria, and acetic acid bacteria. Significant changes in pH, temperature, and moisture occur during cocoa fermentation and drying processes that influence the type and quantity of flavor precursor compounds produced by enzymatic action. A summary of cocoa bean enzymes and their substrates and optimum pHs is provided in this chapter. The ultimate goal of biochemical changes during fermentation is to produce cocoa beans with desirable flavor and color characteristics. Unlike that in cocoa fermentation, there is no defined microbial succession that occurs with coffee maturation and fermentation. Although serving somewhat different purposes, microbial fermentation plays a critical role in the production of both cocoa and coffee. Coffee and cocoa are no exceptions, and it is the proper control of the fermentation process that largely determines the color and flavor qualities of the final products. Consequently, understanding the microbiology and biochemistry of cocoa and coffee production, as well as the factors that influence them, is critical to quality control.

This chapter provides a overview of the scientific principles of the brewing industry. Conversion of starch into simple sugars happens during mashing, and the changes during malting are limited to the breakdown of cell walls and the protein matrix in which the starch granules are embedded, but such modification of the grain is necessary for hydrolysis of the starch during mashing. It is now recognized that hops have an important antimicrobial, particularly antibacterial, effect, and it is presumed that the medieval brewers realized that hopped beers maintained their quality for longer periods of time than did beers with other flavorings. Formerly, the actively fermenting yeasts of the fermentation industries, both culture yeasts and common contaminant “wild yeasts,” were classified as different species of Saccharomyces. Most of these species are now classified officially by yeast taxonomists as a single species, S. cerevisiae, but still it is convenient in the brewing industry to distinguish the different types by their former specific names.Enterobacteria (including Obesumbacterium, the most important of that group in the brewery environment) cause turbidity and off-flavor and often produce indole, phenols, diacetyl, hydrogen sulfide, and dimethyl sulfide, but grow well in the early stages of fermentation until inhibited by the falling pH and increasing ethanol content. Megasphaera (cocci) and Pectinatus (rods) species are recently discovered strictly anaerobic gram-negative bacteria which form acetic, butyric, and propionic acids, hydrogen sulfide, dimethyl sulfide, and turbidity and have become troublesome only because of modern advances in maintaining very low dissolved oxygen levels in beer.

This chapter focuses on the occurrence, growth, and significance of microorganisms in winemaking. It covers wines produced only from grapes and includes table wines, sparkling wines, and fortified wines. The grapes are harvested at an appropriate stage of maturity which determines the chemical composition of the juice extracted from them. Particularly important are the concentrations of sugars and acids which are the major constituents of the juice and impact its fermentation properties. Yeasts are significant in winemaking because they carry out the alcoholic fermentation and they can cause spoilage of the wine. Their autolytic products may affect sensory quality and influence the growth of malolactic and spoilage bacteria. Wines with pHs exceeding 3.5 tend to have mixed microfloras consisting of Oenococcus oeni and various species of Pediococcus and Lactobacillus. Species of Pediococcus and Lactobacillus are more tolerant of higher concentrations of sulfur dioxide than is Oenococcus oeni and are more likely to occur in wines with larger amounts of this substance. Some factors that affect the growth of Oenococcus oeni in wine and successful completion of malolactic fermentation include excessive growth of molds and acetic acid bacteria on grapes, yeast species and strains responsible for the alcoholic fermentation, and bacteriophages. The microbial ecology and biochemistry of base wine production are essentially the same as those of table wine production.

The best-known prebiotics are fructo-oligosaccharides derived from food sources. The current potential for prebiotics rests largely with compounds that can be extracted from foods and investigation of their impact on the residing intestinal microbiota and health-based markers. Probiotic microorganisms designed for delivery in food or dairy products, via supplementation or fermentation, are usually members of the Lactobacillus or Bifidobacterium genus. The primary health targets for animal probiotics are enhancement of animal growth, attainment of weight gains, and reduction in the number of enteric pathogens. Bacterial probiotics are generally effective in chickens, pigs, and preruminant calves, whereas fungal probiotics have shown better results in adult ruminants. One's understanding of the impact of probiotics on the intestinal microbiota will be enhanced considerably by the use of molecular methods that can more accurately reflect those changes occurring within the entire gastrointestinal microbiota. The developments in molecular techniques over the past decade have removed many of the key issues which previously hindered scientific progress in probiotics.

The history of biotechnology reveals that scientific discoveries lead to technological advances and these are usually followed by a phase of research and development to find applications for the technologies. The authors are currently in another application phase, where new technologies such as real-time PCR (rtPCR), DNA microarrays, and biosensors are providing very sophisticated tools for use in diagnostics. This chapter therefore includes discussions on these "next-generation technologies," which will have an impact on the way one can test for pathogens and toxins in foods. All identification assays require a pure culture of the unknown bacteria, which is then identified most often by its biochemical characteristics. It is a lengthy, labor-intensive, and media-consuming process. The introduction of miniaturized biochemical kits, which provide a quick biochemical profile of bacteria at a great savings in cost, labor, and time, has simplified the process of bacterial identification and continues to be important in regulatory testing of foods. Many of the new methods use next-generation technologies such as rtPCR, biosensors, and DNA chips, which may be more rapid, more sensitive, and capable of multitarget testing and hence are well suited for use in screening large numbers of samples in compliance or food security surveillance programs. Furthermore, comparative evaluation by with standard methods or validation of rapid methods is critical to document their efficacy in detecting foodborne pathogens and toxins.

This chapter outlines the basic concepts underlying genomics, proteomics, and microarray technologies of foodborne microorganisms. The heart of all genomics research lies in DNA sequencing. DNA sequencing method utilizes normal DNA replication with a template strand, a primer, DNA polymerase, and a mix of deoxynucleotide triphosphates (dNTPs). DNA microarray analysis is a relatively new technology that allows investigators to take a genome-wide approach to biological systems. While the details of the Gad system were described primarily by experiments with Escherichia coli, genomics and bioinformatics have enabled researchers to identify and study the effects of these genes in other microorganisms. Foods and their microenvironments could potentially be better designed and formulated to minimize the expression of undesirable pathogenic traits (e.g., acid tolerance, virulence, and toxin formation) or to optimize the expression of beneficial properties in desirable microorganisms (e.g., cryoprotection, acidification rates, and adherence to intestinal tissues). The nature of food microbiology has changed dramatically from its historical emphasis on microbial phenotypic properties and behavior to a new perspective dominated by genomic and comparative genomic information. The food microbiologists of the future will become increasingly reliant on genomics and the other omics technologies in their efforts to understand and control microorganisms associated with foods.

This chapter deals with advances in both microbiological modeling and risk assessment. The abundance of data acquired has led to major advances in the ability to study food safety microbiology in a quantitative manner. The importance of environmental contamination, recontamination of heat-processed foods, and the numerous potential pathways for cross contamination has long been recognized; however, few investigators have attempted to quantify or model cross contamination. Numerous nonlinear and multiple-phase models have been used to describe inactivation kinetics, with the Weibull model being used by an increasing number of modelers. Risk managers determined that grouping by moisture level was most appropriate because the classification of cheeses for regulatory purposes is by moisture level. The risk management process also includes managing communication and transparency by creating outside expert panels, conducting public meetings, requesting independent reviews of the finished risk assessment, and disseminating the results in appropriate forums. Various techniques can be used with the risk assessment to determine which of the many parameters make the greatest contribution to the average value, the variation, or the uncertainty of the output distributions. The application of risk assessment techniques to better link microbiological criteria to public health goals is an area that is actively being pursued by regulatory agencies, international intergovernmental organizations and the food industry.

The systematic approach to food safety embodied by hazard analysis and critical control point (HACCP) system is based on seven principles. The approach taken in this chapter is that HACCP system plans should address only significant food safety hazards. However, for a sharper focus for this chapter, the authors have limited the examples of the application of HACCP to the food-manufacturing segment. Certain preliminary steps must be undertaken before beginning the hazard analysis. Just as microbiological testing is not a good tool for monitoring, microbiological criteria are typically not useful as critical limits in a HACCP program. As HACCP targets process control, factors that lend themselves to real-time monitoring and quick feedback should be identified as control measures. Thus, the critical limits used in a HACCP program do not typically relate to a criterion that is directly associated with microbiological testing of products or ingredients. Validation usually focuses on the process of ensuring that the plan reflects identification of all significant hazards, that the control measures identified are appropriate, that the designated critical limits are adequate to control the hazard, and that the rest of the features of the plan are satisfactory. The HACCP concept provides a systematic, structured approach to ensuring the safety of food products. However, there is no blueprint or universal formula for putting together the specific details of a HACCP plan.

This chapter focuses on subtyping for molecular epidemiology. Properties of methods commonly used for molecular subtyping of foodborne pathogens are discussed in the chapter. Subtyping methods includes plasmid profile analysis, restriction fragment length polymorphism (RFLP) methods and ribotyping. Although the focus of the chapter is on molecular methods, it is important to consider them in the context of earlier phenotypic methods such as serotyping, phage typing, biotyping, and antimicrobial susceptibility typing. Perhaps the most important reason for using molecular methods is that they do not require specialized reagents or expertise and can thus be performed in almost any laboratory with basic molecular biology capabilities. This same ease of performance is also the most obvious drawback to molecular subtyping, as it allows every laboratory to perform and interpret subtyping according to their own criteria, making interlaboratory comparisons more difficult unless laboratories can agree to standardized protocols. Specific applications of ribotyping in general and automated ribotyping using the RiboPrinter are discussed. The application of pulsed-field gel electrophoresis (PFGE) to specific foodborne pathogens is described. Applications of amplified fragment length polymorphism (AFLP) to specific foodborne bacteria are discussed in the chapter. For the past several years, PFGE has also been the gold standard for molecular subtyping of Listeria monocytogenes.