Food Microbiology: Fundamentals and Frontiers, Fourth Edition

This chapter addresses three issues: (i) the ability of bacteria to use different biochemical pathways to generate the energy required to grow under adverse conditions in foods; (ii) the interaction of bacteria and foods in ecosystems in which the cells may exist in a variety of physical and physiological states and in which the roles of intrinsic and extrinsic factors and (iii) the kinetics of microbial growth. It talks about microbial physiology and metabolism, and glycolytic pathways such as Embden-Meyerhof-Parnas Pathway and Entner-Doudoroff Pathway. It also discusses the heterofermentative catabolism, homofermentative catabolism, tricarboxylic acid cycle, aerobes, anaerobes, the regeneration of NAD, and respiration, bioenergetics. The food ecosystem is composed of intrinsic factors, which are inherent to the food (i.e., pH, water activity [aw], and nutrients) and extrinsic factors, which are external to it (i.e., temperature, gaseous environment, and the presence of other bacteria). In addition, the chapter focuses on the physiological and genetic responses that bacteria utilize in osmoregulation. Bacteria are classified as psychrophiles, psychrotrophs, mesophiles, and thermophiles according to how temperature influences their growth. Microbial growth in foods is a complex process governed by genetic, biochemical, and environmental factors.

Antimicrobials are widely used in human medicine, agricultural production, and food processing and have been essential for ensuring human and animal health as well as the safety of our food supply. This chapter discusses the resistance to distinct agents such as antibiotics, food antimicrobials, and disinfectants. A section briefly discusses the types of antibiotics and mechanism of antimicrobial resistance (AR). Depending on the mode of action, antibiotics can be categorized into bactericidal and bacteriostatic agents. Various mechanisms have been adopted by bacteria to provide resistance to different types of antibiotics. AR due to genetic changes can be transmitted to progeny by vertical transmission and to other susceptible bacteria by horizontal gene transmission (HGT) mechanisms. In the past decade, a variety of antibiotic-resistant (ART) foodborne pathogens have been isolated and characterized, indicating that the food chain can serve as an avenue for transmitting ART bacteria to humans. The chapter focuses on several important foodborne pathogens as well as nonpathogenic Escherichia coli strains and their resistance to antibiotics. Overall, microbial resistance to food antimicrobials, sanitizers, and disinfectants is less well understood and more difficult to characterize and quantify than resistance to antibiotics, and standardized methods have not been developed, nor are surveillance systems in place. The chapter ends with a discussion on the critical issues, gaps, and future directions of AR.

This chapter describes the fundamental basis of sporulation and the problems that spores present to the food industry. Throughout sporulation, gene expression is ordered not only temporally but also spatially, as some genes are expressed only in the mother cell or the forespore. This chapter highlights the state of knowledge of molecular mechanisms of sporulation, spore dormancy, germination, and outgrowth. The sporulating bacteria discussed in this chapter form heat-resistant endospores that contain dipicolinic acid (DPA) and are refractile or phase bright under phase-contrast microscopy. Spores are metabolically dormant, catalyzing no metabolism of endogenous or exogenous compounds. The major cause of this dormancy is undoubtedly the low water content of the spore core, which precludes protein mobility and enzyme action. Three species of sporeformers, Clostridium botulinum, Clostridium perfringens, and Bacillus cereus, are well known to produce toxins that can cause illness in humans and animals, and many species of sporeformers cause spoilage of food. Certain other species of Bacillus such as Bacillus licheniformis, Bacillus subtilis, and Bacillus pumilus have also been reported to sporadically cause foodborne diseases through production of toxins, and rare strains of Clostridium butyricum and Clostridium baratii produce type E and F botulinal toxins, respectively. With the global increase in population and food consumption, technologies to prevent spoilage would help to alleviate food shortages and spoilage and contribute to food security.

The concepts and principles for the establishment of microbiological criteria were elaborated in the mid-1980s by the International Commission on Microbiological Specifications for Foods (ICMSF). Microbiological criteria have traditionally been developed around significant pathogens, relevant commensals, and hygiene indicators as reflected in the ICMSF cases. They are widely used today to discriminate between acceptable and unacceptable lots of food products. The evolution of the traditional metrics, including microbiological criteria, to include additional risk-based metrics has taken place over recent years. This includes a better understanding of the performance and limitations of microbiological criteria. This chapter provides a framework for the microbiological risk management (MRM) process to Codex Alimentarius Commission (CAC) as well as to CAC members and member organizations. It also provides guidance to the food industry and other stakeholders who design, validate, and implement control measures ensuring the manufacture of safe food that consistently achieves the targets defined in the MRM metrics. The food safety objectives (FSO) metric expresses the maximum frequency and/or concentration of a pathogen in a food item at the time of consumption that provides or contributes to the appropriate level of protection defined by a government. A section illustrates the limitations of sampling plans for finished products to ensure their safety. It emphasizes the need to place microbiological testing in the broader framework of the overall food safety management system.

This chapter examines the risk of intentional food contamination, primarily with microbiological agents. It provides an overview of current methods for evaluating risk and defining appropriate interventions. It lists a sampling of microbial pathogens and toxins of concern, along with some of the potential chemical agents of concern provided for comparison; several references that go into significant detail on the agents of concern and illness progression and food protection more broadly are available. The basic scales used in operational risk management (ORM) as they could be applied to the food system are noted in a table. Importantly, successful utilization of ORM does not require sensitive details on potential agents. As applied to the food and agriculture critical infrastructure, detection and diagnostic tools have the potential to prevent or contain intentional contamination events. Approaches to harden the food system against intentional contamination and enhance its inherent biosecurity are being adapted from ORM, CARVER, CARVER+Shock, and other systems from military and law enforcement experience and new concepts in intelligent adversary modeling, among others. While similar to the continual improvement approach dictated by food safety systems to prevent unintentional microbiological contamination of food products, food system defense represents an additional set of challenges.

This chapter provides an overall presentation of microbiological issues associated with all muscle foods, which is then followed by individual sections addressing, in sequence, specific spoilage and safety issues and their control for meat, poultry, and seafood. A spoiled food is not necessarily unsafe, if pathogens are absent; therefore, spoilage is considered an economic loss and can lead to loss of consumer confidence. It is important to continuously develop reliable methods for measuring freshness and quality, predicting the shelf life of their products, and for inspection purposes. The most important muscle food safety issues of current worldwide concern are the need to control traditional as well as new, emerging, or evolving pathogens, including those of increased virulence at low infectious doses or resistant to antibiotics or to food processing-associated stresses caused by physical factors (e.g., heat, cold, drying, and radiation) and chemical agents (e.g., acids, salts, and sanitizers). For microbial food safety, the practical issue is whether antibiotic-resistant pathogen strains are of similar or higher resistance to common food-processing treatments compared to sensitive counterparts, as higher resistance of antibiotic-resistant strains is of concern. Decontamination processes are applied to animals and carcasses through a variety of physical and chemical interventions. Additional interventions to help enhance food safety are applied during processing and include heating, chilling, freezing, drying, fermentation, use of chemicals as acidulants or antimicrobials, packaging, proper storage and distribution, and appropriate handling and preparation for consumption.

In this chapter, the discussion of spoilage of milk and dairy products is based on the types of microorganisms associated with various defects. These include gram-negative psychrotrophic microorganisms, gram-positive bacteria including lactic acid bacteria and spore-forming bacteria, yeasts, and molds. The chapter describes the interactions of these microorganisms with dairy foods that lead to commonly encountered product defects. Citrate in milk can be utilized by many microorganisms but is not present in sufficient amount to support significant growth. The major microbial inhibitors in raw milk are lactoferrin and the lactoperoxidase system. As much as milk and dairy products are important components of a healthy diet, the presence of pathogenic microorganisms poses a potential health hazard to the consumers. Proteases of psychrotrophic bacteria cause product defects either at the time they are produced in the product or as a result of the enzymes surviving a heat process. Spoilage of milk and dairy products resulting from growth of acid-producing fermentative bacteria occurs when storage temperatures are sufficiently high for these microorganisms to outgrow psychrotrophic bacteria or when product composition is inhibitory to gram-negative aerobic organisms. Spoilage by spore-forming bacteria can occur in low-acid fluid milk products that are preserved by substerilization heat treatments and packaged with little chance for recontamination with vegetative cells. Yeasts and molds that spoil dairy products can be isolated in the processing plant on packaging equipment, in the air, in salt brines, on manufacturing equipment, and in the general environment.

This chapter focuses on the origin, description, and control of bacterial and fungal spoilage of fruits and vegetables. It talks about some chemical treatments like fungicides and decontamination for fruits and vegetables. Synthetic antimicrobial chemicals are still widely applied to fruits and vegetables after harvest, and decontamination aims at reducing the number of microbial contaminants on the surface of fruits and vegetables, thereby prolonging the time required to develop spoilage. Spoilage of fruits and vegetables is the result of complex interactions between a living plant organ and its microflora, and therefore deals with plant pathology and plant physiology as much as with food microbiology. Control of postharvest spoilage microorganisms largely accounts for these interactions, which could be additionally affected by the global climate change. Today, millions of tons of fruits and vegetables cross seas, oceans, and continents, from the Southern to the Northern hemisphere and from tropical to temperate zones. Developing countries increasingly play a role in this world market. Further development of minimally processed fruits and vegetables will bring new questions as to how to maintain the quality of processed produce, which requires prevention of spoilage, when the produce is often heavily stressed and naturally occurring defenses of the intact tissues have been overwhelmed. Consumer demand for high-quality fruits and vegetables produced under environmentally friendly conditions will probably not decrease. Finding solutions to historical problems associated with the preservation of fruits and vegetables against infection and spoilage by bacteria and fungi will be an ongoing future challenge.

The culinary definition of nuts is very broad and includes botanically defined nuts (e.g.,acorn, chestnut, and filbert), seeds (e.g.,Brazil nut, cashew, pignoli or pine nut, and pumpkin, sesame, and sunflower seeds), legumes (e.g.,peanut), and drupes (e.g.,almond, coconut, macadamia nut, pecan, pistachio, and walnut). This chapter presents an overview of the behavior of microorganisms on nuts, cereals, and products produced from them, with particular emphasis on describing conditions that permit or inhibit growth and treatments that can be used for microbial control or elimination. Worldwide, the production, harvesting, and processing techniques for nuts range from highly mechanized to labor intensive, and methods vary significantly among various types of nuts. The initial microbiota of peanuts, which develop beneath the soil surface, originates from the soil. The aspergilli, especially Aspergillus flavus, are ubiquitous invaders of nuts and can produce the mycotoxin aflatoxin. Processing steps that involve a thermal treatment often play a dual role of altering the texture and appearance of nuts and reducing microbial contamination. Microbiological contamination of cereal grains occurs while the grains are growing in the field. These contaminants can increase in number while the grains are actively growing and after harvesting. Milled cereal grains are produced by a dry-milling process. The resulting flours can be further wet processed, or “hydroprocessed,” in order to separate the gluten and starch components.

In recent years, the rapid advance of DNA/RNA sequencing technology has made whole-genomic sequencing a very promising tool in the investigation of foodborne outbreaks, as it can be used to assess the population structure of highly clonal, outbreak-related pathogens at a single-base resolution and can help identify temporal, geographical, and evolutionary origins of outbreaks. A study using a whole-genome single nucleotide polymorphism-based assay has demonstrated its discriminatory power by being able to distinguish between outbreak-related and non-outbreak-related cases that were associated with a multistate Salmonella Montevideo outbreak originating from salami made with contaminated red and black peppers. The resistance and survival of foodborne salmonellae to inactivation processes and hostile environments are often the reasons underlying many food-associated Salmonella outbreaks. The resistance that salmonellae demonstrate to heat, chemical sanitizers or preservatives, low pH, and water activity (aw) ultimately plays an important role in causing human disease. Enteric fever is a serious human disease associated with Salmonella Typhi and Salmonella Paratyphi, which are mainly transmitted from human to human via the fecal-oral route and are particularly well adapted for invasion and survival within host tissues. Antibiotic resistance in Salmonella spp. has been reported since the early 1960s, when most of the reported resistance was to a single antibiotic. Salmonella spp. continue to be a leading cause of foodborne illness. The situation has persisted because of the widespread occurrence of salmonellae in the natural environment and their prevalence in many sectors of the global food chain.

This chapter highlights the important bacteriological and epidemiologic features related to thermotolerant Campylobacter species (C. jejuni and C. coli, and to a lesser extent C. lari) contamination in the human food chain. The genus Campylobacter belongs to the family Campylobacteraceae together with the genera Arcobacter and Sulfurospirillum. A recent study revealed that culturability and adhesion/invasion of C. jejuni are linearly related. The identification of several putative adhesion factors in C. jejuni was based mainly on experiments in culture cell lines. The application of discriminatory molecular subtyping methods (e.g., multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE)) was useful in clarifying some aspects of host association and source attribution. Evidence from epidemiologic studies and molecular subtyping investigations has identified poultry meat as a major vehicle for foodborne transmission of campylobacter enteritis. Future research and scientific collaborations among the medical, food, and veterinary professions are needed to substantially reduce Campylobacter contamination in the poultry meat chain. Research directions should focus on practical control options that would be appealing to stakeholders in the farm, slaughterhouse, and processing sectors. In addition, there are opportunities for the development of enhanced Campylobacter detection and quantification methods. Methods able to identify highly contaminated samples through online detection would be very useful, as this could help in identifying and excluding highly contaminated samples from the human food chain.

Gastrointestinal pathogenic Escherichia coli includes enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and enterohemorrhagic E. coli (EHEC). This chapter focuses largely on the EHEC group, which among the E. coli strains that cause foodborne illness, is the most significant group based on the frequency of foodborne illness in the United States and the severity of illness. Some types of EPEC are referred to as enteroadherent E. coli (EAEC), based on specific patterns of adherence. The chapter next discusses the characteristics of E. coli O157:H7, first identified as a foodborne pathogen in 1982. The serious nature of the symptoms of hemorrhagic colitis and hemolytic-uremic syndrome (HUS) caused by E. coli O157:H7 places this pathogen in a category apart from other foodborne pathogens, which typically cause only mild symptoms. 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. Cattle are a major reservoir of E. coli O157:H7. E. coli O157:H7 is still by far the most important serotype of Shiga toxins (Stx)-producing E. coli (STEC) in North America. The increased availability in clinical laboratories of techniques such as testing for Stxs or their genes and identification of other virulence markers unique for EHEC will continue to enhance the detection of disease attributable to non-O157 EHEC.

This chapter reviews the basic biology, ecology, phylogenetic characterization, virulence factors, pathogenicity, and epidemiology of the emerging opportunistic foodborne pathogen, Cronobacter Species. The Cronobacter Species is categorized in the family Enterobacteriaceae and like most species in this family is considered an opportunistic pathogen. Nevertheless, advances have been made in the molecular characterization of Cronobacter species using amplification and sequencing of the 16S rRNA gene, pulsed-field gel electrophoresis (PFGE), ribotyping, and plasmid typing. The chapter explains susceptibility to physical and chemical treatments that include temperature, water activity, biological inactivation, chemical inactivation and competitive exclusion/probiotics. Cronobacter has been recovered from clinical specimens such as cerebrospinal fluid (CSF), blood, sputum, throat, nose, stool, gut, skin, wounds, bone marrow, eye, ear, stomach aspirates, anal swabs, and the breast abscess of infected patients. Cronobacter has been mainly associated with necrotizing enterocolitis (NEC), septicemia, and meningitis. Neurological sequelae are commonly reported and include brain abscess and infarction, ventricle compartmentalization due to necrosis of brain tissue and liquefaction of white cerebral matter, and cranial cystic changes, as well as hemorrhagic and nonhemorrhagic intercerebral infarctions leading to cystic encephalomalacia. Nazarowec-White and Farber tested the antibiotic resistance of seventeen strains of Cronobacter and found four antibiotic susceptibility patterns (antibiograms). Cronobacter species has become a growing concern for government regulatory agencies, health care providers (especially those in neonatal intensive care units), and powdered infant formula (PIF) manufacturers.

Yersinia pestis is transmitted to its host via flea bites or respiratory aerosols, whereas Y. pseudotuberculosis and Y. enterocolitica are foodborne pathogens. These three species share a number of essential virulence determinants that enable them to overcome the innate defenses of their hosts. Given that Y. pestis is incapable of infecting the intestinal tract directly and not pathogenic when ingested and that the role of most other Yersinia species in disease is uncertain, this chapter focuses on Y. enterocolitica and Y. pseudotuberculosis. Y. pseudotuberculosis is a relatively homogenous species, which is subdivided into serotypes according to its lipopolysaccharide (LPS) O antigens. Y. enterocolitica is far more heterogenous than Y. pseudotuberculosis, being divisible into a large number of subgroups according to biochemical activity and LPS O antigens. Infection with the enteropathogenic yersiniae typically manifests as nonspecific, self-limiting diarrhea but may produce a variety of suppurative and autoimmune complications, the risk of which is determined partly by host factors, in particular age and underlying immune status. Indeed, Y. enterocolitica is one of the most important causes of fatal bacteremia following transfusion with packed red blood cells or platelets. Explanations for the link between yersiniosis and autoimmunity include antigen persistence, molecular mimicry, impaired immune responsiveness, and infection-induced presentation of normally cryptic cellular antigens.

Foodborne infections with Shigella species are an important source of illness in both economically developed and developing countries. This chapter presents to food microbiologists important features of Shigella spp., the disease they cause, and the impact that these pathogens have with respect to food safety. It discusses the diagnosis, epidemiology, ecology, modes of transmission, and examples of recent foodborne outbreaks, along with the current understanding of the genetics of Shigella pathogenesis, the genes involved in causing disease, and how they are regulated. Shigella can be transmitted by consumption of raw or processed food. Generally, poor personal hygiene practices by food workers at the point of final preparation and food service are the major factors for food contamination. The single most effective means of preventing secondary transmission of Shigella is hand washing with soap and chlorination of water. While foodborne infections due to members of the Shigella spp. may not be as frequently reported as those caused by other foodborne pathogens, they have the potential for explosive spread due to the low infectious dose that can cause overt clinical disease.

The presence and distribution of Vibrio species in coastal waters are dependent upon a variety of environmental factors such as temperature, salt concentration, pH, and nutrients. Vibrios tend to be more common in warmer waters, especially when temperatures rise above 17°C, and depending on the species, they tolerate a range of salinities. Vibrios, which are generally the predominant bacterial genus in estuarine waters, are associated with a great variety of seafood. This chapter discusses isolation and identification of Vibrio species before going on to explain Vibrio enumeration in seafood and Vibrio susceptibility to physical/chemical treatments such as irradiation and high pressure. Various Vibrio species – V. cholerae, V. parahaemolyticus, V. vulnificus, V. fluvialis, V. hollisae, and V. alginolyticus are individually discussed in this chapter.

Botulism is a neuroparalytic disease in humans and animals resulting from the actions of botulinum neurotoxins produced by Clostridium botulinum and rare strains of C. butyricum and C. baratii. Botulinogenic clostridia are widely dispersed in nature by virtue of their ability to form resistant endospores. Since botulism is a true toxemia and botulinum neurotoxin is solely responsible for the illness, foodborne, infant, and wound botulism are clinically similar. The major treatment of botulism is supportive nursing care, with specific attention given to respiratory ability and the need for mechanical ventilation. The current good safety record for commercial foods is due, in large part, to the diligence of food manufacturers in formulating, processing, and controlling temperature during distribution of foods. Foodborne botulism is the class of botulism that can most readily be prevented through proper food processing, preservation, and temperature control. Chlorine and related compounds are among the most effective chemicals for destruction of spores. In general, botulinal neurotoxins are not affected by freezing, particularly in the presence of proteins and organic acids at pH values of 5 to 6.5. Brining is the most common practice for reducing water activity (aw) in food preservation. Temperature is commonly used to prevent C. botulinum growth in foods. Temperature abuse is one of the most common mishandling practices that result in botulinum neurotoxin production and botulism outbreaks. Botulinum neurotoxin is absorbed through mucous membranes, and three cases of botulism were documented in laboratory workers who apparently inhaled the toxin.

This chapter focuses on Clostridium perfringens type A food poisoning. C. perfringens is a gram-positive, rod-shaped, encapsulated, nonmotile anaerobe that causes a spectrum of human and veterinary diseases. The virulence of this bacterium largely results from its prolific toxin-producing ability, including several toxins (e.g., C. perfringens enterotoxin [CPE] and β-toxin) with activity on the human gastrointestinal (GI) tract. C. perfringens growth in food is affected by a variety of environmental factors, including temperature, Eh, pH, and water activity (aw). Current knowledge of the reservoir(s) for C. perfringens type A food poisoning isolates remains deficient, which is unfortunate because it impairs efforts to rationally control/reduce outbreaks of C. perfringens type A food poisoning. Epidemiologic studies provided strong initial evidence that CPE plays a pivotal role in C. perfringens type A foodborne illness. Everyone is susceptible to C. perfringens type A food poisoning; however, this illness tends to be more serious in elderly, debilitated, or medicated individuals. Additional studies may also lead to the development of agents capable of blocking CPE expression or activity. Finally, continued research on the mechanism of CPE activity may lead to the development of potent new anticancer agents or to better delivery of therapeutic agents.

The Bacillus cereus group presently consists of seven Bacillus species, i.e., B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis, and the most recently recognized member of the group, B. cytotoxicus, which is thermotolerant. 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. The most recently discovered B. cereus enterotoxin, cytotoxin K (CytK), is similar to the β-toxin of Clostridium perfringens (and other related toxins) and was the causative agent in a severe outbreak of B. cereus foodborne illness in France in 1998. The two types of B. cereus foodborne illness are caused by very different types of toxins including emetic toxin and enterotoxins. Expression of the B. cereus toxins Hb1, Nhe, and CytK is regulated by the PlcR quorum-sensing system. The spore of B. cereus is an important factor in contributing to foodborne illness. The B. cereus spore is more hydrophobic than spores from any other Bacillus spp., which enables it to adhere to several types of surfaces. B. cereus foodborne illness is likely to be highly underreported because of its relatively mild symptoms with short duration. However, increased consumer interest for precooked, chilled food products with extended shelf lives may be well suited for B. cereus survival and growth. Such foods could increase the prominence of B. cereus as a foodborne pathogen.

The significance of Listeria monocytogenes as a foodborne pathogen is complex. The severity and case-fatality rate of the disease listeriosis require appropriate preventive measures, but the characteristics of the microorganism are such that it is unrealistic to expect all food to be Listeria-free. This dilemma has generated an ongoing debate concerning both the various strategies for prevention of listeriosis and the regulation of L. monocytogenes in foods. Epidemiologic investigations of outbreaks have helped identify the vehicles of transmission and have led to an expanding list of ready-to-eat (RTE) foods that have been associated with outbreaks. Basic research on the genetics, molecular biology, and immunologic response of animals and humans to L. monocytogenes has provided detailed insights into the virulence characteristics of this fascinating pathogen. Concurrent infection can also influence susceptibility to listeriosis. FbpA behaves as a chaperone for two important virulence factors, listeriolysin O (LLO) and internalin B (InlB), probably preventing their degradation. Public health surveillance, outbreak investigations, and applied and basic research conducted during the past 30 years have helped to characterize the disease listeriosis, define the magnitude of its public health problem and its impact on the food industry, identify the risk factors associated with disease, and develop appropriate and targeted control strategies.

The etiological agents of Staphylococcal food poisoning (SFP) are members of the genus Staphylococcus, predominantly Staphylococcus aureus. This form of food poisoning is considered intoxication; it does not involve infection by, and growth of, the bacteria in the host. This chapter primarily addresses SFP; however, in regard to the staphylococcal enterotoxins (SEs), there is significant overlap in the natural histories of both diseases. Humans are the main reservoir for staphylococci involved in human disease, including S. aureus. S. aureus is known for acquiring genetic resistance to heavy metals and antimicrobial agents used in clinical medicine. SFP occurs as either isolated cases or outbreaks affecting a large number of people. Biochemical and structural studies of SEs have revealed that some SEs are dependent on zinc ions to be functional and to be able to properly bind major histocompatibility complex class II (MHCII). SEs and other superantigens (SAgs) interact with a characteristic repertoire of T-cell receptors (TCR) sequences. The TCR specificity of each SE is determined by toxin residues in the shallow cavity at the top of the molecule. The structural aspects of SEs that enable them to survive degradation by pepsin and other enzymes in the gastrointestinal tract are required for the toxins to induce SFP. Progress has been made toward understanding the molecular aspects relevant to SFP. S. aureus has some unique properties that promote its ability to produce foodborne illness.

This chapter provides an introduction to epidemiology and epidemiologic methods as they are applied to problems of foodborne diseases. An understanding of epidemiology is important, because despite all of our best efforts to prevent foodborne diseases, humans remain the ultimate bioassay for low-level or sporadic contamination of our food supply. Epidemiologic methods of foodborne disease surveillance are needed to detect outbreaks, identify their causes, and assess the effectiveness of control measures. Epidemiologic data are also important in establishing food safety priorities, allocating food safety resources, stimulating public interest in food safety issues, establishing risk reduction strategies and public education campaigns, and evaluating the effectiveness of food safety programs. 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. Models such as PulseNet provide opportunities to conduct multinational surveillance for at least the major bacterial foodborne disease agents. As foodborne disease problems imported into one country may represent disease problems endemic to the food-producing country, growing awareness of these problems could stimulate investment in interventions that improve the health of both countries.

This chapter talks about specific mycotoxins that are produced only by specific fungi, usually by only a few species. A particular species of fungus may produce more than one mycotoxin, though never more than one of the major compounds described here. A section deals with the most important mycotoxins that include aflatoxins, ochratoxin A, fumonisins, deoxynivalenol, and zearalenone. The chapter discusses the chemical characterization, fungal sources, genetics, ecology, toxicity, chemical analysis, occurrence and regulations, and control of the mycotoxins. In addition, it deals with the analysis of aflatoxins, occurrence and regulation of aflatoxins in foods, control of aflatoxins in foods, and risk characterization. The most important general observation to be made about Fusarium mycotoxins is that all Fusarium species grow only at high (>0.9) water activities, so that toxin production in crops occurs only before harvest or during early stages of drying. The major source of fumonisins in foods is maize, though other small grains may have low levels at times. With the discovery that Aspergillus niger may produce fumonisins, the range of foodstuffs where fumonisins may be found has become much wider. Genomics, the study of entire genomes, provides basic information to build the knowledge base of gene function that will assist in understanding mycotoxin formation and reduction in crops. Studies on economically important fungi at the genomic level will assist in understanding mycotoxin biosynthesis and also help to understand the biology, evolution, biochemical function, and genetic regulation of the genes in these fungal systems.

Human enteric viruses have properties that are unique from those of bacterial foodborne pathogens. Viruses are usually species-specific and tissue-tropic, meaning that the human enteric viruses are believed to infect only humans. From an epidemiologic perspective, human norovirus (NoV) and hepatitis A virus (HAV) are the two most important. From a foodborne disease standpoint, three types of commodities are commonly associated with viral disease outbreaks, namely, (i) molluscan shellfish contaminated by feces-impacted growing waters; (ii) fresh produce items contaminated by human feces during production or packing, usually through workers’ hands or contact with contaminated water; and (iii) ready-to-eat (RTE) and prepared foods contaminated by infected food handlers as a result of poor personal hygiene. The major steps for the detection of viruses in foods can be designated as follows: (i) virus concentration and purification, (ii) nucleic acid extraction, (iii) detection of amplicons, and (iv) confirmation of amplicon identity. Epidemiologic evidence reveals that foods play an important role in the transmission of human enteric viruses. Improved and more widespread reporting and investigation of foodborne viral disease outbreaks, as well as targeted epidemiologic studies to identify the risk factors for acquiring viral gastroenteritis, would improve our understanding of source attribution.

Bovine spongiform encephalopathy (BSE), widely known as "mad cow disease" is a subacute degenerative disease affecting the central nervous system (CNS) of cattle. This chapter talks about characteristics of the BSE agent, including typical and atypical BSE, stability of the infectious agent, and bodily distribution of infectivity. Much research has gone into defining the pathogenesis of BSE, the most important of which is studies in which cattle were infected orally and their tissues were examined at various times thereafter, up to and including the fully developed stage of illness. It is necessary to use caution in the interpretation of bioassay results from all nonbovine species, particularly if the results are based on ultrasensitive prion amplification techniques or bioassays in transgenic mice overexpressing the PrP gene. The chapter explains the characteristics of BSE in cattle (affected animals develop a progressive degeneration of the nervous system) and of variant form of Creutzfeldt-Jakob disease (vCJD) in humans (the young age at onset of illness with many adolescents afflicted). It is important that regulatory policies – protection of animal health and human health – be modified in accord with advances in experimental and epidemiologic knowledge to minimize adverse consequences to both animal and human health. In particular, the development of preclinical diagnostic tests may vastly improve the precision of proactive measures to minimize risks to animal and human health.

There are four meat-borne helminths of medical significance: Trichinella species, Taenia solium, and Taenia asiatica, which occur primarily in pork; and Taenia saginata, which is found in beef. There are a variety of reasons for this, including animal management systems that perpetuate infection, inadequate or poorly enforced inspection requirements for slaughtered animals, new sources of infection, and demographic changes in human populations that introduce new culinary practices of preparing meats. Lower infectivity of other Trichinella species for the domestic pig diminishes their importance in the domestic cycle. As cooking, freezing, and other processing methods kill Trichinella larvae in meat, most human infections have resulted from instances where meat preparation was not adequate. Pork products such as fresh sausage, summer sausage, and dried or smoked sausage have all been implicated as sources of human trichinellosis. Diagnosis is based on a history of eating infected meat, symptoms, laboratory findings, and recovery of larvae from muscles. After the mid-1980s, pigs are putatively identified as the intermediate host for a unique taeniid (similar to T. saginata and T. solium) that had a predilection for liver tissue in a multitude of experimental hosts. For this reason, both T. asiatica and T. saginata asiatica have appeared and continue to appear in the literature as the scientific names identifying this organism. Although most cases of taeniasis are asymptomatic, up to one-third of patients complain of nausea or abdominal “hunger” pain that is often relieved by eating.

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. This chapter discusses helminths acquired from finfish and shellfish. The majority of human infections acquired in the United States have been associated with dishes prepared at home, which suggests that the skill and care of the food handler and the selection of fish prepared for consumption may be important risk factors. It then talks about helminths acquired from vegetables. Fresh vegetables grown in areas where night soil (human waste) is used as fertilizer are frequently contaminated and, thus, may facilitate transmission of any of a number of geohelminths. The chapter also discusses helminths acquired from invertebrates in drinking water, and helminths acquired from other invertebrates including snails, ants, fleas and beetles. Diagnosis of infection is presumptive and usually not confirmed until the worm is removed and identified through histological sectioning. Inadequate washing of produce or poor hygiene among food handlers can result in a variety of helminthic infections. A variety of geohelminth species utilize the fecaloral route for person-to-person and animal-to-person transmission.

Protozoan parasites have long been associated with foodborne and waterborne outbreaks of disease in humans. Difficulties arise with the inactivation of these organisms because of their resistance to environmental stresses. 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, Sarcocystis hominis and S. suihominis, and Isospora belli frequently inhabit the intestinal mucosa and produce diarrheal illnesses in humans. The life cycle stages of apicomplexan parasites are produced intracellularly in the host. For Cyclospora, Toxoplasma, and Isospora, sporogony typically occurs outside the host, requiring the passage of time before oocysts are infective to a new host, while Cryptosporidium oocysts excreted already are sporulated and are infectious when shed. Cryptosporidiosis is acquired after ingesting food or water contaminated with infective Cryptosporidium oocysts. Cyclosporiasis is characterized by mild to severe nausea, anorexia, abdominal cramping, mild fever, and watery diarrhea. The only successful antimicrobial treatment for Cyclospora is trimethoprim-sulfamethoxazole (TMP-SMX). Toxoplasmosis can be acquired by ingestion of lamb, poultry, horse, and wild game animals. Patients with muscular sarcocystosis present with musculoskeletal pain, fever, rash, cardiomyopathy, bronchospasm, and subcutaneous swelling. Inactivation of the protozoan parasites has been a challenging task. Molecular tools such as PCR, restriction fragment length polymorphism, and variations of these techniques are being developed to improve the sensitivity and specificity of detection and identification processes.

Food preservation methods were originally developed to extend the shelf life of food by protecting the product from microbiological, chemical, and physical changes that could lead to spoilage. The microbiological changes are prevented by eliminating spoilage microorganisms or simply suppressing their metabolic activity. Modern preservation methods are designed not only to extend the shelf life of food, but also to ensure its safety by inactivating pathogenic microorganisms and viruses of concern, or in some cases just preventing their growth in the product. The most commonly used preservation methods are physical in nature. Treatment of food with heat (i.e., thermal processing) inactivates spoilage-initiating microorganisms and enzymes, as well as disease-causing microorganisms. Removal of heat to refrigerate or freeze food suppresses microbial metabolism and multiplication, and the process also may inactivate a fraction of the food microbiota. Decreasing water availability is effectively used in preserving many foods through concentration or drying or by addition of water activity (aw) modifiers. Most of the alternative technologies to thermal processing are considered physical preservation methods. These include gamma radiation, which is gradually gaining acceptance as an effective preservation method. Use of ultrahigh pressure to preserve prepackaged value-added food is increasing. Emerging preservation approaches also include using pulsed electric fields, UV light, and ultrasound, with the aim of ensuring food safety while minimizing adverse impacts of processing on product quality. Many of these physical treatments are addressed in this chapter, with emphasis on engineering background, microbiological considerations, and applications in food processing.

This chapter talks about antimicrobial compounds that are divided into two classes: traditional and naturally occurring. 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, or (iii) are produced by synthetic processes. Next, the chapter discusses the factors affecting activity and traditional antimicrobials and ester derivatives of some weak organic acids. Many fatty acid esters exhibit antimicrobial activity in foods, with glycerol monolaurate being one of the most effective. Traditional antimicrobials and natural food antimicrobials are important tools for preserving food from microbiological spoilage and the growth of pathogens. Despite the extensive research already completed on the sources of antimicrobials, their spectra of activity, and the levels required for successful inhibition of foodborne bacteria and fungi, more research is still needed to better elucidate the mechanisms of antimicrobial activity of many of the chemicals discussed in the chapter. In addition to validating the activity and elucidating the mechanistic features of antimicrobials, they will have to be proven toxicologically safe. Demonstrating the efficacy of antimicrobial compounds in food products at concentrations that do not have adverse sensory effects, as well as controlling the cost of these interventions, are likely the greatest hurdles to their future application.

This chapter provides an overview of the biologically based preservation technologies termed "biopreservation". The first part of the chapter covers acid production by lactic acid bacteria (LAB) in temperature-abused foods (controlled acidification). 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. Then the chapter discusses some LAB produce antimicrobial proteins, called bacteriocins, that inhibit spoilage and pathogenic bacteria without changing the physicochemical nature of the food. The largest section of this chapter deals with bacteriocins. Bacteriocins are ribosomally synthesized antimicrobial peptides of bacterial origin that are not lethal to the host. Many bacteriocins inhibit foodborne pathogens of serious concern such as Listeria monocytogenes, which is recalcitrant to traditional preservation methods. The chapter presents general characteristics, methodological considerations, bacteriocin applications in foods, genetics of LAB bacteriocins, and resistance of bacteriocins of each of these conditions in detail. The use of bacteriophages to control pathogens in food has “shown promise” for decades. But, perhaps due to the difficulty of obtaining reproducible results in foods, they have not gained widespread use. The chapter closes by examining the use of bacteriophages as biocontrol agents.

The fermented dairy products category contains products with a diversity of flavors, textures, and appearances, all of which are directly dependent on microbial metabolism. The enzymes and metabolites required to produce these products are provided by a diverse set of microorganisms, including molds, yeasts, and bacteria. Of these organisms, homofermentative lactic acid bacteria (LAB) are of the greatest importance, as the manufacture of fermented dairy products is directly dependent on their primary metabolic end product, lactic acid. This chapter describes the potential for production of diacetyl and carbon dioxide from lactose metabolism in lactococci with reduced lactic acid dehydrogenase activity. The main volatile flavor components of fermented milks are acetic acid, acetaldehyde, and diacetyl. Proteolytic systems in LAB contribute to their ability to grow in milk and are necessary for the development of flavor in ripened cheeses. Peptides and amino acids formed by proteolysis may impart flavor directly or serve as flavor precursors in fermented dairy products. The dairy industry has employed improved sanitation regimens, utilized sophisticated starter culture propagation vessels, developed starter culture systems to minimize the impact of phage infection, and isolated and constructed starter strains with enhanced bacteriophage resistance. The power of recombinant DNA approaches is that strains can be constructed that differ in a single defined genetic alteration, e.g., inactivation of a specific gene.

The wide variety of fermented foods can be classified by the products of the fermentation, such as alcohol (beer, wine); organic acids, including lactic acid and acetic acid (vegetables, dairy); carbon dioxide (bread); and amino acids or peptides from protein (fish fermentations and others). Food fermentation is one of the earliest technologies developed by humans. The primary retail fermented vegetable products produced in the United States and Europe are cucumber pickles, olives, and sauerkraut. In Asia, a variety of fermented vegetable products are available, including pickles and fermented cabbage, notably kimchi in South Korea. The fermentation process for vegetables can result in nutritious foods that may be stored for extended periods, one year or more, without refrigeration. Prior to fermentation, fresh fruits and vegetables harbor a variety of microorganisms, including aerobic spoilage microflora such as Pseudomonas, Erwinia, and Enterobacter species, as well as yeasts and molds. Brining vegetables for fermentation results in the production by lactic acid bacteria (LAB) of organic acids and a variety of antimicrobial compounds. With the advent of whole-genome sequencing, it has become apparent that the LAB present in vegetable fermentations have relatively small genomes compared with many other mesophilic organisms. LAB isolated from vegetable fermentations frequently contain plasmids. Plasmid-borne genes encoding proteins involved in bacteriocin production, lactose utilization, and citric acid utilization have been isolated from several Leuconostoc species; however, none of these functions appears to be present in the ATCC 8293 plasmid.

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. This chapter talks about factors affecting color, texture, flavor, and appearance of fermented meats. Incorporation of sodium chloride, sodium or potassium nitrite and/or nitrate, glucose, and homofermentative lactic acid starter cultures in sausage formulas dramatically alters the ecology of the culture environment and chemical characteristics of finished products. The chapter also talks about chemical characteristics of fermented dry and semidry sausage products including cervelat, capicola and salami. Fish fermentation involves minimal bacterial conversion of carbohydrates to lactic acid but entails extensive tissue degradation by proteolytic and lipolytic enzymes derived from viscera and muscle tissues. Sauces have a predominantly salty taste and are derived from decanting or pressing fermented fish or shrimp after a 9-month to 1-year fermentation. The use of starter cultures in fermented meat products is a relatively recent practice compared with their use in fermented dairy foods and alcoholic beverages. More recent trends have been focused on starter cultures not only as fermentation tools but also for functional food purposes, to capitalize on their flavor-enhancing, bioprotective, and health-beneficial properties. The advantage of microbial activity is the reduction of nitrates, thus removing excess nitrate/ nitrite from the meat.

This chapter focuses on the more comprehensive role of fermentation in cocoa curing and to a lesser extent on the role of fermentation in the production of coffee. The two principal objectives of fermentation are to remove mucilage, thus provoking aeration during fermentation of the beans and facilitating drying later on; and to provide heat and acetic acid necessary for inhibiting germination, which ensures proper curing of the beans. Approximately one-half of the world crop is fermented in some type of box, and the remaining half is fermented by using heaps or other primitive methods. The progress of the fermentation is assessed by the odor and the external and internal color changes in the beans. Fermentation begins immediately after beans are removed from the pods, as they become inoculated with a variety of microorganisms from the pod surface, knives, laborers’ hands, containers that are used to transport the beans to the fermentary, dried mucilage on surfaces of the fermentation box (tray, platform, or basket) from the previous fermentation, insects, and banana or plantain leaves. The actual production of chocolate flavor precursors occurs within the cocoa bean and is primarily the result of biochemical changes that take place during fermentation and drying. There are several environmental factors: pH, temperature, and moisture, in the fermenting mass that influence cocoa bean enzyme reactions. 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.

This chapter overviews the scientific principles of the brewing industry. The malting process occurs in three stages: steeping, germination, and kilning. Wort is the sugary solution prepared from malt, either alone or with sugar (e.g., glucose, sucrose, or maltose crystals or syrups) or unmalted cereal adjunct if appropriate, after the grist is extracted with warm water. The traditional decoction mashing process for Bavarian and Czech beers has origins predating the invention of the thermometer. Certainly 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. The chapter deals with postfermentation treatments such as conditioning, filtration, pasteurization and packaging. The basic principle of high-gravity brewing is that it is theoretically possible to double the production of a brewery, without the expense of additional brewhouse or fermentation capacity, by fermenting double-strength wort. Recently, however, there has been renewed interest in continuous fermentation, now using immobilized-cell technology.

Winemaking is a bioprocess that has its origins in antiquity. Scientific understanding of the process commenced with the studies of Louis Pasteur, who demonstrated that wines were the product of alcoholic fermentation of grape juice by yeasts. Microorganisms are fundamental to the winemaking process. To understand their contribution, it is necessary to know (i) the taxonomic identities of the species and strains associated with the process; (ii) the kinetics of their growth and survival throughout the entire production chain; (iii) the biochemical, physiological, and genomic responses of these species and their effects on the physical and chemical properties of the wine; (iv) the influence of winemaking practices upon the microbial response; and (v) the linkage between microbial action, sensory quality, and consumer acceptability of the wine. 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 chapter describes the details of the process of winemaking, and emphasizes grape wines, although it is recognized that wines from other fruits are regionally popular. The microorganisms involved in the winemaking are yeasts, lactic acid bacteria (LAB), acetic acid bacteria (AAB), molds and other bacteria.

Probiotic bacteria have long been believed to influence general health and well-being through their association with the gastrointestinal tract (GIT) and its normal microbiota. The microbiotas of humans, animals, and fowl vary considerably with the architecture of their GITs. Species of microorganisms are located at different locations throughout the GIT and include strains that are either harmful or beneficial to the host depending on the circumstances and specific strains involved. Probiotic microorganisms typically designed for delivery in dairy foods are most often members of the Lactobacillus or Bifidobacterium genus. This chapter discusses the effects of probiotics on GIT ecology, and deals with the appropriateness, technological suitability, competitiveness, and performance and functionality, as the criteria for selection of probiotic cultures. Prebiotics stimulate the growth and activity of beneficial bacteria in an individual’s intestinal microbiota. The best-known prebiotics are fructo-oligosaccharides derived from food sources. Production of designer prebiotics can offer multiple activities in retarding undesirable microorganisms, better promoting the native desirable microbiota, or stimulating the growth or activity of synbiotic cultures. Expansion of avenues for incorporation into appropriate food vehicles and improved stimulation of beneficial microfloras are some of the aspects that are good targets for development of prebiotics.

Foods are teeming with microorganisms that may be innocuous, pathogenic threats, spoilage agents, or beneficial microorganisms driving fermentations or acting as biocontrol agents. This chapter outlines the basic concepts underlying genomics, proteomics, and associated technologies. With the number of completely sequenced bacterial genomes increasing rapidly, one powerful approach to defining unique or conserved gene content and understanding how Lactobacillus plantarum microorganisms evolved is comparative genomics, via an in silico analysis. The discipline of functional genomics deals with defining the roles of genes in their appropriate organisms. This chapter utilizes comparative and functional genomics and proteomics to demonstrate the role of SpaC in mucin binding and potentially its importance to the retention of some lactobacilli in the gastrointestinal tract. The principles behind DNA microarray technology make it very applicable to many different uses that include comparative genomics and global gene expression analysis. While the details of the Gad system were elucidated primarily by experiments in Escherichia coli, genomics and bioinformatics have enabled researchers to identify and study the effects of these genes in other organisms. The contribution of genome sequencing and functional genomics has greatly facilitated our understanding of the pathogenicity of Listeria monocytogenes. Differences between the pathogenic and nonpathogenic Listeria species appear most strongly in the secretory proteome.

Predictive microbiology focuses on the quantitative description and prediction of the behavior (growth, survival, and inactivation) of pathogenic and spoilage microorganisms in food products. A first section of this chapter focuses on modeling trends up to now. The classical primary and secondary model approach, used to describe growth and inactivation, as well as probabilistic models used to describe the growth/no growth (G/NG) boundary, are discussed. In the following section, contemporary and future modeling trends are listed and the extension of existing models is discussed, including (i) the trend for the incorporation of multiple environmental factors and (ii) the incorporation of the specific aspect of food structure. To move from the macroscopic to the meso- and microscopic levels, the concepts of metabolic networks and individual-based models (IbM) have been introduced. The chapter provides a short overview of mesoscopic models, i.e., models that describe the dynamics of the population as a combination of different compartments. The last section deals with the transfer of predictive microbiology as a tool for food safety and food quality from academia to industry. Specifically, a series of software tools is listed. In this context, lactic acid bacteria are increasingly being investigated, not only because of their ability to inhibit outgrowth of pathogens and spoilage microorganisms in fermented foods but also for their potential to act as protective cultures in minimally processed foods.

This chapter introduces the basic concepts of microbial risk assessment and provides an overview of the methodology and applications to food safety. Risk analysis is used to develop an estimate of risks, to identify and implement measures to control the risks, and to communicate with stakeholders about the risks and measures applied. A distinct consideration when developing a microbial risk assessment model is the need to account for the changes in the concentration and prevalence of the hazard, as microbes grow and/or numbers decline throughout the food supply chain. Numerous papers and guidelines have described the basic approaches and methods for conducting microbial risk assessments. According to the Codex Alimentarius, the four steps of a risk assessment are hazard identification, exposure assessment, hazard characterization, and risk characterization. Risk assessments can be either qualitative or quantitative. Risk assessment also can focus on a specific segment(s) of the food chain or encompass the entire food continuum. The chapter focuses on stochastic sensitivity analysis; however, less detailed methods to conduct deterministic and worst-case sensitivity analysis are also described. The link between a deterministic risk assessment and food safety objective (FSO) is pretty straightforward but might not be entirely realistic because deterministic models do not account for uncertainty and variability, which are inevitable when conducting a microbial risk assessment.

The complexity of food production, processing, and preparation requires a systematic approach that simultaneously provides a common framework for managing microbial food safety risks and the flexibility and practicality needed to deal with the diversity of hazards, ingredients, and technologies. This is achieved almost globally with the application of two systems: good hygienic practices (GHPs) and hazard analysis critical control point (HACCP). The HACCP concepts that emerged and were ultimately expanded to include all types of foods and hazards focused on a more qualitative approach to hazard identification, identifying critical control points (CCPs), and establishing critical limits (CLs). Adherence to seven principles is recognized as being needed to achieve consistent control. A number of preliminary steps are necessary in order to acquire the key resources and information needed to initiate of the development of a HACCP program. The National Advisory Committee on Microbiological Criteria for Foods (NACMCF) advises that the process of conducting a hazard analysis involves two stages: hazard identification and hazard evaluation. Since the inception of HACCP there has been a dramatic shift from hazard based food safety systems to risk-based systems. The focus of HACCP has been predominately associated with the food manufacturing phase of the food chain.

Molecular subtyping can be used to study the population structure of a particular bacterial species, to determine the possible evolution of the subject microorganism, or to study the molecular epidemiology of a microbe. The types of methods used for subtyping and the approaches to data analysis and interpretation may vary greatly with the reason for specific subtyping. This chapter focuses almost entirely on subtyping for molecular epidemiology. Molecular epidemiology can be applied to identifying the source of a particular outbreak or to a broader understanding of the role of certain foods or processes in outbreak-related or sporadic infections. Perhaps the most easily appreciated reason for molecular subtyping is to facilitate the identification and investigation of foodborne disease outbreaks. Although the focus of this 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. Most of these phenotypic methods have long and successful histories of use in subtyping for the same purposes for which molecular methods are now used. Although molecular methods typically provide greater strain discrimination than phenotypic methods, this is not always the case, and it is only one reason why molecular methods are generally preferred. In recent years, the main focus of subtyping method development has been on DNA sequence-based methods. Sequence-based approaches to subtyping of bacteria, such as multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA), are already being widely implemented in the surveillance of foodborne infections.