Food Microbiology: Fundamentals and Frontiers, 5th Edition

Understanding the behavior of microorganisms in food is essential for promoting their growth when needed, inhibiting growth-associated spoilage and toxin production, and eliminating infectious pathogens through processing. Therefore, three behavioral modes are addressed: growth, survival, and death. The first part of the chapter describes growth in relation to its phases, quantification, kinetics, and applications in food. Additionally, selected growth-related phenomena are discussed, including quorum sensing and biofilm formation. Considering that metabolism is dependent intimately on growth, this topic is addressed briefly. The second section focuses on the survival behavior of microorganisms and the relevance of this behavior state to food safety. The discussion includes physiological changes leading to this state and survival-associated phenomena such as stress adaptation, persistence, dormancy, and the viable-but-nonculturable state. Implications of these phenomena for safety and quality of food are presented. The third section addresses various aspects of microbial death in food. The discussion includes unmediated microbial death, in which microorganisms die during food storage without the application of external lethal treatments, and programmed cell death, due to the application of certain stresses. Furthermore, the section covers death kinetics when bacterial populations are exposed to lethal factors, phases of the death curve, and the consequences of pathogens’ persistence during the tailing phase of that curve.

Members of the Gram-positive genera Bacillus and Clostridium and some closely related genera respond to slowed growth or starvation by initiating the process of sporulation, and the resultant spores can cause practical problems in food microbiology as well as human disease. The molecular biology of sporulation and spore resistance and germination in Bacillus subtilis has been extensively studied for many years, and there is detailed knowledge of these processes and many of the regulatory mechanisms involved. With the recent availability of many genome sequences as well as methods for genetic manipulation of clostridia of industrial and medical importance, a detailed molecular understanding of sporulation and spore germination in clostridia is also being developed. This chapter describes the fundamental bases of sporulation, spore germination, and spore resistance and the problems that spores present to the food industry.

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. These concepts have been used to develop recommendations for criteria for foods in international trade or for criteria specific to pathogens, such as Listeria monocytogenes, in foods. They have also been the basis of the Codex Alimentarius Commission document “Principles and Guidelines for the Establishment and Application of Microbiological Criteria Related to Foods,” first issued in 1997 and revised in 2013. This document defines a microbiological criterion as a risk management metric which indicates the acceptability of a food or the performance of either a process or a food safety control system following the outcome of sampling and testing for microorganisms, their toxins and metabolites, or markers associated with pathogenicity or other traits at a specified point of the food chain.

Preservation technologies subject bacterial cells to different levels of stress, which in the most effective cases lead to their inactivation and death. The term “stress” can refer to any extracellular influence that threatens the ability of microorganisms to perform their living functions. The food preservation technologies designed to rapidly inactivate microbial cells include thermal processes, irradiation, high-pressure processing, and the use of strong oxidant compounds. Other technologies accomplish the preservation of foods by inhibiting growth; the most extensively used are low-temperature storage (refrigeration and freezing), reduction of moisture content (concentration and drying), control of ox-redox potential (use of controlled atmospheres and vacuum packaging), and acidification (fermentation and addition of organic acids). In nature, microorganisms are constantly exposed to similar changes in temperature, oxygen, moisture, light, pH, and chemical composition. Bacteria are able to survive thanks to a wide array of molecular responses that provide cellular protection against stresses. Bacteria are protected from changes in pH, temperature, oxidative conditions, solute concentrations, and pressure by a network of sophisticated global genetic regulatory systems and molecular stress responses specific to individual chemical or physical threats. The most important general regulators and specific genetic systems reported in representative foodborne pathogenic bacteria are highlighted in this chapter.

Dairy-associated microbes are important determinants of food quality and safety and are essential for the production of fermented dairy products. Built upon over 130 years of dairy food microbiology research, this chapter focuses on the microbial ecology and systems biology of dairy products from the perspective of culture-independent metagenomics research. Recent studies have provided new perspectives on the microbial composition in raw and processed fluid milk from bovine, goat, and other animal sources and the introduction and succession of those microbes on the farm and at processing facilities. Also discussed are microbiotas in cheese and cheese-associated environments. The diversity of cheese varieties is possible because of those microorganisms and the metabolic processes they perform. The bacteria contained in milk and found on processing equipment, as well as starter cultures, bacteriophages, enzymes and other ingredients, and production and ripening conditions, have interconnected effects, resulting in different cheese varieties with distinct organoleptic properties. Lastly, the microbial composition of other fermented dairy products is presented, including fermented milk beverages such as kefir, yogurt, koumiss, kurut, nunu, and tarag. High-throughput “-omics” approaches have revolutionized our understanding of the ecology and molecular capacities of dairy-associated microbes. With continued methodological and technical advances, these methods will propel improvements in dairy quality and safety assurance and will accelerate a fundamental understanding of complex microbial ecosystems.

Meat and poultry are important commodities in the United States and worldwide. Given the popularity of these commodities and their high consumption rate, it is critical to understand the sources of contamination and the means to prevent cross-contamination to protect public health. Contamination of meat and poultry by microorganisms occurs naturally as a result of procedures necessary to produce foods of animal origin. In general, most foodborne illnesses and outbreaks are due to undercooking or underprocessing of these products, cross contamination, or improper handling of cooked meat and poultry. As these are highly perishable commodities, particularly fresh meat and poultry, temperature controls are critical to prevent contamination. Given the broad spectrum of variables that can cause foodborne illnesses linked to meat and poultry products, this chapter discusses microbiological issues related to these commodities and their control measures.

Concerted efforts of scientists to enhance food productivity and nutrition are important to mitigate hunger and malnutrition. At the same time, it is essential that crops be microbiologically safe. This chapter addresses various microbiological issues associated with several types of agricultural crops, including fruits, vegetables, nuts, and grains. One common attribute of such crops is that they are primarily grown in open fields, where sources of microbial contamination may be difficult to control. Hence, the microbiological profile of a crop will vary by the plant type, the region in which it is grown, and the management and processing practices applied to it. To systematically address this topic, this chapter first provides a short description of the food groups discussed. After reviewing some of the major microbial groups associated with each crop group, the chapter addresses (i) quality and safety repercussions associated with microbial contamination of these food groups; (ii) sources of contamination; (iii) detection of contamination; (iv) interventions available to reduce microbial contamination, including physical, chemical, biological, and hurdle treatments; and (v) quantitative microbial risk assessment. Over the past decade, considerable research has been directed toward understanding and mitigating spoilage and pathogen risks associated with these crops, which should lead to extended shelf lives and reduced risk of illnesses associated with these plant-based foods.

Humans are the ultimate bioassay for low-level or sporadic contamination of our food supply. Epidemiologic methods of foodborne disease surveillance are powerful tools because they take advantage of events that are occurring throughout the population. This population-based lens, focused by advances in molecular subtyping and information technology available to public health laboratories, is particularly well suited to dealing with foodborne diseases associated with mass-produced and widely distributed food products. Whole-genome sequencing (WGS) is becoming the standard for public health surveillance. It offers greater sensitivity for outbreak detection and greater specificity for outbreak investigation than previous methods of analysis. WGS links between cases and foods or environmental samples will require exposure assessment. Public health officials will be challenged to provide the epidemiologic resources to investigate and solve the anticipated growth in small outbreaks. Better outbreak investigation methods, including environmental assessments, will provide more useful data to evaluate the effectiveness of food safety policies and improve food attribution models. Improving the science base for policy development should lead to more effective food control activities. Since national food supplies are rapidly becoming global in origin, the need for an international system for foodborne disease surveillance exists as well. Because 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 public health in both countries.

Salmonella is a ubiquitous rod-shaped member of the family Enterobacteriaceae. Thanks to the ability to swiftly adapt to diverse environments, this human pathogen can infect a multitude of hosts, animals, plants, and protozoa and can colonize diverse environments. This characteristic makes this pathogen a major public health threat that cannot be eradicated, only identified and contained. In developed and industrialized countries, Salmonella spp. contaminate mainly animal products and produce, whereas in developing countries, waterborne transmission and person-to-person transmission play a more important role. There are as many as 130 million cases of nontyphoidal salmonellosis worldwide each year, and of those, about 80 million are foodborne. In the United States alone, nontyphoidal Salmonella is responsible for approximately 1.2 million illnesses and more than 450 deaths each year. These estimates make nontyphoidal Salmonella serovars the leading cause of bacterial foodborne illness. This chapter provides a detailed historical perspective on the discovery, nomenclature, and characterization of this microorganism. The discussion covers the classification of this complex taxon based on the recent concepts of core genomes and pangenomes and the most prevalent isolation and identification methods currently used. The most recent understandings of virulence mechanisms and antibiotic resistance prevalence are also covered to present a complete overview of this important human pathogen.

Campylobacter is regarded as a leading cause of bacterial foodborne infection in many areas of the world. Campylobacter jejuni and, to a lesser extent, Campylobacter coli are important causes of human diarrheal illnesses, even surpassing Salmonella in importance in many countries. Although human illnesses are usually self-limiting, the associated morbidity and cost are significant. In Europe, the burden of human campylobacteriosis is estimated to be between 8 and 100 times higher than the annually reported number of cases, which has approximated 200,000 in recent years. Such a high incidence of Campylobacter-related diarrhea has significant socioeconomic impact, making this pathogen a priority for public health and food safety researchers. This chapter highlights the important bacteriological and epidemiologic features of contamination thermotolerant Campylobacter spp. (C. jejuni and C. coli) in the human food supply chain.

This chapter summarizes the latest information available for Shiga toxin-producing Escherichia coli (STEC). STEC was first recognized as a human pathogen in 1982, when E. coli O157:H7 was identified as the cause of two outbreaks of hemorrhagic colitis. Since then, many other serogroups of E. coli, such as O26, O111, O145, O45, O113, O121, and sorbitol-fermenting O157:NM, have also been associated with cases of hemorrhagic colitis and have been classified as STEC. However, serotype O157:H7 is the predominant cause of STEC-associated disease in the United States and many other countries. Production of Shiga toxins (Stxs) by E. coli O157:H7 was subsequently associated with a severe and sometimes fatal condition, hemolytic-uremic syndrome. E. coli organisms of many different serotypes can produce Stxs, with more than 600 serotypes being identified so far, including approximately 160 O serogroups and 50 H types. However, only strains that cause hemorrhagic colitis are considered enterohemorrhagic E. coli (EHEC), and there are at least 130 EHEC serotypes that have been recovered from human patients. Major non-O157 EHEC serogroups identified in the United States include O26, O45, O103, O111, O121, and O145. Here, we discuss many aspects of their biology, reservoirs, virulence, genomics, and antimicrobial resistance, as well as some recommendations for reduction of their numbers in different foods. We also present some web tools that could help us identify other uncommon causes or vehicles and track down the source of contamination in the future.

Foodborne infections caused by Shigella species remain an important source of diarrheal illness worldwide in both economically developed and developing countries. This chapter presents important features of Shigella spp., the disease they cause, and the impact that these pathogens have with respect to food safety. Diagnosis, epidemiology, ecology, modes of transmission, and examples of recent foodborne outbreaks are presented, along with current understanding of the genetics of Shigella pathogenesis, the genes involved in causing disease, and how they are regulated.

The genus Vibrio contains 130 confirmed species, of which a dozen have been demonstrated to cause infections in humans. As vibrios are natural inhabitants of aquatic environments, infections are usually associated with wound exposure to seawater or consumption of raw seafood. As estimated by the Centers for Disease Control and Prevention, vibriosis causes approximately 80,000 illnesses and 100 deaths in the United States every year, mostly during the summer months, when water temperatures are warmer, and in contrast to infections caused by other major foodborne pathogens, the number of Vibrio infections is steadily increasing. Several reports have recently indicated that human Vibrio illnesses are increasing worldwide, as well as the species responsible for these infections. Besides “the big four” (Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio alginolyticus), additional Vibrio species [Vibrio fluvialis, Vibrio mimicus, Grimontia (Vibrio) hollisae, Vibrio metschnikovii, Vibrio metoecus, and Vibrio furnissii] have recently been associated with food consumption. These 10 Vibrio species are the subject of this chapter.

The genus Cronobacter currently consists of seven species, including Cronobacter sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. dublinensis, C. universalis, and C. condimenti. This genus is now regarded as an opportunistic group of pathogenic bacteria with remarkable versatility in their abilities to cause disease in humans. It is now realized that infections due to Cronobacter can affect susceptible individuals, including neonates, infants, and elderly individuals, and it continues to attract attention in national and international media. Cronobacter species are recognized as being considerably more ecologically widespread than was once thought, and they have been found to be associated with many low-water-activity foods and environments, including powdered infant formula (PIF) and follow-up formulas, as well as in filth and stable flies, PIF and milk powder production facilities, household environments, and water. Pathogen-specific virulence factors have been discovered that adversely affect a wide range of eukaryotic cell and host processes, including protein synthesis, cell division, and proinflammatory host responses. A variety of mobile genetic elements, such as plasmids, transposons, and pathogenicity islands, have been identified, and this genomic plasticity implies ongoing microevolution with the possible acquisition of new virulence factors that will undoubtedly complicate efforts to classify these organisms into various subgroups or into sharply delineated genomopathotypes. This chapter describes the dynamic nature of Cronobacter, which is a highly diverse, versatile, opportunistic pathogen that will continue to present challenges for the global food safety and public health communities in terms of diagnosis, treatment, and prevention of infections.

Gram-negative Aeromonas species are ubiquitous in both aquatic and terrestrial environments. Their adaptability to various ecosystems has resulted in their isolation from a wide variety of organisms, spanning mammals to teleosts. As the awareness of this genus grows, its prevalence and economic impact continue to increase. Because of their aquatic nature, aeromonads have been isolated from most agricultural food products, whether directly or as a result of contamination within the food processing system. Additionally, seafood, especially finfish, is vulnerable to Aeromonas-associated diseases. Most food- and water-related human illnesses caused by aeromonads are due to the species Aeromonas hydrophila, A. veronii, A. caviae, and A. dhakensis. This genus has demonstrated its pathogenic nature in conditions ranging from gastroenteritis to wound infections to severe life-threatening septicemia due to a myriad of virulence factors, including adhesion molecules (i.e., lateral flagella and pili), capsules, cytotonic and cytotoxic enterotoxins (i.e., Alt, Ast, Act and AerA), hemolysins, and degradative enzymes, as well as the formation of biofilms. Thanks to their ubiquitous nature, in combination with overuse of antibiotics agriculturally and clinically, aeromonads have acquired an alarming resistance to a plethora of antibiotics. Therefore, this genus can serve as biological reservoirs of antibiotic resistance genes; intergenus gene exchange between members of the Enterobacteriaceae and the Aeromonadaceae has been documented. Together, Aeromonas spp. present multiple risks: they are foodborne pathogens; they impose economic burdens on the food industry due to contamination, resulting in food spoilage; and they act as reservoirs of antibiotic resistance, resulting in clinical infections that are more resilient to treatment.

This chapter discusses Yersinia enterocolitica, a zoonotic pathogen that causes yersiniosis in humans and animals. Yersinia enterocolitica is a Gram-negative, non-spore-forming, non-toxin-producing, rod-shaped bacterium. It is an aerobic bacterium but can grow anaerobically and is a good competitor with other bacteria. An important property of this bacterium is its ability to multiply at temperatures near 0°C, which allows it to survive in many chilled foods. Growth temperatures for Y. enterocolitica range from 0 to 44°C. This bacterium belongs to the family Enterobacteriaceae, whose members are often isolated from clinical specimens. Since Y. enterocolitica is widely distributed in nature, it has the potential to contaminate foods, especially surfaces of produce items. Contamination of foods with this pathogen and its ability to survive at low temperatures in storage make it a concern for both food manufacturers and consumers. This chapter covers the persistence, survival, and growth of yersiniae in foods; the detection and identification of Yersinia in foods; the incidence of outbreaks of foodborne yersiniosis, its pathogenesis, and outbreak surveillance; and finally zoonosis virulence and pathogenesis. Possible routes of transmission and conditions necessary for survival and growth in food systems are discussed as well. Finally, we suggest that an improved method for detection and characterization of this pathogen is needed to effectively distinguish genotypes among strains isolated from humans and food systems.

Listeriosis is an atypical foodborne illness of major public health concern because of the severity of the disease (meningitis, septicemia, and spontaneous abortion), the high case fatality rate (approximately 20 to 30%), the long incubation time (up to 70 days), and a predilection for individuals who have an underlying condition that leads to impairment of T-cell-mediated immunity. Control of L. monocytogenes in foods represents a significantly greater challenge than most foodborne pathogens in that it is widely distributed in the environment, is resistant to diverse environmental conditions, including low pH and high NaCl concentrations, and is facultatively anaerobic and psychrotrophic. The various ways in which L. monocytogenes can enter food processing plants, its capacity for prolonged survival in the environment (soil, plants, and water), on foods, and in food processing plants, and its ability to grow at low temperatures (2 to 4°C) and to survive in biofilms or on/in foods and food contact surfaces for prolonged periods under adverse conditions have made this bacterium a major concern of the agrifood industry for more than 25 years.

Botulism is a neuromuscular paralytic disease in humans and animals resulting from the actions of botulinum neurotoxins (BoNTs), which are produced by Clostridium botulinum and rare strains of Clostridium butyricum and Clostridium baratii. BoNTs are the most poisonous toxins known for humans and vertebrate animals and are almost unique among foodborne toxins in being highly toxic by the oral route. C. botulinum produces endospores that are resistant to many food processing conditions and to antimicrobials in foods. Under permissive conditions, C. botulinum can grow and form BoNT, which on consumption causes botulism. C. botulinum produces seven serotypes of BoNTs (A, B, C1, D, E, F, and G), which are distinguished by neutralization of toxicity in mice using homologous antisera prepared against the purified toxins. In the United States, C. botulinum and BoNTs are categorized as tier 1 select agents, the most dangerous group of biological agents, and high-security laboratory facilities and rigorously trained personnel are required to experiment with C. botulinum and quantities (>1 mg) of BoNTs that qualify it as a select agent. Outbreaks of botulism are considered a public health emergency and lead to rapid regulatory and industry responses.

Significant progress has been made in the last few years towards our understanding of the epidemiology, pathogenicity, and control of foodborne disease, including food poisoning, caused by Clostridium perfringens strains. Despite this, a significant burden of food poisoning illnesses still occurs in the United States every year, with nearly a million cases reported. C. perfringens food poisoning commonly occurs as outbreaks in institutions where food is prepared in large quantities. Prophylactic measures to prevent food poisoning should focus on restricting multiplication of vegetative cells in cooked foods. Cooking at the proper temperature and for the right time, along with rapid cooling after cooking with subsequent refrigeration, is the most effective action to control the multiplication of C. perfringens and thus avoid food poisoning outbreaks. Processors can take advantage of multiple food formulation factors or hurdles in foods (e.g., water activity, pH, and added preservatives) to restrict growth from spores in cooked foods. Predictive models have been developed to estimate growth under conditions that are relevant to food processing operations. Recent advances in molecular techniques have enabled researchers to characterize C. perfringens virulence factors, toxins, sporulation, spore heat resistance, to carry out epidemiologic trace-back of foodborne illness and toxigenic typing methods, etc. Future research efforts should be directed towards efficient tracing of C. perfringens strains of public health significance, multiple hurdles in formulated foods, proper processing of ready-to-eat foods, and consumer awareness of handling of such foods.

The Bacillus cereus group currently consists of nine Bacillus species, i.e., B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis, B. cytotoxicus, B. bombysepticus, and the most recent member of the group, B. toyonensis. The species within the B. cereus group are very closely related, and their toxicity ranges from avirulent strains used as probiotics to highly toxic strains responsible for severe illness and fatalities. B. cereus causes two different types of foodborne illness: the diarrheal type caused by enterotoxins and the emetic type caused by a small heat-stable emetic toxin. For both these types of foodborne illness, the food involved has usually been heat treated, and surviving spores are the source of the food poisoning. The members of the B. cereus group are common soil saprophytes and are easily spread to many types of foods, especially those of plant origin (rice and pasta), but are also frequently isolated from dairy products. Some strains of the B. cereus group are able to grow at refrigeration temperature. These variants raise concerns regarding the safety of cooked, refrigerated foods with extended shelf lives. Foodborne B. cereus illness is probably greatly underreported, as both types of illness are usually mild and last for less than 24 hours. However, more severe forms of B. cereus foodborne illness, including fatalities, are reported occasionally.

Staphylococcal enterotoxins (SEs) are causative agents of staphylococcal food poisoning (SFP). Advances in genome sequencing and recent monkey feeding assays have continuously expanded the list of SEs expressed by Staphylococcus aureus and other staphylococci. Although the implementation of Hazard Analysis and Critical Control Points systems and active foodborne illness outbreak surveillance programs have greatly reduced SFP, most instances of SFP are attributable to improper food handling practices in the retail industry. Additional knowledge of the molecular aspects of staphylococcal survival and growth, SE production in food, and the molecular mechanism of emesis caused by SEs will enable further development of effective ways to prevent SFP. In addition to emetic activity, all SEs have superantigenic properties that modulate host immune responses to cause toxic shock syndrome, as well as causing transient immunosuppression by inducing regulatory T cells. These suggest that the harmful effects of SEs are not merely emesis to allow S. aureus to exit the host but also immunosuppression to survive in and persistently colonize its many human and animal hosts.

Mycotoxins are fungal metabolites which can cause disease or death in humans and domestic animals. Since the discovery of mycotoxins, some species of fungi have emerged as a significant health issue because of their potential to produce mycotoxins, thereby resulting in a public health concern. The most important mycotoxins are aflatoxins, ochratoxin A, fumonisins, deoxynivalenol, and zearalenone. This chapter provides an overview of the fungal sources of these mycotoxins, their ecology, the occurrence in foods, the significance in human health, methods for their analysis, and strategies to manage the risk from these contaminants.

Human enteric viruses are responsible for substantial morbidity worldwide. Transmitted predominantly by the fecal-oral route and exclusively in association with human feces and/or vomitus, these viruses come into contact with humans by a variety of routes, including the consumption of contaminated foods. From an epidemiologic perspective, the most significant of these are human noroviruses, which are the most common cause of acute gastroenteritis worldwide and are now recognized as the leading causes of foodborne illness. Enteric viruses can be transmitted directly by person-to-person contact or indirectly by consumption of contaminated food or water or contact with fomites. The usual source of enteric virus contamination is human fecal matter, which can easily harbor up to 1010 genomic RNA copies per gram when shed by infected individuals. Human enteric viruses have properties that are distinct 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. Since they must resist the enzymatic conditions and pH extremes encountered in the gastrointestinal tract, enteric viruses are also resistant to a wide range of commonly used food-processing, preservation, and storage treatments.

Foodborne parasites pose a risk to human health in virtually all regions of the world. In addition to the direct effect that these parasites have on human health, zoonotic parasites found in food animals often serve as trade barriers for countries where these parasites occur. A considerable body of legislation has been developed for the purpose of preventing and controlling zoonotic parasites in food animals, including very costly meat inspection programs. 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. Despite the availability of sensitive, specific diagnostic tests, veterinary public health programs (meat inspection), and effective chemotherapeutic agents for human tapeworm carriers, these parasites continue to be a threat to public health in many parts of the world. 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. Current control and preventive procedures are often inadequate, and more effective control measures are needed to ensure safe meat for human consumption.

Protozoan parasites have been linked to human illnesses since antiquity and have been associated with outbreaks of foodborne and waterborne illness. Microorganisms such as amoebas, flagellates, ciliates, and coccidians are the most common in this group. Toxoplasma and Sarcocystis are most frequently associated with consumption of undercooked meats, whereas Cryptosporidium, Cyclospora, Cystoisospora, Giardia, and the amoebas are mostly associated with consumption of contaminated water and fresh produce (vegetables and fruits). Foodborne transmission of Trypanosoma cruzi has also been reported, particularly in South and Central America, when infected insects or their feces are consumed in fruit juice preparations. Cyclospora cayetanensis, a coccidian parasite, is endemic to various locations worldwide and was responsible for a number of U.S. foodborne-illness outbreaks in the 1990s as a result of consumption of imported berries and in the past 5 years after consumption of imported salad greens and cilantro. Sarcocystis infection has been diagnosed in ill travelers complaining of muscular pains; transmission was associated with consumption of improperly cooked meats from Southeast Asia. Most parasites are inactivated by heating at high temperatures and in some instances by prolonged storage under freezing conditions, but parasites are highly resistant to chemical disinfection. Increased travel to tropical areas and consumer demands for off-season produce have resulted in globalization of the food supply. Along with the advantages of globalization, the incidental spread of foodborne parasites has also increased. The biology, epidemiology, inactivation, treatment, and control of foodborne parasites are addressed in this chapter.

There is a growing consumer demand for safe foods that are free of additives, minimally processed, and fresh-like in appearance and taste. While conventional thermal processing methods can address microbial safety considerations, the severity of processing invariably lowers the quality of food. To address these needs, numerous nonthermal processing methods are under investigation. In this chapter, we highlight some of these physical, nonthermal processing technologies and discuss their mechanisms of action, their benefits over conventional technologies, and their potential limitations.

Food antimicrobials are chemical preservatives added to or present in foods that retard growth of or kill microbes, but they do not include therapeutic antibiotic-type compounds used for growth promotion or disease treatment in food animals. These compounds are divided into the traditional and the naturally occurring. Antimicrobials are classified as traditional when they (i) have been used for many years, (ii) are approved by many countries and/or regulatory bodies for inclusion in foods as antimicrobial agents, or (iii) are produced by synthetic processes as opposed to being natural extracts (e.g., industrial fermentation of nisin from Lactococcus lactis subsp. lactis). Ironically, many synthetic and traditional food antimicrobials are found in nature; examples include acetic acid from vinegar and lactoperoxidase in fluid milk. The use of natural antimicrobials will likely continue to grow in popularity. Additional research is needed to determine the levels of natural antimicrobials required for successful inhibition of foodborne pathogens, their mechanisms of action, and their safety. The development of novel applications of existing antimicrobials, including encapsulation, incorporation into edible polymers and the use of combinations of antimicrobials capable of synergistic inhibition of foodborne microorganisms, is being investigated. Major challenges in future applications include 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.

This chapter provides an overview of current and emerging methods of food preservation using live microorganisms and biologically derived substances. This field of study is rapidly evolving, as interest in so-called “natural” and “organic” products has grown exponentially among the public in the past 25 years. Biological methods of preservation are of particular interest, again, due to their natural/biological origins and safety compared with commonly used chemical preservatives and physical treatments. Broadly speaking, biological preservatives can be divided into several distinct groups: (i) live microorganisms, (ii) plant derivatives, (iii) proteinaceous compounds of both eukaryotic and prokaryotic origins, and (iv) bacteriophages. Each group presents its own set of pros and cons, which must be carefully considered and balanced for every individual food application. The safety of biopreservatives is paramount and is the final basis on which a treatment may be chosen for application in the food industry, regardless of the efficacy demonstrated in laboratory studies. Impact on the quality of food is also a concern, as any loss of quality will result in an undesirable product. Quality concerns can be addressed through various techniques aimed at decreasing the amount of preservatives needed while increasing efficacy. These include novel modes of delivery, as well as synergistically acting combination treatments, both of which show promise.

Novel trends in consumer demands and the global threat of antibiotic-resistant bacteria have generated the need for natural preservation techniques to reduce the use of preservatives in food production and to provide alternatives to aid safe food production. Bacteriophages, the natural killers of bacteria, provide alternative biological solutions for control of foodborne pathogens covering the entire food chain. Bacteriophages are obligate parasites that are specific to bacteria, thus being harmless to humans, animals, and plants. Phages are highly specific and leave the remaining microbiota untouched, another property that favors phages over conventional methods that may affect the beneficial microbiota of the food. Furthermore, phages have low inherent toxicity and are already present in foods as well as the human and animal gut. Finally, phages can be used along the entire food chain, including phage therapy for reduction of pathogen colonization of animals in primary production and phage biocontrol during food production. In this chapter, we explain the principles and mechanisms behind the use of phages for biological control of foodborne pathogens, as well as the rationale and outcome of using phages for therapy and biocontrol, including the challenges and limitations of such applications. In terms of future prospects, we discuss the technical and regulatory challenges of widespread industrial use of phages for biological control of foodborne pathogens.

The production of fermented foods and beverages is one of the oldest biotechnological practices, dating back 7,000 years or more. Throughout human history, many different substrates of animal or plant origin have been fermented, including milk, meat, fish, various vegetables, and soybeans. The manufacture of fermented foods, although rooted in practices developed over many thousands of years, is today a major biotechnological activity which is valued at many billions of dollars. The success of such a large and diverse process is critically dependent on the activity of microorganisms, particularly the lactic acid bacteria (LAB). These are the microbes for which the term “starter culture” was specifically coined, as they are added deliberately to a substrate to start a given fermentation process. This chapter provides a detailed examination of the LAB, outlining their key functional properties. The historical development of starter cultures, their composition, the formats in which they are commercially available, and the particular issues pertaining to the use of starters in a modern, high-throughput industrial setting are discussed. The more recent advances in the analysis of phage-host interactions is also reviewed. Finally, the challenges relating to the use of starters in the production of fermented foods are examined, particularly as they relate to strain diversity and the generation of new products with modified flavor, texture, and health attributes.

This chapter details well-established ideas within the microbiome field relating to the interplay between our microbiota and diet and their associations with long-term health over the course of our lives. The chapter details what the microbiota and microbiome are, how the microbiota is established, and how nutrition plays a major role in the progression of the microbiota throughout our lives. Initially the chapter details the establishment of the microbiota and the influence of early-life diet. It characterizes the progression to a more adult-like microbiota due to dietary changes and places the adult microbiota in the context of the different dietary and lifestyle choices we make as adults, the influence these have on our body mass index, and how this may influence our health. Finally, we detail the effect of diet and the microbiota on our health as we reach an advanced age and how the microbiota may be maintained or modulated through diet.

Probiotics, live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, and prebiotics, substrates that are selectively utilized by host microorganisms conferring a health benefit, make up a group of highly researched substances targeted at influencing microbiota-mediated functions for the benefit of the host. Evidence that probiotics and prebiotics can benefit human and animal health continues to build and fuel basic research on mechanisms, genomics, and genetic improvement of these substances. This chapter explores basic mechanisms of probiotic and prebiotic function, next-generation probiotics and prebiotics, comparative genomics, taxonomy and molecular identification, health benefits for humans and animals, and safety and regulatory issues.

In fermented foods, microbial and enzymatic conversions determine and maintain food safety and quality. The deliberate use of microorganisms for food production is one of the oldest unit operations in food processing. In this chapter, products, production processes, and fermentation microbiotas are described for major products from tubers, cereals, beans, milk, fish, and meat. In addition, the origins and properties of fermentation microbiota are described, and metabolic pathways that contribute to the quality of fermented foods are discussed.

The rapid surge of antibiotic resistance has raised serious public health concerns and led to the enforcement of policies to limit the uses of antibiotics in food animal production and human medicine. However, recent scientific breakthroughs present a much more comprehensive picture of antibiotic resistance ecology. The identification of new risk factors in antibiotic resistance development, enrichment, dissemination, and persistence demands innovative strategies for effective mitigation. This chapter discusses important concepts as well as major shifts in research scope and methods based on microbiota instead of individual pathogens. The chapter also discusses corresponding findings on the abundance of antibiotic-resistant bacteria and resistance-encoding genes in the food chain, the major avenues of dissemination of antibiotic-resistant bacteria to hosts through food and feed, mechanisms of antibiotic resistance and persistence, and the impact of antibiotic administration on resistance ecology, gut microbiota, and modern diseases. Discoveries related to foodborne antibiotic-resistant pathogens are illustrated. Specifically, the chapter examines the mainstream practice of oral antibiotic administration in both food animal production and human medicine as a key driver for massive antibiotic resistance in the ecosystem and gut microbiota dysbiosis in animal and human hosts. These breakthroughs in knowledge have laid a solid foundation for targeted controls and opened the doors for innovative and effective mitigation of the major challenges of antibiotic resistance and modern diseases associated with gut microbiota dysbiosis. Future directions for research and paradigm changes in policy and practices in the food chain essential to improve food safety and human health are outlined.

As in other fields of science, food microbiologists are relying more and more on genomic techniques to understand the microbiota associated with our foods. We are looking to whole-genome sequences to decipher the genetic content of foodborne pathogens, industrially relevant strains, and potentially beneficial microbes. Metagenomic and metatranscriptomic techniques are now also being used to determine both the composition and function of microbes in a complex food matrix. Additionally, in recent years, laboratory-based global initiatives have been implemented that use genomics in place of traditional culturing techniques for identification and surveillance of strains involved in outbreaks of foodborne illness. There is little doubt that genomics has emerged as an important tool and will play a large role in the future of food microbiology.

This chapter focuses on the understanding and application of metagenomics as it applies to meat and poultry research. Key concepts for understanding metagenomics include how to conduct a metagenomic study from experimental design to analysis, the difference between shotgun metagenomics and 16S rRNA amplicon studies, and data visualization. Special consideration is also given to discussions on rarefaction and normalizing data. Application of metagenomics in meat and poultry production from both a food safety and a food quality standpoint is discussed in relation to the current body of work published and possible future directions. Meat and poultry food safety is framed around reduction and detection of pathogens, while food quality includes shelf life and packaging differences. The conditions in which animals are reared and the processing environments they are harvested in are considered in the context of impact on end food quality and safety. Additionally, the public heath and regulatory implications of using shotgun metagenomics as a tool for pathogen detection and reduction are addressed.

This chapter takes a brief look at the new frontier of microbiological food safety, nutrition, and immunology. With the use of metagenomic profiles of food and food ecologies, we can now better describe risk factors for contamination by human pathogens along the farm-to-fork continuum. We have also begun to organize data that may also improve our understanding of the more comprehensive prebiotic, probiotic, and antibiotic impact of food on the human microbiomes and how this may be relevant to nutrition, immunology, and chronic disease. We describe case studies with fresh produce such as tomatoes, sprouts, and leafy greens and highlight new approaches to pathogen detection using quasimetagenomics.

Molecular subtyping is an instrumental tool for foodborne illness surveillance and outbreak investigation. The term “molecular epidemiology” in the context of foodborne bacteria is usually applied to the subtyping of bacteria that cause foodborne disease and the ways in which such subtyping data contribute to understanding the transmission of those bacteria to humans. Molecular subtyping techniques 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. Advances in sequencing technology over the last two decades have made whole-genome sequencing (WGS)-based subtyping approaches the method of choice for many foodborne pathogens. Routine application of WGS in laboratory surveillance and monitoring of foodborne pathogens is transforming public health microbiology. With the increasing international trade of food and food animals, it is crucial that molecular subtyping methods for foodborne pathogens be harmonized worldwide to facilitate the rapid comparison of strains isolated in different countries. This method harmonization for comparison is best done in the framework of surveillance networks. The ongoing implementation of WGS provides an unprecedented opportunity to establish universal global standards for subtyping foodborne bacteria that will result in easily exchangeable data and global nomenclature.

Quantitative microbial modeling of foods represents a proactive approach to food quality and safety by accumulating information on bacterial responses to environmental factors and by summarizing the responses through mathematical models and in databases. The first model for food-related processes, documented in the scientific literature in the 1920s, describes the relationship between heat treatment and inactivation of Clostridium botulinum spores. However, it was not until the 1980s that growth and survival of microorganisms in food started to receive more focused attention. During the last 30 to 40 years, predictive microbiology has achieved status as a scientific discipline within food microbiology. In the past decade, the food safety discipline has addressed the assessment of hazards in foods within the framework of risk analysis, a science-based paradigm intended to ensure human health protection. The objectives of risk analysis are to estimate the risk to human health of a hazard associated with food consumption and, most importantly, to assess appropriate management strategies in the food chain capable of reducing the risks. Risk analysis represents a structured decision-making process with two closely connected components: risk management and risk assessment. This chapter focuses on methods of describing the unique aspects of microbial behavior in food systems using predictive microbiology and risk assessments.

Producing safe food, all the time, must be a foundational goal for any individual or corporation in the business of making food. It is critical to understand the risks associated with food and take actions to mitigate those risks. Food safety management systems (FSMSs) include multiple, interrelated elements to help ensure that risks are mitigated to an acceptable level. Good manufacturing practices and hazard analysis and critical control point (HACCP) programs are key elements of an FSMS, but there are other elements based in policy and culture that are also important. A robust FSMS that is effectively implemented, continuously monitored, verified, and subject to continuous improvement is essential for the success of food manufacturers.

Water is an essential part of human life; however, it is also used for a number of other purposes. Based on climate change and an anticipated growth in population, the demand for water is expected to increase and strain our limited water resources, especially in arid regions. Treating wastewater to produce reclaimed water provides a sustainable alternative to the finite supply of fresh water. In addition to the standard treatments applied to wastewater, this chapter discusses both advanced and low-cost treatments that are available to produce reclaimed/reconditioned water. Included in the discussion is an overview of the regulations and guidelines that are available to ensure that microbiological contaminants are reduced to a level that is deemed safe for the water’s intended use. Critical to the implementation of these guidelines is the availability of reliable tools to detect the microbiological contaminants that may contribute to human illness. Within the food industry, the two main uses of reclaimed water include irrigation of agricultural crops and reuse within the processing sector for operations not contacting ready-to-eat food products. Several limitations to the use of reclaimed water in these applications are examined; the primary ones include exacerbation of antibiotic-resistant microorganisms, regrowth of enteric pathogens not completely eliminated during treatment, and the public's negative opinion associated with the idea of recycling sewage. To offset these limitations and provide perspective on the level of risk associated with an application involving reclaimed water, quantitative microbial risk assessments are now routinely being conducted.

The microbiological safety of food has always been essential to ensuring the public’s well-being; however, over the past decade, many issues have emerged or continue to be of concern. Four of these topics are addressed in this chapter. The application of whole-genome sequencing to enhance the identification and investigation of outbreaks of foodborne illness through its use as a standard tool for “fingerprinting” pathogens has revolutionized surveillance of foodborne-illness outbreaks and source tracking. Implementation of the U.S. Food and Drug Administration’s recently adopted rules of the Food Safety Modernization Act is another factor that should contribute to the enhanced safety of foods, and a brief discussion of the intricacies of this law is provided, highlighting features that are likely to have the greatest impact. Although the food industry generally has a remarkable record of producing safe foods, it is increasingly challenged by consumers’ demand for “natural” foods that are free of synthetic antimicrobials. In addition, an ever-increasing percentage of foods consumed in the United States and in many developed countries are imported, of which a major portion originate from developing countries whose sanitary practices in production and processing are often inferior to those employed in developed countries. The food safety landscape is not likely to remain static; however, improvements will need to be balanced against competing nutritional, cost, and sustainability priorities.