Food-Borne Microbes: Shaping the Host Ecosystem

The oral cavity environment is anatomically, physiologically, and microbiologically diverse. Oral microbial communities exist primarily as multispecies biofilms on the surfaces present in the oral cavity, although large numbers of organisms are also present in the fluid phase of the saliva. The microbial biofilm communities that develop in the oral cavity are clearly intrinsically driven by bacterial interactions, but they are also host driven. Factors such as age, immune status, hormone levels, salivary flow rate, smoking, and dental hygiene standards impact on oral microbial community formation and composition. Dietary factors and antibiotic usage both have major influences. Better understanding of population shifts and what causes them will inform future strategies that may include attempting to recolonize disease sites with more healthy communities. Despite the fact that most oral disease conditions are associated with oral bacteria, the communities that develop in the mouth do largely retain harmonious relationships with the host over long periods. The chapter talks about effect of diet on oral microbial communities. The antibiotic resistance gene pool within the oral microflora and the ability of oral bacteria to readily exchange genetic information within the close confines of biofilms reflect the potential for widespread transfer of antibiotic resistance genes across commensal, pathogenic, and food-borne bacteria.

This chapter complements recent reviews by providing an overview of how the human gut microbiome develops, with a specific emphasis on the colon; some insights into life cycle assessment of gut microbiome structure-function relationships; and description of specific roles that foods might play in modulating these responses. The gastrointestinal (GI) microbiome in many carnivores is, in effect, in competition with the digestive and absorptive processes of the host animal, though the commensal microflora does serve to protect the host from pathogens, principally by colonization resistance. The chapter focuses on distal regions of the human GI tract. The greater abundance of staphylococci in the feces of breast-fed than in those of formula-fed infants may reflect a continuous inoculation of the GI tract of breast-fed infants. Phylogenetic studies employing cloning and sequencing of 16S rRNA genes amplified by PCR have greatly expanded one's knowledge of the diversity and composition of the adult gut microbiome. As noted by Peterson et al., advances in one's abilities to examine gut microbiomes at the RNA, protein, and metabolite levels of investigation will further the mechanistic understanding of microbiome function. In a human trial, Abell et al. noted that the inclusion of resistant starch in the diet significantly increased butyrate and acetate concentrations. The recent advances in genomics and metagenomics provide the opportunity to develop a better understanding of human gut microbiome structure-function relationships and their role in human health and disease, including modulating effects.

The microbial ecosystem of fresh foods is not as complex as that of the corresponding preharvest products, and this is due in large part to the removal of many microbes from meats by animal carcass washing in which hot water, steam, and/or organic acids are applied. Assuming that fresh foods are harvested, processed, packaged, and stored according to acceptable procedures, the fate of each group of microbes can be predicted if one knows how the foods are packaged along with the time and temperature of storage. While some fresh foods are inhibited by specific toxic products of others, some are inhibited by as-yet-unknown means, and this is discussed further in a section on nonspecific microbial interference. When fresh foods that contain a typical microbiota undergo refrigerator spoilage, some or all of the microbial growth parameters come into play. A number of studies on the fate of S. aureus in chicken pot pies, in macaroni and cheese dinners, and in laboratory culture media were published by researchers at the Campbell Soup Co. in the 1960s. Overall, this research demonstrated the inability of this pathogen to compete with naturally occurring organisms under various conditions. The microbial ecosystem of fresh foods is composed of a large number of bacterial genera and species, with most consisting of Gammaproteobacteria and Firmicutes. Whatever the mechanism of microbial interference, both the direct and demonstrated mechanisms, along with the less well-defined mechanisms, interact to affect microbial progression in fresh foods.

This chapter suggests ways in which knowledge of the microbial ecology of the human bowel can be obtained using modern technologies. Probiotics aimed at altering the composition of oral and vaginal ecosystems have been developed or are under development, most products target bowel health. There is little doubt that lacto-bacilli transit the digestive tract following consumption of probiotic products, but in order to evaluate the probiotics phenomenon, one must consider the microbial ecology of the human gut. Some members of the medical profession, as well as the laity, have greeted with enthusiasm the use of current probiotic products as prophylaxis for atopic diseases (allergies), inflammatory bowel diseases (Crohn’s disease [CD] and ulcerative colitis [UC]), and pouchitis. Regulatory mechanisms generated within the ecosystem (autogenic factors) and by external forces (allogenic factors) permit the episodic persistence of some bacterial populations but the elimination of others in a classical biological succession. The best evidence for the efficacy of probiotics in inflammatory conditions of the bowel comes from studies of the maintenance of remission of pouchitis. Metabolomics is the nontargeted, holistic, quantitative analysis of changes in the complete set of metabolites in the cell (the metabolome) in response to environmental or cellular changes. The study of the metabonome might contribute to a full systems biology approach to understanding and maintaining bowel health. The primary aim of the research will be to understand how all of the heterogeneous parts are integrated, with a supplementary aim of identifying biomarkers of health or disease.

This chapter reviews the current knowledge of microbial diversity in different aquatic environments, with a focus on general bacterial populations. This broader understanding of the composition of microbial communities is primarily a result of 16S rRNA gene sequence analysis. The case of antibiotic resistance is used to demonstrate how host ecosystems are influenced by external microbial communities and, in turn, how hosts influence microbial ecology. The chapter discusses knowledge gaps and suggests future research directions to address the link between microbial populations in the aquatic environment and the human microbiota. A better understanding of the microbial diversity of drinking water is necessary to design innovative and effective control strategies that will ensure safe and high-quality drinking water. The vast majority of antibiotic resistance genotyping studies have focused on tetracycline resistance, most in animal waste lagoons, with a couple of studies from alternative aquatic environments such as rivers and drinking water. Antibiotic resistance and the risks of gene exchange accelerated through water treatment are growing concerns. The recent findings of increased abundance of antibiotic resistant bacteria (ARB) and antibiotic resistance genes in surface water and drinking water strengthen these concerns. The interaction between environmental microbes and the human microbiota will likely reshape one's thinking on the relevance of environmental microbiology to public health.

Biofilms are structured microbial communities of cells differentiated to play specific roles in the maintenance of the community and its structure. In this chapter, the discussion of biofilm development is organized into three sections: initiation (reversible and irreversible attachment), structure development (maturation), and dispersal. Although there is a general process for how biofilms develop, mechanisms underlying the process differ among microorganisms. The chapter talks about the biofilm matrix, and biofilm ecosystem. Research on food processing biofilms has centered on the ability of pathogenic and spoilage microorganisms to grow or survive in these environments, with emphasis on the influence of sanitation procedures. Researchers isolated two types of rough colony variants of Listeria monocytogenes from biofilms, distinguished by short-chain and long-chain cell morphologies. Both types of rough variants exhibited enhanced biofilm formation, with the variants exhibiting increased cell chain length (filamentous growth) when grown as biofilms. The predominant microflora of water system biofilms can be characterized as having low physiological activity and as being difficult to culture using conventional plating methods. Biofilms containing mainly commensal microorganisms can form on roots, leaves, and the internal vascular tissues of edible plants. Cells in biofilms are more difficult to inactivate by application of antimicrobial chemicals and physical stresses than their planktonic counterparts. The major contributor of biofilm microorganisms in our diet is most likely fresh produce, since biofilms form on these foods before harvest, postharvest growth is likely, and the products are consumed without heat treatment.

The advances from at least two major research areas, biofilms and bacterial quorum sensing, have led us to begin to appreciate the concept that bacteria can organize into groups, form well-organized communities, and communicate with each other for coordinated activities or social life that was once believed to be restricted to multicellular organisms. Bacteria with altered physiological activities (biofilm phenotypes) are known to result largely from bacterial social behaviors controlled by quorum sensing or other mechanisms when they are living in biofilms. Understanding bacterial social behaviors and their molecular mechanisms in the development of biofilms will greatly facilitate the development of novel strategies in the prevention and treatment of biofilm infections. In 1998, researchers first described the role of las quorum sensing in biofilm formation of Pseudomonas aeruginosa. The biofilms formed by the mutant were also dispersed by the addition of the detergent sodium dodecyl sulfate. This finding suggests that quorum sensing plays an important role in the development of bacterial biofilms. More importantly, this study suggests an inextricable connection between two bacterial social behaviors, quorum sensing and biofilm formation. In P. aeruginosa organism, quorum sensing is highly complex and consists of two interlinked N-acyl-homoserine lactone (AHL) dependent regulatory circuits, which are modulated by numerous regulators acting at both the transcriptional and posttranscriptional levels. The chapter discusses how might quorum sensing signal molecules function in biofilms. Quorum sensing is emerging as an integral component of bacterial global gene regulatory networks responsible for bacterial adaptation in biofilms.

This chapter describes the most important global regulator systems in representative food-borne pathogens and reviews the individual molecular mechanisms of survival against specific food-related stresses. It talks about representative gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus) and gram-negative (Escherichia coli, Salmonella, and Campylobacter) food-borne pathogens which have been thoroughly studied. Weber et al. have classified all the RpoS-regulated genes into six major categories based on their functions: metabolism, regulation, transport, adaptation to stress, protein processing, and unknown. Gram-negative and gram-positive bacteria use global strategies in their response to osmotic stress as well as some unique species-specific responses. The chapter reviews the current knowledge on the molecular response to osmotic stress as manifested by a few food-borne pathogens. In most bacteria, glycine betaine is the most effective osmoprotectant, as it increases the volume of available water in the cytoplasm. Low-temperature storage of foods is an extremely successful preservation technology. Advancement of one's knowledge about the molecular basis for bacterial survival under stressful conditions is critical to the assurance of safe and palatable foods, whether using traditional or novel preservation technologies. The advent of genomics-, proteomics-, and metabolomics-based techniques has accelerated one's knowledge of the components involved in novel stress responses. In addition, the availability of an increasing number of fully sequenced bacterial genomes should facilitate further advances in the field of bacterial stress responses.

Food fermentation is one of humankind’s oldest methods of preservation. It is used to preserve, enhance, and add flavor to many different types of foods. In this chapter, the author examines the roles of microbes in fermentation and the general principles involved in fermentations, including how the primary fermentation organisms interact with the other microbes present in the fermentation and the roles these other microbes have in both product quality and safety. The types of substrate and fermented products can vary greatly, from fermented milk products that contain ethanol such as koumiss to the production of distilled beverages such as whiskey. A section focuses on beer and wine, two of today's most popular fermented products. The fermentation process in the case of cereals differs from the processes involved in vegetable, wine, and dairy fermentations in that it is conducted in order to create a more functional product, whereas the other fermentations are primarily conducted to increase the shelf life of the substrate. Interestingly, it was found that as with the bacterial populations, there were two separate phage-host populations, with phage from the heterolactic segment of the fermentation unable to infect bacteria from the succeeding homolactic fermentation.

This chapter provides an overview of bile composition and conjugation mediated by the normal flora and its aforementioned antimicrobial effects. It discusses known molecular mechanisms behind bile resistance and the role bile has in altering the virulence of enteric pathogens. Fasting and malnourishment have been shown to decrease the amount of bile in the intestine and, consequently, leave individuals vulnerable to bacterial pathogens. Similar to commensal enteric bacteria, pathogenic Listeria monocytogenes contains bile salt hydrolases (BSH) genes thought to confer bile resistance and successful colonization and disease manifestation. Probiotics researchers suggest that subsequent limitations to enterohepatic circulation in the presence of unconjugated bile acids cause enhanced fecal loss of bile salts. The chapter talks about the effect of bile on pathogenic bacteria. Adaptability to the harsh effects of bile acids is a critical component of survival for gastrointestinal pathogens. Both conjugated and unconjugated bile salts increased expression of the CmeABC efflux pump, while other antimicrobials, including chloramphenicol, ethidium bromide, and erythromycin, did not affect transcription of cmeABC. Surface plasmon resonance provided evidence that bile salts were capable of inhibiting binding of CmeR to the cmeABC promoter, leading to increased pump expression and elevated bile resistance. Interestingly, the presence of bile salts in culture media enhanced the resistance of Campylobacter to multiple antibiotics, including cefotaxime, novobiocin, and fusidic acid. As long as the integrity of the normal microbial flora is maintained, compounds that are mentioned in the chapter could be manufactured to target known factors contributing to bile resistance.

Helminths are powerful modulators of host mucosal and systemic immunity. Helminths induce mucosal T cells to make Th2 and regulatory cytokines that participate in the protective process. Helminths affect pathways of innate immunity and induce various regulatory Tcell subsets that limit immune reactivity. The chapter explores the implications of expanding industrialization on the demographics of immunological diseases worldwide. The ‘‘hygiene hypothesis’’ states that immunological diseases may be an unanticipated consequence of improvements in public health and hygiene. Deworming of children and adults, associated with improved public hygiene, could be one of the factors leading to the rise in immunological diseases. People carrying helminths can show immune bias away from the Th1 response normally elicited with tetanus vaccination or in vitro mitogen stimulation. Animal experimentation shows that helminths can prevent the onset of various immunological diseases and reverse ongoing pathology. A case control study in Ethiopia showed a lower frequency of asthma in people infected with hookworm than in uninfected people. Colonization with intestinal helminths protects mice from trinitrobenzene sulfonic acid (TNBS)-induced colitis, which is a Th1-driven disease. It appears that helminths trigger various immunoregulatory pathways to control aberrant inflammation. Helminth colonization can have negative consequences. Some worms can cause disease. Recent experiments using murine and larger animal models suggest that worm infection can interfere with vaccine efficacy. An underlying helminth infection may interfere with the accurate diagnosis of infectious status or leave some animals more susceptible to some serious pathogen-induced enteric infections.

In the years between the introduction of antibiotic therapy and the mid-1970s, antibiotic-resistant bacteria were generally restricted to the hospital setting and epidemic diseases which were not major issues in much of the industrialized world. In this chapter, the development of tetracycline-resistant (Tcr) fish pathogens and bacteria associated with aquaculture environments is discussed. Antibiotic residues may be found in a variety of foods produced around the world. Currently, unlabeled but high levels of cephalosporins (ceftiofur) are allowed in some foods. Rules exist which aim to minimize the level of antibiotic residues found in food products; however, not all foods are tested, nor is it clear that the standards required by farms in North America and Europe are followed when the food is produced for overseas consumption. The majority of the novel genes were mobile elements carrying antibiotic resistance and virulence genes. Enterococci are normal inhabitants of the intestinal flora of most mammals, birds, and humans, as well as from soil, surface waters, plants, vegetables, raw foods such as milk and meat, and fermented meats such as Italian salami or raw sausage. Studies have shown that fish foods, even unlabeled ones, may contain antibiotic-resistant bacteria and/or antibiotic residues. This chapter also illustrates a few examples in which specific antibiotic resistance genes, regions of DNA, and/or plasmids have been found in bacteria from very different ecosystems, from different parts of the world, and in bacteria that are host species-specific.

This chapter focuses on the occurrence of antimicrobial-resistant (AMR) phenotypes among selected food-borne bacteria, with emphasis on isolates recovered from foods of animal origin and the potential public health consequences. Once an AMR determinant becomes widely disseminated in different ecosystems, it is difficult to conduct trace studies to identify particular origins. It may be possible, however, to track the spread of resistant organisms after the introduction of a new animal antimicrobial drug. Continuous monitoring of antimicrobial susceptibility/ resistance profiles in food-borne pathogens is needed in order to identify important trends that signify a need to amend antimicrobial use practices. Bacterial isolates recovered from ill people are sent to the Centers for Disease Control and Prevention (CDC) Coordinating Center for Infectious Diseases in Atlanta, GA, by participating state and local health departments. Enterococci were readily isolated from all retail meats tested, ranging, on average, from 88%of pork chops to 98% of all chicken breasts sampled, which is consistent with other studies from the United States. The enteric habitat of Escherichia coli in animals provides an easy source of contamination for animal-derived meats at slaughter and at points downstream in the food production process. Transmission of resistance genes from normally nonpathogenic species to more virulent organisms within the animal or human intestinal tract may be an important mechanism for acquiring clinically relevant AMR organisms. The evolution and propagation of transmissible plasmids in Salmonella and E. coli carrying resistance to eight or more antimicrobials are particularly worrisome.

This chapter describes the molecular mechanisms and major routes in the dissemination and persistence of foreign genes in microbial ecosystems. Emphasis is placed on commensal bacteria with respect to the emergence, circulation, and enrichment of antibiotic resistance (AR) in the natural environment, the food chain, and the animal or human host. For the microbial consortium, AR is nothing more than another trait offering a survival advantage in adverse environments. Therefore, improved understanding of the basic molecular mechanisms and critical control steps involved in horizontal gene transmission (HGT) in microbial ecosystems will shed light on the development of strategies for control of AR dissemination. Increasing evidence from recent ecological analyses indicates that certain bacteria might play more important roles than others in HGT in microbial ecosystems. Probiotics as a business has grown rapidly in the last couple of decades based on the belief that consumption of certain lactic acid bacteria and bifidobacteria is beneficial for the maintenance of a healthy gut microflora. Expanded coverage, particularly the quantitative assessment of the AR gene pools in commensal bacteria, could provide a more precise picture of the AR status in the environmental, food, animal, and human microbial ecosystems, enabling prediction of the forthcoming risks associated with AR in targeted pathogens.

This chapter discusses how antimicrobial resistance impacts bacterial fitness and how bacteria adapt to restore fitness in the absence of antibiotic selection pressure. This topic has been studied for many different bacterial pathogens, but in the chapter the authors have reviewed information only on food-borne bacteria, with a particular emphasis on Escherichia coli, Salmonella, and Campylobacter. Mutation-mediated antibiotic resistance often occurs in genes encoding products that are involved in vital cellular processes (e.g., DNA gyrase and 23S rRNA), and the resistance-conferring mutations often affect the normal physiological functions of the products, leading to reduced growth rates. Bacterial resistance to actinonin is usually mediated by mutations in the fmt gene, encoding methionyl-tRNA formyltransferase, or the folD gene, encoding an enzyme involved in the production of 10-formyl-H4-folate. Gene amplification (increased copy number) is also involved in the evolutionary process of fitness restoration. In order to effectively control the persistence and transmission of antimicrobial resistance in food-borne bacteria, we must have a better understanding of if and how antibiotic resistance affects bacterial adaptation and evolution and, in particular, how antibiotic-resistant bacteria interact with their environments and animal hosts in the absence of selection pressure.

This chapter provides a review of one's knowledge of immune defense mechanisms, with special emphasis on the recent emergence of community-associated antibiotic resistant strains. Two families of molecules have an especially important role in the colonization of human body surfaces: (i) bacterial surface-bound proteins that bind to human matrix proteins for a tight interaction with epithelial tissues and (ii) ion transporters that cope with the high-salt and low-pH environment of the skin. It is important to stress that innate host defense is most likely by far the most important part of host defense with the task of eliminating invading Staphylococcus aureus. S. aureus may survive in phagocytes over certain periods without lysing them. The immune evasion strategies described so far are rather ‘’passive’’; i.e., they either provide the pathogen with molecules necessary to survive under the hostile conditions encountered on or inside the human body, are aimed to hide from the immune system in a relatively benign fashion, or finally, sabotage mechanisms of the host that would kill S. aureus. First reported to occur in children in the northwestern United States, CA-MRSA has spread with astounding speed. The molecular reason for which community associated methicillin-resistant S. aureus (CA-MRSA) and especially USA300 are so much more infective than their hospital-associated counterparts is still puzzling researchers.

The Mycobacterium avium complex includes the closely related Mycobacterium avium subsp. avium, Mycobacterium avium subsp. paratuberculosis, and Mycobacterium intracellulare, as well as the wood pigeon bacillus. In recent years, M. avium complex strains have assumed greater importance in human medicine, largely because of intractable Mycobacterium avium complex infections in AIDS patients and also because of the possible association of M. avium subsp. paratuberculosis with Crohn’s disease. M. avium subsp. paratuberculosis is the causative agent of Johne’s disease (or paratuberculosis), a debilitating chronic enteritis in ruminants. Current research based on understanding of the genomic diversity among M. avium subsp. paratuberculosis strains is now enabling elucidation of mechanisms of survival in the environment, host specificity, and the association of specific genotypes with overt disease versus subclinical states. The infected macrophages play a critical role in the pathogenesis of the disease and dictate the disease outcome. In this chapter the authors have investigated the nature of macrophage M. avium subsp. paratuberculosis interactions. Contaminated domestic water, pasteurized milk, contaminated meat or other food, environmental sources, and direct animal exposures have all been suggested as possible mechanisms of human exposure to M. avium subsp. paratuberculosis. Comprehensive analysis of these survival mechanisms at the transcriptome and proteome levels is expected to improve one's understanding of host-pathogen and pathogen-environment interactions of M. avium subsp. paratuberculosis.

This chapter provides a survey of established molecular approaches as well as emerging ones for studying microbial communities in gastrointestinal systems. Whole-genome typing methods are cost-effective alternatives for assessing genetic relatedness. These molecular techniques provide more specificity than phenotypic identification of culturable microbes and include pulsed-field gel electrophoresis (PFGE), rep-PCR, multilocus sequence typing (MLST), and octamer-based genome scanning (OGBS). The genotyping method used in recent years for Enterobacteriaceae has been a PCR-based technique using primers targeting repetitive sequences (rep-PCR), which also produces numerous DNA fragments that are separable by electrophoresis. MLST is another PCR-enabled genotyping method that has found widespread use for subtyping a variety of pathogens. The technique is an extension of multilocus enzyme electrophoresis and was first described in 1998 using Neisseria meningitidis as an example. OBGS, a powerful method enabled by genome sequence information, was developed as a result of determining the complete genome sequence of Escherichia coli. Researchers using molecular community fingerprinting methods can rapidly infer the genetic composition or community structure following the analysis of a region of amplified DNA. All community fingerprinting methods begin with extraction of total genomic DNA from the sample. Direct DNA extraction followed by PCR amplification and subsequent restriction digestion is the basis for (terminal restriction fragment length polymorphism (T-RFLP). The greatest challenge to microbial community analysis is linking the community structure at the phylogenetic level with its metabolic potential and subsequent functionality in the environment being studied.

This chapter presents interesting examples of the use of mathematical models in microbial ecology, since comprehensive coverage is not possible. The examples were also chosen to introduce various types of mathematical models to provide some guidance on the choice of suitable types of models to address a particular problem. The common thread running through the variety of examples and models presented is how spatial structure can change the interactions of microbes with each other and the host. To this end, the author begins with a model of a well-mixed system, the chemostat, as a reference point and then moves to spatially structured systems, including chemostats with wall growth, plug flow reactors (PFRs), colonies on agar plates, and finally biofilms. The discussion of chemostat dynamics focuses on the steady state, although more interesting dynamics such as damped or sustained oscillations can occur even in single-substrate, single-species chemostats, e.g., due to the slow induction of a transporter causing a delayed response. The indigenous flora of the intestinal tract is quite diverse and stable, implying that many species have been able to colonize the gut and coexist for a long time, yet the resident flora is resistant to colonization by new invaders. The common explanation that microbes form biofilms because they provide better growth conditions than the bulk liquid neglects the intense competition for resources diffusing into the biofilm, allowing only the top layer of cells to grow.