Genomes of Foodborne and Waterborne Pathogens

Although all Shigella species share similar pathogenic properties, each species exhibits peculiar epidemiological characteristics. S. dysenteriae, S. flexneri, and S. boydii are most common in developing countries, whereas S. sonnei is more prevalent in developed countries. A remarkable difference between Shigella and E. coli genomes is the presence of pseudogenes in the former. The second remarkable feature of the Shigella genome is the presence of enormous copies of IS elements, which are likely the cause of genomic rearrangements, including deletions, inversions, and translocations that may effectively disrupt the colinearity among different Shigella genomes. After the acquisition of the ancestral forms of the virulence plasmid by Shigella/enteroinvasive E. coli (EIEC), genome reduction by inactivation of the pathway-specific antivirulence loci (AVL) is vital for adaptation in the cytosolic niche. Similar to obligate pathogens, reduced selection pressure might have played an important role in Shigella genome reduction, which may have further accelerated terminal evolution and resulted in the increased host specificity. Numerous genes responsible for cell motility cell envelope, carbohydrate transport, and metabolism that are present in E. coli were frequently lost in Shigella. A combination of various strategies using the basic information provided by genomic research will be helpful in efficient control and prevention of Shigella infections.

Campylobacteraceae is composed of three main genera, Campylobacter, Sulfurospirillum, and Arcobacter, and is represented currently by 43 described or proposed species. Unlike Campylobacter spp., that are associated with warm-blooded avian and mammalian hosts, members of the Sulfurospirillum and Arcobacter genera are non-host-associated, free-living, environmental organisms. Arcobacter spp. can grow aerobically, unlike campylobacters, which are restricted to microaerobic and/or anaerobic environments. The organisms whose genomes have been or are being sequenced are (i) host-associated or free-living; (ii) nonpathogenic, human commensals, or human pathogens (oral or gastrointestinal); or (iii) food or environmental isolates. Analysis of these genomes alongside comparative genomics will provide clues pertaining to the genetic nature of pathogenicity, host association, environmental adaptation, and evolution. The chapter addresses the topics related to the Campylobacter and Arcobacter genomes. The coding sequences (CDSs) with assigned function are fairly consistent across the sequenced Campylobacteraceae genomes, with an assigned function/total CDSs average of 45%. It is possible that many Campylobacter species are composed of multiple host-associated phylogenetic clusters. Comparative genomics of the Campylobacter and Arcobacter genomes has not identified any pathogenicity islands containing toxin-encoding genes or other virulence determinants. Campylobacter and Arcobacter species provide fertile ground for comparative genomics. Therefore, comparative genomics of Campylobacter and Arcobacter should provide insights into pathogenicity, host adaptation and specificity, evolution, and environmental adaptation and survival. The degree of variation within Campylobacter and Arcobacter is an important consideration.

This chapter talks about Vibrio parahaemolyticus that can cause fatal septicemia in immune-compromised hosts. The Kanagawa phenomenon (KP) has long been recognized as an effective marker for discriminating pathogenic from nonpathogenic V. parahaemolyticus strains and is used extensively as a clinical indicator for the virulence of this bacterium. Thermostable direct hemolysin (TDH) is considered an important virulence factor for V. parahaemolyticus. Genes classified in transcriptional regulation have specific roles in response to environmental changes. The most notable finding of the genome sequencing of V. parahaemolyticus was the presence in the genome of two sets of genes for the type III secretion system (T3SS). T3SS2 may also be useful for novel preventive and therapeutic methods, for example, as a target for vaccines or drugs against V. parahaemolyticus infections. The novel preventive and therapeutic methods discussed here are highly selective for this pathogen and may therefore contribute to therapies that are distinct from conventional treatments based on the administration of antibiotics. Studies on V. parahaemolyticus have mainly focused on the role of virulence factors TDH and thermostable direct related hemolysin (TRH), but genome sequencing of this bacterium has shed light on novel aspects of this organism. A notable finding in this context is the discovery of functional T3SS genes in the genome of V. parahaemolyticus, which are not found in the genomes of cholera-toxin-producing V. cholerae and V. parahaemolyticus.

The study of the Y. pestis genome indirectly reveals enteric features of Y. pseudotuberculosis (and Y. enterocolitica). This chapter describes the salient features of postgenomic studies on Y. enterocolitica and Y. pseudotuberculosis and relates this to our understanding of the diversity and evolution of virulence for these species. The enteropathogenic yersiniae are ubiquitous in the environment, and are a common cause of animal infections, and they have been isolated from cattle, sheep, pigs, domesticated animals, and avian species. Various other adhesion- and invasion-associated factors such as Ail and the Myf fimbriae have also been reported in enteropathogenic yersiniae. B12 is an essential cofactor for several reactions, including 1,2-propanediol degradation. Although the true significance of tetrathionate respiration operon in Y. enterocolitica is unknown, in Salmonella, 1,2-propanediol is an important source of energy, and mutants unable to make B12 are significantly attenuated in their ability to grow in macrophages. With respect to the enteropathogenic yersiniae, the natural deselection highlights many known genes required for the enteropathogenicity by Y. pseudotuberculosis (and Y. enterocolitica), but also highlighted many previously unsuspected candidates. Perhaps the most striking aspect of the evolution of Yersinia is the extremely rapid emergence of Y. pestis from Y. pseudotuberculosis, and genome analysis shows how this has happened.

Analyses of over 400 strains suggested that Listeria monocytogenes populations adapted to different niches exist. This study identified one L. monocytogenes 1/2a strain that was dominant among the strains collected from the food-processing plants. It is clear that the patterns and markers identified by these different genomic techniques in L. monocytogenes strains are an invaluable basis for developing powerful tools for rapidly tracing listeriosis outbreaks and for conducting effective surveillance of food-processing environments. The ongoing sequencing projects aimed at determining the complete genome sequence of one representative of each species of the genus Listeria by the Institut Pasteur and the German PathoGenomiK network. The determination of the complete genome sequence of an additional 18 Listeria strains of by the Broad Institute can be the driving force for understanding the function of the many factors encoded by the genome, whether involved in virulence or not, and to understanding strain-specific differences in niche adaptation and virulence. The sequencing of hundreds of genomes and communities, like those resident in food production environments, will allow us to move from population genetics to population genomics.

Bacillus cereus is the putative ancestor of all Bacillus anthracis strains, which obtained two virulence plasmids (pXO1 and pXO2) and at least one chromosomal mutation that inactivated the plcR gene. Identification of the molecular signals that trigger germination and the spore surface receptors involved is critical to understanding the pathogenesis of B. anthracis. Sporulation and pathogenesis are opposite processes, while germination and virulence gene expression are synergistic, since transition from dormant spores to vegetative cells is essential for the virulence of B. anthracis. One of the enzymes within the basal layer of B. anthracisspores is an alanine racemase capable of converting the spore germinant L-alanine to the germination inhibitor D-alanine. Comparative genomic studies have contributed significantly to our understanding of the virulence properties, host specificity, ecology, and adaptations of B. anthracis and other species comprising the B. cereus group. While all Banthracis strains examined so far contain four prophages of the lambda family inserted at defined loci, most of the other sequenced B. cereus group genomes do not contain homologous prophages inserted at these sites. With the advent of next-generation sequencing technologies, which promise to deliver even more sequence data over shorter periods of time, and with metagenomics and community genomics approaches on the rise, the future of pathogen genomics is bright and growing fast.

Clostridium perfringens is one of the most frequent causative agents of human food-poisoning worldwide. Strains of C. perfringens are divided into five distinct toxin types on the basis of their differential production of four major C. perfringens extracellular toxins. The published genomes of C. perfringens ATCC 13124, SM101, and strain 13 contain single circular chromosomes of 3,256,683, 2,897,393, and 3,031,430 bp, respectively. The genome sequence of C. perfringens strain 13 was examined to identify catabolic pathways that may utilize such sugar substrates as energy sources. Importantly, the fermentative pathways were predicted to lead to the production of CO2 and H2 gasses that may be involved in the establishment of an anaerobic environment within an infected host suitable for C. perfringens growth. One of the hallmark characteristics of clostridial species is their capacity to form endospores, which are essentially highly modified dormant cells resistant to extremes of heat and radiation. In C. perfringens strain 13, 49 genes involved in capsule production were found to be organized into a single large gene cluster. Beyond broadening one's appreciation for toxin genes, the genome sequences highlighted additional factors that may facilitate the pathogenic potential of this organism, for example, genes involved in capsule production and in sporulation. Future studies will explore the precise functional roles of these genes in the virulence of C. perfringens and may yield avenues to combating C. perfringens infections.

The noroviruses (NVs) cause approximately one illness for every 30 persons per year on a worldwide basis and are now recognized as the number one cause of foodborne illness in the United States, causing about 9 million foodborne infections per year. Human calciviruses fall into two major genera, Norovirus and Sapovirus. Sapoviruses also cause gastroenteritis, principally in infants, and are less commonly associated with foodborne transmission than noroviruses (NVs). NVs are nonenveloped, icosahedral viruses ranging from 27 to 35 nm in diameter. The icosahedral virion has T=3 symmetry and is composed of 90 dimers of the single capsid protein VP1. The first 225 amino acids form the shell domain, which is critical for icosahedron formation. One or two copies of VP2 are believed to be within the capsid. The high isoelectric point of VP2 suggests the possibility of an interaction between the positively charged virus protein and the viral RNA during virus assembly. STAT-1 mice are deficient for interferon production and interferon-mediated responses. On the surface, this information suggests that the innate immune response may be critical for successful defense against NV. For a protective immune response in normal mice inoculated with MNV-1, it has recently been shown that both adaptive CD4 and CD8 T-cell responses are critical for a protective immune response. The high degree of variability of different NV strains suggests that there is significant evolutionary immune selection on NVs. Currently, outbreaks from virus strains from the GII.4 cluster account for approximately 80% of outbreaks worldwide.

Food microbiologists often overlook the importance of fungi as a threat to food safety and security even though fungi produce a wide array of toxic compounds. Aspergillus flavus is known to produce over 14 described mycotoxins. It is common for strains of A. flavus to produce both aflatoxin and cyclopiazonic acid (CPA) and for commodities to contain both mycotoxins. Other than A. flavus and A. minisclerotigenes, no other fungal genera are known to produce aflatrem; however, structurally related tremorgenic mycotoxins, such as penitrem from Penicillium spp., are prevalent in other fungi and likely share common enzymes. Fungi have the capacity to produce many diverse secondary metabolites, and over 300 fungal secondary metabolites are described as mycotoxins. It is now possible to predict whether potential toxins produced by secondary metabolism clusters may be present in food. With respect to the regulation of aflatrem biosynthesis, researchers have shown that the gene called veA, previously shown to control aflatoxin and sclerotial production in A. parasiticus was found to not only be necessary for the production of aflatoxins B1 and B2 and sclerotia, but also regulated the synthesis of the mycotoxins cyclopiazonic acid and aflatrem. New tools for genomewide gene profiling and functional analysis will surely reveal additional information on aflatoxin production and the regulation of the process. This knowledge will empower researchers to find effective strategies for controlling aflatoxin contamination of food and feed. Gradually, newly developed next-generation sequencing technologies will become common research tools for functional genomics studies.

This chapter provides an update on Giardia genome research and highlights studies that utilized genomics and functional genomics to understand the phylogeny, biochemistry, molecular biology, and epidemiology of Giardia lamblia. Earlier observations of a highly compact G. lamblia genome containing a disproportionate number of genes coding for variant surface proteins (VSPs), structural proteins, and ribosomal RNA have been confirmed by genomewide sequence analysis. Many cellular processes of G. lamblia (e.g., DNA replication, transcription, and RNA processing), as well as metabolic pathways, are simple compared to higher eukaryotes. To ensure its survival inside and outside its host, G. lamblia must exert precise control over the transcription of genes involved in encystation, excystation, and trophozoite replication. RNA polymerases and transcription initiation factors are similar in some respects to those in eukaryotes but also contain peptides unique to Giardia. G. lamblia is of special interest to those attempting to understand eukaryotic evolution. Transfection of CHO cells with a second cyst wall protein (cwp2) promoter region fused to the gene for firefly luciferase was used to study regulation of cwp2 transcription by sterol regulatory element binding proteins (SREBPs). This study showed that, in the absence of cholesterol, cwp2 transcription was increased and that SREBP binds to the cwp2 promoter to activate transcription of this cyst wall protein. A third cyst wall protein (cwp3) was identified by searching the G. lamblia database with amino acid sequence of a first cyst wall protein (cwp1) and cwp2 leucinerich repeat regions.

This chapter provides an assessment of the extent to which the Toxoplasma gondii genome has fulfilled its promise. It describes the available genomic resources, and provides an overview of those aspects of toxoplasmosis research that have grown most as a consequence of genomic resources. To provide an estimate and description of the "attributable fraction" of progress owed to genomic biology, most of this chapter reviews the hundred (or so) papers that acknowledge several key developments in Toxoplasma gondii genomics. The chapter discusses some additional perspectives intended to expand upon and supplement the documented record of developments made possible by genomic biology, including a discussion of literature that may only indirectly establish that causal link. Finally, the chapter considers the impact that genomics has made on our understanding of T. gondii toxoplasmosis, and broader issues in eukaryotic Microbiology. Recombinant vaccine candidates for N. caninum have been identified by searching T. gondii genomic and expressed sequence tags (ESTs) databases. A genomewide survey of SNP polymorphism provided a means to more fully substantiate and explain earlier observations, that most isolates of T. gondii studied to date can be classified as members of one of three multilocus types, and that only a few ancestors could have contributed all of the diversity represented among them. In the case of T. gondii, the genomic data have encouraged the engagement of a creative and inquisitive cadre of research scientists.