Weather factors, soil microbiome, and bacteria-fungi interactions as drivers of the epiphytic phyllosphere communities of romaine lettuce.

Lettuce is associated with seasonal outbreaks of Shiga toxin-producing Escherichia coli (STEC) infections. Little is known about how various biotic and abiotic factors affect the lettuce microbiome, which in turn impacts STEC colonization. We characterized the lettuce phyllosphere and surface soil bacterial, fungal, and oomycete communities at harvest in late-spring and -fall in California using metagenomics. Harvest season and field type, but not cultivar, significantly influenced the microbiome composition of leaves and surface soil near plants. Phyllosphere and soil microbiome compositions were correlated with specific weather factors. The relative abundance of Enterobacteriaceae, but not E. coli, was enriched on leaves (5.2%) compared to soil (0.4%) and correlated positively with minimum air temperature and wind speed. Co-occurrence networks revealed seasonal trends in fungi-bacteria interactions on leaves. These associations represented 39%-44% of the correlations between species. All significant E. coli co-occurrences with fungi were positive, while all negative associations were with bacteria. A large proportion of the leaf bacterial species was shared with those in soil, indicating microbiome transmission from the soil surface to the canopy. Our findings provide new insight into factors that shape lettuce microbial communities and the microbial context of foodborne pathogen immigration events in the lettuce phyllosphere.

Development and validation of an improved method for the detection of Salmonella in cinnamon bark and oregano leaves using the adsorbent beta zeolite in the pre-enrichment media.

The detection of Salmonella in spices is challenging due to the presence of antibacterial components. In this study, we evaluated the use of an adsorbent beta zeolite in pre-enrichment media to improve the recovery of Salmonella from cinnamon bark and oregano leaves. Samples (25 g) were spiked with varying levels of S. Montevideo or S. Senftenberg. After 2 weeks of stabilization at RT, betazeolite was added to cinnamon and oregano samples prior to the addition of 225 mL or 475 mL of pre-enrichment media, respectively. Detection sensitivity and rate of the test method were compared to the FDA Bacteriological Analytical Manual (BAM) method which requires the use of 2.5 L pre-enrichment broth. While Salmonella could not be detected in the test method using the reduced volume of pre-enrichment media alone, the addition of beta zeolite resulted in a positivity rate of 62% and 72.6% for cinnamon bark and oregano leaves respectively (all spike levels and both serovars combined). Furthermore, while there were differences in the LOD50 compared to the BAM method, there was no significant difference in the minimum level of detection between the betazeolite and the BAM methods. Our results demonstrate that the use of betazeolite in the pre-enrichment media offers a method with reduced media volumes without compromising on the sensitivity or efficiency of Salmonella detection in cinnamon bark and oregano leaves.

Insular Microbiogeography: Three Pathogens as Exemplars.

Traditional taxonomy in biology assumes that life is organized in a simple tree. Attempts to classify microorganisms in this way in the genomics era led microbiologists to look for finite sets of 'core' genes that uniquely group taxa as clades in the tree. However, the diversity revealed by large-scale whole genome sequencing is calling into question the long-held model of a hierarchical tree of life, which leads to questioning of the definition of a species. Large-scale studies of microbial genome diversity reveal that the cumulative number of new genes discovered increases with the number of genomes studied as a power law and subsequently leads to the lack of evidence for a unique core genome within closely related organisms. Sampling 'enough' new genomes leads to the discovery of a replacement or alternative to any gene. This power law behaviour points to an underlying self-organizing critical process that may be guided by mutation and niche selection. Microbes in any particular niche exist within a local web of organism interdependence known as the microbiome. The same mechanism that underpins the macro-ecological scaling first observed by MacArthur and Wilson also applies to microbial communities. Recent metagenomic studies of a food microbiome demonstrate the diverse distribution of community members, but also genotypes for a single species within a more complex community. Collectively, these results suggest that traditional taxonomic classification of bacteria could be replaced with a quasispecies model. This model is commonly accepted in virology and better describes the diversity and dynamic exchange of genes that also hold true for bacteria. This model will enable microbiologists to conduct population-scale studies to describe microbial behaviour, as opposed to a single isolate as a representative.

Global transcriptomic response of Listeria monocytogenes during growth on cantaloupe slices.

Understanding a pathogen's response to food environments is imperative to develop effective control strategies as well as to elucidate the impact of foods on virulence potential. The purpose of this study was to assess transcriptional response of Listeria monocytogenes after growth in cantaloupe, as well as its impact on survival in synthetic gastric fluid (SGF). The transcriptional profiles of L. monocytogenes grown in cantaloupe or Brain Heart Infusion (BHI) under refrigeration were compared by a custom-designed microarray. A total of 286 and 175 genes were significantly up- and down-regulated, respectively, in L. monocytogenes grown in cantaloupe as compared to BHI (fold change ≥ 2.5 and adj. P < 0.05). The majority of upregulated genes belonged to functions related to amino acid and nucleotide metabolism, flagellar biosynthesis, and iron acquisition, while most downregulated genes belonged to carbohydrate metabolism. Notably, the branched chain amino acid (BCAA: leucine, isoleucine, valine) biosynthesis operon was shown to be highly upregulated as well as the purine and pyrimidine biosynthesis pathways. Transcript levels of several stress- and virulence-related genes were significantly altered, implying an impact of growth in cantaloupe on the virulence potential of L. monocytogenes. Enhanced survival of L. monocytogenes in SGF following growth in cantaloupe further demonstrated the impact of cantaloupe-associated growth on the pathogen's subsequent response to a host relevant stress.

A Vegetable Fermentation Facility Hosts Distinct Microbiomes Reflecting the Production Environment.

Fermented vegetables are highly popular internationally in part due to their enhanced nutritional properties, cultural history, and desirable sensorial properties. In some instances, fermented foods provide a rich source of the beneficial microbial communities that could promote gastrointestinal health. The indigenous microbiota that colonize fermentation facilities may impact food quality, food safety, and spoilage risks and maintain the nutritive value of the product. Here, microbiomes within sauerkraut production facilities were profiled to characterize variance across surfaces and to determine the sources of these bacteria. Accordingly, we used high-throughput sequencing of the 16S rRNA gene in combination with whole-genome shotgun analyses to explore biogeographical patterns of microbial diversity and assembly within the production facility. Our results indicate that raw cabbage and vegetable handling surfaces exhibit more similar microbiomes relative to the fermentation room, processing area, and dry storage surfaces. We identified biomarker bacterial phyla and families that are likely to originate from the raw cabbage and vegetable handling surfaces. Raw cabbage was identified as the main source of bacteria to seed the facility, with human handling contributing a minor source of inoculation. Leuconostoc and Lactobacillaceae dominated all surfaces where spontaneous fermentation occurs, as these taxa are associated with the process. Wall, floor, ceiling, and barrel surfaces host unique microbial signatures. This study demonstrates that diverse bacterial communities are widely distributed within the production facility and that these communities assemble nonrandomly, depending on the surface type.IMPORTANCE Fermented vegetables play a major role in global food systems and are widely consumed by various global cultures. In this study, we investigated an industrial facility that produces spontaneous fermented sauerkraut without the aid of starter cultures. This provides a unique system to explore and track the origins of an "in-house" microbiome in an industrial environment. Raw vegetables and the surfaces on which they are handled were identified as the likely source of bacterial communities rather than human contamination. As fermented vegetables increase in popularity on a global scale, understanding their production environment may help maintain quality and safety goals.

Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia sp. Cryptic Lineage 1 Strain 7v Harbors a Hybrid Plasmid.

UNLABELLED: Hybrid isolates of Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) encoding heat-stable enterotoxin (ST) are being reported with increasing frequency from a variety of sources. However, information regarding the plasmids that these strains harbor is scarce. In this study, we sequence and characterize a plasmid, p7v, from the STEC/ETEC hybrid strain 7v. Whole-genome phylogenetic analyses of STEC/ETEC hybrid strains and prototype E. coli isolates of other pathotypes placed 7v in the Escherichia sp. cryptic lineage 1 (CL1) clade. The complete plasmid, p7v, was determined to be 229,275 bp and encodes putative virulence factors that are typically carried on STEC plasmids as well as those often carried on ETEC plasmids, indicating that the hybrid nature of the strain extends beyond merely encoding the two toxins. Plasmid p7v carries two copies of sta with identical sequences, which were discovered to be divergent from the sta sequences found in the prototype human ETEC strains. Using a nomenclature scheme based on a phylogeny constructed from sta and stb sequences, the sta encoded on p7v is designated STa4. In silico analysis determined that p7v also encodes the K88 fimbria, a colonization factor usually associated with porcine ETEC plasmids. The p7v sequence and the presence of plasmid-encoded virulence factors are compared to those of other STEC/ETEC CL1 hybrid genomes and reveal gene acquisition/loss at the strain level. In addition, the interrogation of 24 STEC/ETEC hybrid genomes for identification of plasmid replicons, colonization factors, Stx and ST subtypes, and other plasmid-encoded virulence genes highlights the diversity of these hybrid strains. IMPORTANCE: Hybrid Shiga toxin-producing Escherichia coli/enterotoxigenic Escherichia coli (STEC/ETEC) strains, which have been isolated from environmental, animal, and human clinical samples, may represent an emerging threat as food-borne pathogens. Characterization of these strains is important for assessing virulence potential, aiding in the development of pathogen detection methods, and understanding how the hybrid strains evolve to potentially have a greater impact on public health. This study represents, to our knowledge, both the first characterization of a closed plasmid sequence from a STEC/ETEC hybrid strain and the most comprehensive phylogenetic analysis of available STEC/ETEC hybrid genomes to date. The results demonstrate how the mobility of plasmid-associated virulence genes has resulted in the creation of a diverse plasmid repertoire within the STEC/ETEC hybrid strains.

FDA Escherichia coli Identification (FDA-ECID) Microarray: a Pangenome Molecular Toolbox for Serotyping, Virulence Profiling, Molecular Epidemiology, and Phylogeny.

UNLABELLED: Most Escherichia coli strains are nonpathogenic. However, for clinical diagnosis and food safety analysis, current identification methods for pathogenic E. coli either are time-consuming and/or provide limited information. Here, we utilized a custom DNA microarray with informative genetic features extracted from 368 sequence sets for rapid and high-throughput pathogen identification. The FDA Escherichia coli Identification (FDA-ECID) platform contains three sets of molecularly informative features that together stratify strain identification and relatedness. First, 53 known flagellin alleles, 103 alleles of wzx and wzy, and 5 alleles of wzm provide molecular serotyping utility. Second, 41,932 probe sets representing the pan-genome of E. coli provide strain-level gene content information. Third, approximately 125,000 single nucleotide polymorphisms (SNPs) of available whole-genome sequences (WGS) were distilled to 9,984 SNPs capable of recapitulating the E. coli phylogeny. We analyzed 103 diverse E. coli strains with available WGS data, including those associated with past foodborne illnesses, to determine robustness and accuracy. The array was able to accurately identify the molecular O and H serotypes, potentially correcting serological failures and providing better resolution for H-nontypeable/nonmotile phenotypes. In addition, molecular risk assessment was possible with key virulence marker identifications. Epidemiologically, each strain had a unique comparative genomic fingerprint that was extended to an additional 507 food and clinical isolates. Finally, a 99.7% phylogenetic concordance was established between microarray analysis and WGS using SNP-level data for advanced genome typing. Our study demonstrates FDA-ECID as a powerful tool for epidemiology and molecular risk assessment with the capacity to profile the global landscape and diversity of E. coli IMPORTANCE: This study describes a robust, state-of-the-art platform developed from available whole-genome sequences of E. coli and Shigella spp. by distilling useful signatures for epidemiology and molecular risk assessment into one assay. The FDA-ECID microarray contains features that enable comprehensive molecular serotyping and virulence profiling along with genome-scale genotyping and SNP analysis. Hence, it is a molecular toolbox that stratifies strain identification and pathogenic potential in the contexts of epidemiology and phylogeny. We applied this tool to strains from food, environmental, and clinical sources, resulting in significantly greater phylogenetic and strain-specific resolution than previously reported for available typing methods.

Application of metagenomic sequencing to food safety: detection of Shiga Toxin-producing Escherichia coli on fresh bagged spinach.

Culture-independent diagnostics reduce the reliance on traditional (and slower) culture-based methodologies. Here we capitalize on advances in next-generation sequencing (NGS) to apply this approach to food pathogen detection utilizing NGS as an analytical tool. In this study, spiking spinach with Shiga toxin-producing Escherichia coli (STEC) following an established FDA culture-based protocol was used in conjunction with shotgun metagenomic sequencing to determine the limits of detection, sensitivity, and specificity levels and to obtain information on the microbiology of the protocol. We show that an expected level of contamination (∼10 CFU/100 g) could be adequately detected (including key virulence determinants and strain-level specificity) within 8 h of enrichment at a sequencing depth of 10,000,000 reads. We also rationalize the relative benefit of static versus shaking culture conditions and the addition of selected antimicrobial agents, thereby validating the long-standing culture-based parameters behind such protocols. Moreover, the shotgun metagenomic approach was informative regarding the dynamics of microbial communities during the enrichment process, including initial surveys of the microbial loads associated with bagged spinach; the microbes found included key genera such as Pseudomonas, Pantoea, and Exiguobacterium. Collectively, our metagenomic study highlights and considers various parameters required for transitioning to such sequencing-based diagnostics for food safety and the potential to develop better enrichment processes in a high-throughput manner not previously possible. Future studies will investigate new species-specific DNA signature target regimens, rational design of medium components in concert with judicious use of additives, such as antibiotics, and alterations in the sample processing protocol to enhance detection.

Re-examination of the taxonomic status of Enterobacter helveticus, Enterobacter pulveris and Enterobacter turicensis as members of the genus Cronobacter and their reclassification in the genera Franconibacter gen. nov. and Siccibacter gen. nov. as Franconibacter helveticus comb. nov., Franconibacter pulveris comb. nov. and Siccibacter turicensis comb. nov., respectively.

Recently, a taxonomical re-evaluation of the genus Enterobacter, based on multi-locus sequence typing (MLST) analysis, has led to the proposal that the species Enterobacter pulveris, Enterobacter helveticus and Enterobacter turicensis should be reclassified as novel species of the genus Cronobacter. In the present work, new genome-scale analyses, including average nucleotide identity, genome-scale phylogeny and k-mer analysis, coupled with previously reported DNA-DNA hybridization values and biochemical characterization strongly indicate that these three species of the genus Enterobacter are not members of the genus Cronobacter, nor do they belong to the re-evaluated genus Enterobacter. Furthermore, data from this polyphasic study indicated that all three species constitute two new genera. We propose reclassifying Enterobacter pulveris and Enterobacter helveticus in the genus Franconibacter gen. nov. as Franconibacter pulveris comb. nov. (type strain 601/05(T) = LMG 24057(T) = DSM 19144(T)) and Franconibacter helveticus comb. nov. (type strain 513/05(T) = LMG 23732(T) = DSM 18396(T)), respectively, and Enterobacter turicensis in the genus Siccibacter gen. nov. as Siccibacter turicensis comb. nov. (type strain 508/05(T) = LMG 23730(T) = DSM 18397(T)).

Genomic anatomy of Escherichia coli O157:H7 outbreaks.

The rapid emergence of Escherichia coli O157:H7 from an unknown strain in 1982 to the dominant hemorrhagic E. coli serotype in the United States and the cause of widespread outbreaks of human food-borne illness highlights a need to evaluate critically the extent to which genomic plasticity of this important enteric pathogen contributes to its pathogenic potential and its evolution as well as its adaptation in different ecological niches. Aimed at a better understanding of the evolution of the E. coli O157:H7 pathogenome, the present study presents the high-quality sequencing and comparative phylogenomic analysis of a comprehensive panel of 25 E. coli O157:H7 strains associated with three nearly simultaneous food-borne outbreaks of human disease in the United States. Here we present a population genetic analysis of more than 200 related strains recovered from patients, contaminated produce, and zoonotic sources. High-resolution phylogenomic approaches allow the dynamics of pathogenome evolution to be followed at a high level of phylogenetic accuracy and resolution. SNP discovery and study of genome architecture and prophage content identified numerous biomarkers to assess the extent of genetic diversity within a set of clinical and environmental strains. A total of 1,225 SNPs were identified in the present study and are now available for typing of the E. coli O157:H7 lineage. These data should prove useful for the development of a refined phylogenomic framework for forensic, diagnostic, and epidemiological studies to define better risk in response to novel and emerging E. coli O157:H7 resistance and virulence phenotypes.

Evaluation of the Increased Genetic Resolution and Utility for Source Tracking of a Recently Developed Method for Genotyping Cyclospora cayetanensis.

Cyclospora cayetanensis is a foodborne parasite that causes cyclosporiasis, an enteric illness in humans. Genotyping methods are used to genetically discriminate between specimens from cyclosporiasis cases and can complement source attribution investigations if the method is sufficiently sensitive for application to food items. A very sensitive targeted amplicon sequencing (TAS) assay for genotyping C. cayetanensis encompassing 52 loci was recently designed. In this study, we analyzed 66 genetically diverse clinical specimens to assess the change in phylogenetic resolution between the TAS assay and a currently employed eight-marker scheme. Of the 52 markers, ≥50 were successfully haplotyped for all specimens, and these results were used to generate a hierarchical cluster dendrogram. Using a previously described statistical approach to dissect hierarchical trees, the 66 specimens resolved into 24 and 27 distinct genetic clusters for the TAS and an 8-loci scheme, respectively. Although the specimen composition of 15 clusters was identical, there were substantial differences between the two dendrograms, highlighting the importance of both inclusion of additional genome coverage and choice of loci to target for genotyping. To evaluate the ability to genetically link contaminated food samples with clinical specimens, C. cayetanensis was genotyped from DNA extracted from raspberries inoculated with fecal specimens. The contaminated raspberry samples were assigned to clusters with the corresponding clinical specimen, demonstrating the utility of the TAS assay for traceback efforts.

Divergent Aspergillus flavus corn population is composed of prolific conidium producers: Implications for saprophytic disease cycle.

The ascomycete fungus Aspergillus flavus infects and contaminates corn, peanuts, cottonseed, and tree nuts with toxic and carcinogenic aflatoxins. Subdivision between soil and host plant populations suggests that certain A. flavus strains are specialized to infect peanut, cotton, and corn despite having a broad host range. In this study, the ability of strains isolated from corn and/or soil in 11 Louisiana fields to produce conidia (field inoculum and male gamete) and sclerotia (resting bodies and female gamete) was assessed and compared with genotypic single-nucleotide polymorphism (SNP) differences between whole genomes. Corn strains produced upward of 47× more conidia than strains restricted to soil. Conversely, corn strains produced as much as 3000× fewer sclerotia than soil strains. Aspergillus flavus strains, typified by sclerotium diameter (small S-strains, <400 µm; large L-strains, >400 µm), belonged to separate clades. Several strains produced a mixture (M) of S and L sclerotia, and an intermediate number of conidia and sclerotia, compared with typical S-strains (minimal conidia, copious sclerotia) and L-strains (copious conidia, minimal sclerotia). They also belonged to a unique phylogenetic mixed (M) clade. Migration from soil to corn positively correlated with conidium production and negatively correlated with sclerotium production. Genetic differences correlated with differences in conidium and sclerotium production. Opposite skews in female (sclerotia) or male (conidia) gametic production by soil or corn strains, respectively, resulted in reduced effective breeding population sizes when comparing male:female gamete ratio with mating type distribution. Combining both soil and corn populations increased the effective breeding population, presumably due to contribution of male gametes from corn, which fertilize sclerotia on the soil surface. Incongruencies between aflatoxin clusters, strain morphotype designation, and whole genome phylogenies suggest a history of sexual reproduction within this Louisiana population, demonstrating the importance of conidium production, as infectious propagules and as fertilizers of the A. flavus soil population.

Development of a targeted amplicon sequencing method for genotyping Cyclospora cayetanensis from fresh produce and clinical samples with enhanced genomic resolution and sensitivity.

Outbreaks of cyclosporiasis, an enteric illness caused by the parasite Cyclospora cayetanensis, have been associated with consumption of various types of fresh produce. Although a method is in use for genotyping C. cayetanensis from clinical specimens, the very low abundance of C. cayetanensis in food and environmental samples presents a greater challenge. To complement epidemiological investigations, a molecular surveillance tool is needed for use in genetic linkage of food vehicles to cyclosporiasis illnesses, estimation of the scope of outbreaks or clusters of illness, and determination of geographical areas involved. We developed a targeted amplicon sequencing (TAS) assay that incorporates a further enrichment step to gain the requisite sensitivity for genotyping C. cayetanensis contaminating fresh produce samples. The TAS assay targets 52 loci, 49 of which are located in the nuclear genome, and encompasses 396 currently known SNP sites. The performance of the TAS assay was evaluated using lettuce, basil, cilantro, salad mix, and blackberries inoculated with C. cayetanensis oocysts. A minimum of 24 markers were haplotyped even at low contamination levels of 10 oocysts in 25 g leafy greens. The artificially contaminated fresh produce samples were included in a genetic distance analysis based on haplotype presence/absence with publicly available C. cayetanensis whole genome sequence assemblies. Oocysts from two different sources were used for inoculation, and samples receiving the same oocyst preparation clustered together, but separately from the other group, demonstrating the utility of the assay for genetically linking samples. Clinical fecal samples with low parasite loads were also successfully genotyped. This work represents a significant advance in the ability to genotype C. cayetanensis contaminating fresh produce along with greatly expanding the genomic diversity included for genetic clustering of clinical specimens.

Precision metagenomics sequencing for food safety: hybrid assembly of Shiga toxin-producing Escherichia coli in enriched agricultural water.

Culture-independent metagenomic sequencing of enriched agricultural water could expedite the detection and virulotyping of Shiga toxin-producing Escherichia coli (STEC). We previously determined the limits of a complete, closed metagenome-assembled genome (MAG) assembly and of a complete, fragmented MAG assembly for O157:H7 in enriched agricultural water using long reads (Oxford Nanopore Technologies, Oxford), which were 107 and 105 CFU/ml, respectively. However, the nanopore assemblies did not have enough accuracy to be used in Single Nucleotide Polymorphism (SNP) phylogenies and cannot be used for the precise identification of an outbreak STEC strain. The present study aimed to determine the limits of detection and assembly for STECs in enriched agricultural water by Illumina MiSeq sequencing technology alone, followed by establishing the limit of hybrid assembly with nanopore long-read sequencing using three different hybrid assemblers (SPAdes, Unicycler, and OPERA-MS). We also aimed to generate a genome with enough accuracy to be used in a SNP phylogeny. The classification of MiSeq and nanopore sequencing identified the same highly abundant species. Using the totality of the MiSeq output and a precision metagenomics approach in which the E. coli reads are binned before assembly, the limit of detection and assembly of STECs by MiSeq were determined to be 105 and 107 CFU/ml, respectively. While a complete, closed MAG could not be generated at any concentration, a complete, fragmented MAG was produced using the SPAdes assembler with an STEC concentration of at least 107 CFU/ml. At this concentration, hybrid assembled contigs aligned to the nanopore-assembled genome could be accurately placed in a neighbor-joining tree. The MiSeq limit of detection and assembly was less sensitive than nanopore sequencing, which was likely due to factors including the small starting material (50 vs. 1 µg) and the dilution of the library loaded on the cartridge. This pilot study demonstrates that MiSeq sequencing requires higher coverage in precision metagenomic samples; however, with sufficient concentration, STECs can be characterized and phylogeny can be accurately determined.

Draft Genome Sequences of 18 Streptococcus Strains Isolated from Live Dietary Supplements and Cultured Food Products.

We present the genome sequences of 18 Streptococcus isolates from 8 different dietary supplements and 9 cultured food products. Strains from this species naturally colonize the human mouth and upper respiratory tract. Studies have shown that S. thermophilus and S. salivarius strains confer oral health benefits to their host with little to no risk of pathogenic infection.

Draft Genome Sequences of Nine Bacillus Strains and Two Weizmannia Strains Isolated from Live Dietary Supplements and a Cultured Food Product.

We present the genome sequences of nine Bacillus isolates and two Weizmannia isolates from 10 different dietary supplements and one cultured food product. Strains of these species have been associated with health benefits when ingested by humans, due to their ability to survive the stomach's acidic environment and colonize the intestinal tract.

Phylogenomic Analysis of Salmonella enterica subsp. enterica Serovar Bovismorbificans from Clinical and Food Samples Using Whole Genome Wide Core Genes and kmer Binning Methods to Identify Two Distinct Polyphyletic Genome Pathotypes

Salmonella enterica subsp. enterica serovar Bovismorbificans has caused multiple outbreaks involving the consumption of produce, hummus, and processed meat products worldwide. To elucidate the intra-serovar genomic structure of S. Bovismorbificans, a core-genome analysis with 2690 loci (based on 150 complete genomes representing Salmonella enterica serovars developed as part of this study) and a k-mer-binning based strategy were carried out on 95 whole genome sequencing (WGS) assemblies from Swiss, Canadian, and USA collections of S. Bovismorbificans strains from foodborne infections. Data mining of a digital DNA tiling array of legacy SARA and SARB strains was conducted to identify near-neighbors of S. Bovismorbificans. The core genome analysis and the k-mer-binning methods identified two polyphyletic clusters, each with emerging evolutionary properties. Four STs (2640, 142, 1499, and 377), which constituted the majority of the publicly available WGS datasets from >260 strains analyzed by k-mer-binning based strategy, contained a conserved core genome backbone with a different evolutionary lineage as compared to strains comprising the other cluster (ST150). In addition, the assortment of genotypic features contributing to pathogenesis and persistence, such as antimicrobial resistance, prophage, plasmid, and virulence factor genes, were assessed to understand the emerging characteristics of this serovar that are relevant clinically and for food safety concerns. The phylogenomic profiling of polyphyletic S. Bovismorbificans in this study corresponds to intra-serovar variations observed in S. Napoli and S. Newport serovars using similar high-resolution genomic profiling approaches and contributes to the understanding of the evolution and sequence divergence of foodborne Salmonellae. These intra-serovar differences may have to be thoroughly understood for the accurate classification of foodborne Salmonella strains needed for the uniform development of future food safety mitigation strategies.

Comparative genomics of 28 Salmonella enterica isolates: evidence for CRISPR-mediated adaptive sublineage evolution.

Despite extensive surveillance, food-borne Salmonella enterica infections continue to be a significant burden on public health systems worldwide. As the S. enterica species comprises sublineages that differ greatly in antigenic representation, virulence, and antimicrobial resistance phenotypes, a better understanding of the species' evolution is critical for the prediction and prevention of future outbreaks. The roles that virulence and resistance phenotype acquisition, exchange, and loss play in the evolution of S. enterica sublineages, which to a certain extent are represented by serotypes, remains mostly uncharacterized. Here, we compare 17 newly sequenced and phenotypically characterized nontyphoidal S. enterica strains to 11 previously sequenced S. enterica genomes to carry out the most comprehensive comparative analysis of this species so far. These phenotypic and genotypic data comparisons in the phylogenetic species context suggest that the evolution of known S. enterica sublineages is mediated mostly by two mechanisms, (i) the loss of coding sequences with known metabolic functions, which leads to functional reduction, and (ii) the acquisition of horizontally transferred phage and plasmid DNA, which provides virulence and resistance functions and leads to increasing specialization. Matches between S. enterica clustered regularly interspaced short palindromic repeats (CRISPR), part of a defense mechanism against invading plasmid and phage DNA, and plasmid and prophage regions suggest that CRISPR-mediated immunity could control short-term phenotype changes and mediate long-term sublineage evolution. CRISPR analysis could therefore be critical in assessing the evolutionary potential of S. enterica sublineages and aid in the prediction and prevention of future S. enterica outbreaks.

Genome signatures of Escherichia coli O157:H7 isolates from the bovine host reservoir.

Cattle comprise a main reservoir of Shiga toxin-producing Escherichia coli O157:H7 (STEC). The significant differences in host prevalence, transmissibility, and virulence phenotypes among strains from bovine and human sources are of major interest to the public health community and livestock industry. Genomic analysis revealed divergence into three lineages: lineage I and lineage I/II strains are commonly associated with human disease, while lineage II strains are overrepresented in the asymptomatic bovine host reservoir. Growing evidence suggests that genotypic differences between these lineages, such as polymorphisms in Shiga toxin subtypes and synergistically acting virulence factors, are correlated with phenotypic differences in virulence, host ecology, and epidemiology. To assess the genomic plasticity on a genome-wide scale, we have sequenced the whole genome of strain EC869, a bovine-associated E. coli O157:H7 isolate. Comparative phylogenomic analysis of this key isolate enabled us to place accurately bovine lineage II strains within the genetically homogenous E. coli O157:H7 clade. Identification of polymorphic loci that are anchored both in the chromosomal backbone and horizontally acquired regions allowed us to associate bovine genotypes with altered virulence phenotypes and host prevalence. This study catalogued numerous novel lineage II-specific genome signatures, some of which appear to be associated intimately with the altered pathogenic potential and niche adaptation within the bovine rumen. The presented extended list of polymorphic markers is valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies of this emerging human pathogen.

Seasonality, shelf life and storage atmosphere are main drivers of the microbiome and E. coli O157:H7 colonization of post-harvest lettuce cultivated in a major production area in California.

BACKGROUND: Lettuce is linked to recurrent outbreaks of Shiga toxin-producing Escherichia coli (STEC) infections, the seasonality of which remains unresolved. Infections have occurred largely from processed lettuce, which undergoes substantial physiological changes during storage. We investigated the microbiome and STEC O157:H7 (EcO157) colonization of fresh-cut lettuce of two cultivars with long and short shelf life harvested in the spring and fall in California and stored in modified atmosphere packaging (MAP) at cold and warm temperatures. RESULTS: Inoculated EcO157 declined significantly less on the cold-stored cultivar with short shelf life, while multiplying rapidly at 24 °C independently of cultivar. Metagenomic sequencing of the lettuce microbiome revealed that the pre-storage bacterial community was variable but dominated by species in the Erwiniaceae and Pseudomonadaceae. After cold storage, the microbiome composition differed between cultivars, with a greater relative abundance (RA) of Erwiniaceae and Yersiniaceae on the cultivar with short shelf life. Storage at 24 °C shifted the microbiome to higher RAs of Erwiniaceae and Enterobacteriaceae and lower RA of Pseudomonadaceae compared with 6 °C. Fall harvest followed by lettuce deterioration were identified by recursive partitioning as important factors associated with high EcO157 survival at 6 °C, whereas elevated package CO2 levels correlated with high EcO157 multiplication at 24 °C. EcO157 population change correlated with the lettuce microbiome during 6 °C storage, with fall microbiomes supporting the greatest EcO157 survival on both cultivars. Fall and spring microbiomes differed before and during storage at both temperatures. High representation of Pantoea agglomerans was a predictor of fall microbiomes, lettuce deterioration, and enhanced EcO157 survival at 6 °C. In contrast, higher RAs of Erwinia persicina, Rahnella aquatilis, and Serratia liquefaciens were biomarkers of spring microbiomes and lower EcO157 survival. CONCLUSIONS: The microbiome of processed MAP lettuce evolves extensively during storage. Under temperature abuse, high CO2 promotes a lettuce microbiome enriched in taxa with anaerobic capability and EcO157 multiplication. In cold storage, our results strongly support a role for season and lettuce deterioration in EcO157 survival and microbiome composition, suggesting that the physiology and microbiomes of fall- and spring-harvested lettuce may contribute to the seasonality of STEC outbreaks associated with lettuce grown in coastal California.